**Proteasome Inhibitors in the Treatment of Multiple Myeloma**

Lisa J. Crawford and Alexandra E. Irvine *Centre for Cancer Research and Cell Biology, Queen's University Belfast Northern Ireland* 

#### **1. Introduction**

The ubiquitin proteasome system is responsible for the degradation of proteins involved in a wide range of cellular processes such as the cell cycle, apoptosis, transcription, cell signalling, immune response and antigen presentation. Protein homeostasis is essential for normal cell growth and inhibition of proteasome function has emerged as a viable strategy for anti-cancer treatment. The first proteasome inhibitor to enter clinical practice, bortezomib, was approved by the Food and Drug Administration as a single agent to treat relapsed/refractory Multiple Myeloma in 2003 and expanded to first-line treatment in combination with melphalan and prednisone in 2008. It is now a routine component of Multiple Myeloma therapy and has had a major impact on expanding treatment options in the last few years. Bortezomib exhibits novel action against Multiple Myeloma by targeting both intracellular mechanisms and interactions within the bone marrow environment. Although it demonstrates significant anti-Myeloma activity when used alone, it has been shown to have even greater benefits when used in combination with conventional and novel chemotherapeutic agents. There are currently over 200 clinical trials ongoing or recently completed examining bortezomib alone and in combination in various stages of disease and treatment. The clinical success of bortezomib has prompted the development of a number of second generation proteasome inhibitors with improved pharmacological properties. In this chapter, we review the development of bortezomib as a novel therapeutic agent in Multiple Myeloma and summarize the key observations from recently completed and ongoing studies on the effect of bortezomib both as a single agent and in combination therapies in the setting of newly diagnosed Multiple Myeloma and for relapsed disease. We also discuss the progress of next generation proteasome inhibitors in the clinic.

#### **2. The ubiquitin proteasome system**

The ubiquitin proteasome pathway represents the major pathway for intracellular protein degradation. It is responsible for the degradation of approximately 80% of cellular proteins, including misfolded and mutated proteins as well as those involved in the regulation of development, differentiation, cell proliferation, signal transduction, apoptosis and antigen presentation. Proteins are degraded by the ubiquitin proteasome pathway via two distinct and successive steps: the covalent attachment of multiple monomers of ubiquitin molecules to a protein substrate and degradation of the tagged protein by the 26S proteasome. Tagging

Proteasome Inhibitors in the Treatment of Multiple Myeloma 5

Fig. 1. a. Ubiquitin proteasome pathway mediated degradation. b. Proteasome composition.

(Imajoh-Ohmi et al., 1995; Shinohara et al., 1996; Drexler, 1997) and were active in an *in vivo* model of Burkitt's lymphoma (Orlowski et al., 1998). Further *in vitro* investigations demonstrated that proteasome inhibitors displayed a broad spectrum anti-proliferative and pro-apoptotic activity against haematological and solid tumours. While these studies

of a protein by ubiquitin requires the action of three classes of enzymes – ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3). A single E1 enzyme activates ubiquitin by forming a thiol ester bond between E1 and ubiquitin in an ATP-dependent step. Following activation, ubiquitin is then transferred to an active site residue within an E2 enzyme which shuttles ubiquitin either directly or in concert with an E3 enzyme to a lysine residue in the target protein. There are more than 30 different E2 and over 500 E3 enzymes, which work in cooperation to confer exquisite substrate specificity to the ubiquitin proteasome pathway. The successive conjugation of ubiquitin moieties generates a polyubiquitin chain that acts as a signal to target the protein for degradation by the 26S proteasome (Figure 1a).

The 26S or constitutive proteasome is found in the nucleus and cytoplasm of all eukaryotic cells. It is composed of a core 20S particle capped with a 19S structure at each end. The 20S catalytic core is made up of 28 subunits arranged into four stacked rings, creating a central chamber where proteolysis occurs. The two outer rings are composed of 7 different α subunits, which are predominantly structural and the two inner rings are composed of 7 different β subunits, at least three of which contain catalytic sites (Groll et al., 1997). Catalytic activities of the proteasome are classified into three major categories, based upon preference to cleave a peptide bond after a particular amino acid residue. These activities are referred to as chymotrypsin-like, trypsin-like and caspase-like and are associated with β5, β2 and β1 subunits respectively. The chymotrypsin-like activity cleaves after hydrophobic residues, the trypsin-like activity cleaves after basic residues and the caspase-like activity cleaves after acidic residues (Groll et al., 1999; Heinemeyer et al., 1997). Substrates gain access to the proteolytic chamber by binding to the 19S regulatory particle at either end of the 20S proteasome. Polyubiquitin-tagged proteins are recognised by the 19S particle, where ubiquitin is cleaved off and recycled and the target protein is unfolded and fed into the 20S catalytic chamber (Groll et al., 2000; Navon & Goldberg, 2001). An alternative proteasome isoform known as the immunoproteasome can be formed in response to cytokine signalling. Interferon-γ and tumour necrosis factor – α induce the expression of a different set of catalytic β-subunits and regulatory cap to form the immunoproteasome. Subunits β1i (LMP2), β2i (MECL1) and β5i (LMP7) replace constitutive subunits β1, β2 and β5 and the 19S regulatory cap is replaced with an 11S regulatory structure (Figure 1b). These modifications allow the immunoproteasome to generate antigenic peptides for presentation by the major histocompatability (MHC) class 1 mediated immune response (Rock & Goldberg, 1999). The expression of the immunoproteasome appears to be tissue specific and is particularly abundant in immune-related cells. Immunoproteasomes are highly expressed in haemopoietic tumours such as Multiple Myeloma.

#### **3. Proteasome inhibitors as drug candidates**

As the ubiquitin proteasome pathway plays a critical role in regulating many cellular processes, it is not surprising that defects within this pathway have been associated with a number of pathologies, including neurodegenerative diseases and cancer. Proteasome inhibitors were initially synthesized as *in vitro* probes to investigate the function of the proteasome's catalytic activity. However, as the essential role of the proteasome in cell function was established, the proteasome emerged as an attractive target for cancer therapy. Early studies showed that proteasome inhibitors induced apoptosis in leukaemic cell lines

of a protein by ubiquitin requires the action of three classes of enzymes – ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3). A single E1 enzyme activates ubiquitin by forming a thiol ester bond between E1 and ubiquitin in an ATP-dependent step. Following activation, ubiquitin is then transferred to an active site residue within an E2 enzyme which shuttles ubiquitin either directly or in concert with an E3 enzyme to a lysine residue in the target protein. There are more than 30 different E2 and over 500 E3 enzymes, which work in cooperation to confer exquisite substrate specificity to the ubiquitin proteasome pathway. The successive conjugation of ubiquitin moieties generates a polyubiquitin chain that acts as a signal to target the protein for

The 26S or constitutive proteasome is found in the nucleus and cytoplasm of all eukaryotic cells. It is composed of a core 20S particle capped with a 19S structure at each end. The 20S catalytic core is made up of 28 subunits arranged into four stacked rings, creating a central chamber where proteolysis occurs. The two outer rings are composed of 7 different α subunits, which are predominantly structural and the two inner rings are composed of 7 different β subunits, at least three of which contain catalytic sites (Groll et al., 1997). Catalytic activities of the proteasome are classified into three major categories, based upon preference to cleave a peptide bond after a particular amino acid residue. These activities are referred to as chymotrypsin-like, trypsin-like and caspase-like and are associated with β5, β2 and β1 subunits respectively. The chymotrypsin-like activity cleaves after hydrophobic residues, the trypsin-like activity cleaves after basic residues and the caspase-like activity cleaves after acidic residues (Groll et al., 1999; Heinemeyer et al., 1997). Substrates gain access to the proteolytic chamber by binding to the 19S regulatory particle at either end of the 20S proteasome. Polyubiquitin-tagged proteins are recognised by the 19S particle, where ubiquitin is cleaved off and recycled and the target protein is unfolded and fed into the 20S catalytic chamber (Groll et al., 2000; Navon & Goldberg, 2001). An alternative proteasome isoform known as the immunoproteasome can be formed in response to cytokine signalling. Interferon-γ and tumour necrosis factor – α induce the expression of a different set of catalytic β-subunits and regulatory cap to form the immunoproteasome. Subunits β1i (LMP2), β2i (MECL1) and β5i (LMP7) replace constitutive subunits β1, β2 and β5 and the 19S regulatory cap is replaced with an 11S regulatory structure (Figure 1b). These modifications allow the immunoproteasome to generate antigenic peptides for presentation by the major histocompatability (MHC) class 1 mediated immune response (Rock & Goldberg, 1999). The expression of the immunoproteasome appears to be tissue specific and is particularly abundant in immune-related cells. Immunoproteasomes are highly expressed

As the ubiquitin proteasome pathway plays a critical role in regulating many cellular processes, it is not surprising that defects within this pathway have been associated with a number of pathologies, including neurodegenerative diseases and cancer. Proteasome inhibitors were initially synthesized as *in vitro* probes to investigate the function of the proteasome's catalytic activity. However, as the essential role of the proteasome in cell function was established, the proteasome emerged as an attractive target for cancer therapy. Early studies showed that proteasome inhibitors induced apoptosis in leukaemic cell lines

degradation by the 26S proteasome (Figure 1a).

in haemopoietic tumours such as Multiple Myeloma.

**3. Proteasome inhibitors as drug candidates** 

Fig. 1. a. Ubiquitin proteasome pathway mediated degradation. b. Proteasome composition.

(Imajoh-Ohmi et al., 1995; Shinohara et al., 1996; Drexler, 1997) and were active in an *in vivo* model of Burkitt's lymphoma (Orlowski et al., 1998). Further *in vitro* investigations demonstrated that proteasome inhibitors displayed a broad spectrum anti-proliferative and pro-apoptotic activity against haematological and solid tumours. While these studies

Proteasome Inhibitors in the Treatment of Multiple Myeloma 7

canonical and non-canonical pathways of activation. As MLN2238 is structurally distinct from bortezomib, this suggests that different proteasome inhibitors may exert differential

Apoptosis is regulated by the opposing activities of pro-apoptotic and anti-apoptotic molecules. Cancer cells often have disregulated apoptotic signalling pathways with increased levels of anti-apoptotic proteins which provide a survival advantage and confer resistance to chemotherapeutic agents. Inhibition of proteasome activity by bortezomib is associated with an upregulation of pro-apoptotic factors such as p53, Bik, BIM and NOXA and a related decrease in anti-apoptotic proteins such as Bcl-XL and Bcl-2. Induction of NOXA has been reported to be a key mechanism in bortezomib-mediated apoptosis which is independent of p53 status but dependent on c-Myc (Qin et al., 2005; Gomez-Bougie et al., 2007; Nikiforov et al., 2007; Fennell et al., 2008). Bortezomib-mediated apoptosis is accompanied by induction of c-Jun-NH2 terminal kinase, generation of reactive oxygen species, release of cytochrome c, second mitochondria-derived activator of caspases and apoptosis-inducing factor and activation of the intrinsic caspase-8 pathway and extrinsic

The endoplasmic reticulum plays a central role in protein homeostasis. Proteins are processed and folded in the lumen of the endoplasmic reticulum and misfolded proteins are returned to the cytosol and degraded in the proteasome. Multiple Myeloma cells have a high rate of protein synthesis and this is inherently associated with a high level of misfolded proteins. Accumulation of misfolded proteins in the endoplasmic reticulum triggers the Unfolded Protein Response. This process is mediated through three endoplasmic reticulum transmembrane receptors: ATF6, IRE1 and PERK. In resting cells the endoplasmic reticulum chaperone BiP (GRP 78) maintains these receptors in a resting state; BiP becomes dissociated from the endoplasmic reticulum receptors when unfolded proteins accumulate and triggers

It has been recognised for some time that bortezomib can induce the Unfolded Protein Response in Multiple Myeloma cells and that this contributes to its pro-apoptotic activity (Obeng et al., 2006; Meister et al., 2007). Numerous studies have now shown that treatment of Multiple Myeloma cell lines *in vitro* with bortezomib triggers activation of ATF6, IRE 1 and PERK (Davenport et al., 2007; Gu et al., 2008; Dong et al., 2009). Caspase 2 is believed to act upstream of mitochondrial signalling in this bortezomib ER stress- induced apoptosis (Gu et al., 2008). Similar mechanisms have been implicated in mantle cell lymphoma cell lines (Rao et al., 2010; Roue et al., 2011). It is clear that a greater understanding of the Unfolded Protein Response is fundamental to allow the rational development of

Interactions between Multiple Myeloma cells and bone marrow stroma regulate the growth and survival of Myeloma cells and play a critical role in angiogenesis, bone disease and

effects on the NFκB pathway by blocking either one or both pathways of activation.

**4.2 Apoptosis** 

caspase-9 pathway.

**4.3 Unfolded protein response** 

the Unfolded Protein Response.

combination therapies (Kawaguchi et al., 2011).

**4.4 Bone marrow microenvironment** 

established the potential of proteasome inhibitors as anti-cancer agents, many of the compounds available were limited to laboratory studies due to a relative lack of potency, specificity or stability. This led to the development of a series of dipeptide boronic acids, which were more potent and selective than many previously available inhibitors. These inhibitors were screened *in vitro* against the National Cancer Institute's panel of cancer cell lines and on the basis of its cytotoxicity, the compound bortezomib (PS-341, Velcade®) was brought forward for further testing.

#### **4. Mechanisms of action of bortezomib in multiple myeloma**

Bortezomib is a reversible proteasome inhibitor, primarily of the chymotrypsin-like activity of both the constitutive (β5) and immunoproteasome (LMP7) (Lightcap et al., 2000; Crawford et al., 2006). Initial *in vitro* evaluation of bortezomib demonstrated that it induced an accumulation of intracellular proteins, leading to G2-M arrest and then apoptosis through dual activation of caspase – 8 and caspase - 9 (Adams et al., 1999, Mitsiades et al., 2002). Importantly, bortezomib was also demonstrated to be significantly more toxic to Multiple Myeloma tumour cells than to normal counterparts. Hideshima et al. (2001) demonstrated that Multiple Myeloma cell lines and primary patient cells were 20-40 times more sensitive to bortezomib-induced apoptosis than bone marrow or peripheral blood mononuclear cells from healthy donors. Another novel aspect for bortezomib in Multiple Myeloma was that it was found to act not only on the Multiple Myeloma cells themselves but also on the protective bone marrow microenvironment. In addition, inhibition of proteasome function was found to both sensitize tumour cells to conventional chemotherapy and to overcome chemotherapy resistance. Finally, studies in murine xenograft models demonstrated that bortezomib significantly inhibited Multiple Myeloma cell growth and angiogenesis and prolonged survival (Leblanc et al., 2002). The main mechanisms attributed to bortezomib-induced apoptosis in Multiple Myeloma are outlined below.

#### **4.1 NFκB**

One of the first mechanisms of action attributed to bortezomib in Multiple Myeloma was inhibition of the inflammation associated transcription factor NFκB. NFκB, is constitutively activated in Multiple Myeloma and plays an important role in cell survival, proliferation and resistance to cytotoxic agents. NFκB is bound to its inhibitor IκB in the cytoplasm and is activated by proteasomal degradation of IκB. When activated, this transcription factor induces the expression of cell adhesion molecules (e.g. vascular cell adhesion molecule) and anti-apoptotic proteins (e.g. Bcl-2 and XIAP) and increases interleukin-6 production in bone marrow stromal cells. There are two pathways which activate NFκB, known as the canonical (or classical) pathway and the alternative non-canonical pathway (Gilmore, 2006). Inhibition of proteasome activity was demonstrated to prevent degradation of IκB and subsequent activation and translocation of NFκB to the nucleus to activate downstream pathways (Hideshima et al., 2001; Russo et al., 2001; Sunwoo et al., 2001). However, recent studies are challenging the concept that proteasome inhibitors inhibit NFκB activation and suggest that bortezomib may actually activate upstream NFκB activating kinases via the canonical pathway and increase NFκB activity (Markovina et al., 2008; Hideshima et al., 2009). In contrast, Chauhan et al. (2011) recently assessed the action of the second generation proteasome inhibitor MLN2238 on NFκB and report that this compound inhibits both the canonical and non-canonical pathways of activation. As MLN2238 is structurally distinct from bortezomib, this suggests that different proteasome inhibitors may exert differential effects on the NFκB pathway by blocking either one or both pathways of activation.

#### **4.2 Apoptosis**

6 Multiple Myeloma – An Overview

established the potential of proteasome inhibitors as anti-cancer agents, many of the compounds available were limited to laboratory studies due to a relative lack of potency, specificity or stability. This led to the development of a series of dipeptide boronic acids, which were more potent and selective than many previously available inhibitors. These inhibitors were screened *in vitro* against the National Cancer Institute's panel of cancer cell lines and on the basis of its cytotoxicity, the compound bortezomib (PS-341, Velcade®) was

Bortezomib is a reversible proteasome inhibitor, primarily of the chymotrypsin-like activity of both the constitutive (β5) and immunoproteasome (LMP7) (Lightcap et al., 2000; Crawford et al., 2006). Initial *in vitro* evaluation of bortezomib demonstrated that it induced an accumulation of intracellular proteins, leading to G2-M arrest and then apoptosis through dual activation of caspase – 8 and caspase - 9 (Adams et al., 1999, Mitsiades et al., 2002). Importantly, bortezomib was also demonstrated to be significantly more toxic to Multiple Myeloma tumour cells than to normal counterparts. Hideshima et al. (2001) demonstrated that Multiple Myeloma cell lines and primary patient cells were 20-40 times more sensitive to bortezomib-induced apoptosis than bone marrow or peripheral blood mononuclear cells from healthy donors. Another novel aspect for bortezomib in Multiple Myeloma was that it was found to act not only on the Multiple Myeloma cells themselves but also on the protective bone marrow microenvironment. In addition, inhibition of proteasome function was found to both sensitize tumour cells to conventional chemotherapy and to overcome chemotherapy resistance. Finally, studies in murine xenograft models demonstrated that bortezomib significantly inhibited Multiple Myeloma cell growth and angiogenesis and prolonged survival (Leblanc et al., 2002). The main mechanisms attributed to bortezomib-induced

One of the first mechanisms of action attributed to bortezomib in Multiple Myeloma was inhibition of the inflammation associated transcription factor NFκB. NFκB, is constitutively activated in Multiple Myeloma and plays an important role in cell survival, proliferation and resistance to cytotoxic agents. NFκB is bound to its inhibitor IκB in the cytoplasm and is activated by proteasomal degradation of IκB. When activated, this transcription factor induces the expression of cell adhesion molecules (e.g. vascular cell adhesion molecule) and anti-apoptotic proteins (e.g. Bcl-2 and XIAP) and increases interleukin-6 production in bone marrow stromal cells. There are two pathways which activate NFκB, known as the canonical (or classical) pathway and the alternative non-canonical pathway (Gilmore, 2006). Inhibition of proteasome activity was demonstrated to prevent degradation of IκB and subsequent activation and translocation of NFκB to the nucleus to activate downstream pathways (Hideshima et al., 2001; Russo et al., 2001; Sunwoo et al., 2001). However, recent studies are challenging the concept that proteasome inhibitors inhibit NFκB activation and suggest that bortezomib may actually activate upstream NFκB activating kinases via the canonical pathway and increase NFκB activity (Markovina et al., 2008; Hideshima et al., 2009). In contrast, Chauhan et al. (2011) recently assessed the action of the second generation proteasome inhibitor MLN2238 on NFκB and report that this compound inhibits both the

**4. Mechanisms of action of bortezomib in multiple myeloma** 

brought forward for further testing.

apoptosis in Multiple Myeloma are outlined below.

**4.1 NFκB** 

Apoptosis is regulated by the opposing activities of pro-apoptotic and anti-apoptotic molecules. Cancer cells often have disregulated apoptotic signalling pathways with increased levels of anti-apoptotic proteins which provide a survival advantage and confer resistance to chemotherapeutic agents. Inhibition of proteasome activity by bortezomib is associated with an upregulation of pro-apoptotic factors such as p53, Bik, BIM and NOXA and a related decrease in anti-apoptotic proteins such as Bcl-XL and Bcl-2. Induction of NOXA has been reported to be a key mechanism in bortezomib-mediated apoptosis which is independent of p53 status but dependent on c-Myc (Qin et al., 2005; Gomez-Bougie et al., 2007; Nikiforov et al., 2007; Fennell et al., 2008). Bortezomib-mediated apoptosis is accompanied by induction of c-Jun-NH2 terminal kinase, generation of reactive oxygen species, release of cytochrome c, second mitochondria-derived activator of caspases and apoptosis-inducing factor and activation of the intrinsic caspase-8 pathway and extrinsic caspase-9 pathway.

#### **4.3 Unfolded protein response**

The endoplasmic reticulum plays a central role in protein homeostasis. Proteins are processed and folded in the lumen of the endoplasmic reticulum and misfolded proteins are returned to the cytosol and degraded in the proteasome. Multiple Myeloma cells have a high rate of protein synthesis and this is inherently associated with a high level of misfolded proteins. Accumulation of misfolded proteins in the endoplasmic reticulum triggers the Unfolded Protein Response. This process is mediated through three endoplasmic reticulum transmembrane receptors: ATF6, IRE1 and PERK. In resting cells the endoplasmic reticulum chaperone BiP (GRP 78) maintains these receptors in a resting state; BiP becomes dissociated from the endoplasmic reticulum receptors when unfolded proteins accumulate and triggers the Unfolded Protein Response.

It has been recognised for some time that bortezomib can induce the Unfolded Protein Response in Multiple Myeloma cells and that this contributes to its pro-apoptotic activity (Obeng et al., 2006; Meister et al., 2007). Numerous studies have now shown that treatment of Multiple Myeloma cell lines *in vitro* with bortezomib triggers activation of ATF6, IRE 1 and PERK (Davenport et al., 2007; Gu et al., 2008; Dong et al., 2009). Caspase 2 is believed to act upstream of mitochondrial signalling in this bortezomib ER stress- induced apoptosis (Gu et al., 2008). Similar mechanisms have been implicated in mantle cell lymphoma cell lines (Rao et al., 2010; Roue et al., 2011). It is clear that a greater understanding of the Unfolded Protein Response is fundamental to allow the rational development of combination therapies (Kawaguchi et al., 2011).

#### **4.4 Bone marrow microenvironment**

Interactions between Multiple Myeloma cells and bone marrow stroma regulate the growth and survival of Myeloma cells and play a critical role in angiogenesis, bone disease and

Proteasome Inhibitors in the Treatment of Multiple Myeloma 9

implicated in pro-apoptotic cascades, as well as upregulation of heat shock proteins and ubiquitin proteasome pathway members. Chen et al. (2010) performed a genome-wide siRNA screen in malignant cell lines to evaluate the genetic determinants that confer sensitivity to bortezomib. They found that bortezomib promotes apoptosis primarily by disregulating Myc and polyamines, interfering with protein translation and disrupting DNA damage repair pathways. More recently, Takeda and colleagues (2011) investigated genes affecting the toxicity of bortezomib in the fission yeast *S. pombe* and identified factors involved in the ubiquitin proteasome pathway, chromatin silencing, nuclear/cytoplasmic transportation, amino acid metabolism and vesicular trafficking. Gene expression profiling of Multiple Myeloma patients found that treatment with bortezomib resulted in an upregulation of proteasome genes and that high levels of the proteasome subunit PSMD4 was associated with an inferior prognosis (Shaughnessy et al., 2011). Further investigation into fully understanding the mechanism of action of bortezomib will help to identify therapeutic strategies to overcome resistance to bortezomib and to identify agents to

enhance its efficacy.

Phase 1

Phase 2

**5. Clinical use of bortezomib in Multiple Myeloma** 

findings of the trials are outlined below.

**5.1 Bortezomib therapy for relapsed or refractory Multiple Myeloma** 

trials with bortezomib for the treatment of relapsed, refractory Myeloma.

Following encouraging preclinical results bortezomib was introduced into clinical trials to test for safety and efficacy in relapsed and refractory Multiple Myeloma. These studies established that bortezomib was effective and well-tolerated in Multiple Myeloma and led to approval of bortezomib in patients that had undergone at least two prior therapies. The incorporation of bortezomib into the treatment options for Myeloma represented a significant milestone as being the first proteasome inhibitor to be implemented into clinical use and also as the first novel therapy for Multiple Myeloma in over a decade. The main

Orlowski et al. (2002) conducted a Phase 1 trial evaluating the pharmacodynamics of bortezomib, along with toxicity and clinical responses in 27 patients with advanced refractory haematological malignancies. This study demonstrated that bortezomib could be safely administered, with a tolerable side-effect profile. There was significant evidence of anti-tumour activity in patients with Multiple Myeloma, with all 9 evaluable Multiple Myeloma patients showing some evidence of clinical benefit, including one complete response. Taken together with preclinical data this provided the rationale for Phase 2 clinical

The activity of bortezomib in relapsed and refractory Multiple Myeloma was confirmed with two Phase 2 trials, SUMMIT and CREST. SUMMIT (Study of Uncontrolled Multiple Myeloma managed with proteasome Inhibition Therapy) was a large multi-centre trial that enrolled 202 heavily pre-treated patients (Richardson et al., 2003). An overall response rate of 35% was achieved, including 10% of patients who achieved a complete or near complete response. Median time to progression was 7 months compared with 3 months on previous therapy. Grade 3 toxicities included cyclical thrombocytopenia, fatigue, peripheral

drug resistance. The success of bortezomib in Multiple Myeloma has been attributed not only to direct effects on Myeloma cells but also its effect on the bone marrow microenvironment. Vascular cell adhesion molecule-1 is a major ligand on bone marrow stromal cells that mediates binding of Multiple Myeloma cells via the cell surface molecule very late antigen-4. Early studies on proteasome inhibitors demonstrated that they downregulate cytokine-induced expression of vascular cell adhesion molecule-1 (Read et al., 1995). Hideshima et al., (2001) subsequently reported that treatment with bortezomib decreased binding of Myeloma cells to bone marrow stromal cells by 50% and consequently inhibited the related upregulation of interleukin-6 secretion and paracrine tumour growth.

Bortezomib has also been demonstrated to have both direct and indirect effects on angiogenesis. Initial studies found that bortezomib treatment decreased the secretion of vascular endothelial growth factor from Myeloma cells, thereby decreasing vasculogenesis and angiogenesis (Nawrocki et al., 2002). More recent studies using functional assays including chemotaxis, adhesion to fibronectin and capillary formation demonstrated that bortezomib has direct anti-proliferative effects on vascular endothelial cells. Tamura et al. (2010) demonstrated that bortezomib potently inhibits cell growth of vascular endothelial cells by suppressing the G2/M transition of the cell cycle and increasing permeability, thus acting as a vascular targeting drug.

A critical role of the bone marrow microenvironment in the efficacy of bortezomib in Multiple Myeloma was further established by Edwards et al. (2009). *In vivo* studies demonstrated that bortezomib had a greater effect on tumour burden when Myeloma cells were grown in the bone marrow of mice than when they were grown at sub-cutaneous sites.

#### **4.5 Bortezomib and bone formation**

Osteolytic lesions characterised with activated osteoclast activity accompanied with a reduction in osteoblast activity are a major feature of Multiple Myeloma. Bortezomib exhibits important effects on the development and progression of Myeloma-associated bone disease by reducing osteoclast activity and increasing osteoblast function, therefore reducing bone resorption and stimulating new bone formation. Both preclinical and clinical analysis have demonstrated that bortezomib exerts these effects in part by inhibiting dickkopf-1 and receptor activator of nuclear factor-kappa B ligand and increasing levels of alkaline phosphatase and osteocalcin (Terpos et al., 2006; Heider et al., 2006; Giuliani et al., 2007). However, a recent study by Lund and colleagues (2010) found that the combination of a glucocorticoid such as dexamethasone with bortezomib could inhibit the positive effects of bortezomib on osteoblast proliferation and differentiation, suggesting that bortezomib may result in better healing of osteolytic lesions when used without a glucocorticoid.

#### **4.6 Gene expression studies**

While a number of mechanisms of action of bortezomib have been outlined above, the full mechanism of bortezomib-induced cytotoxicity remains to be elucidated. Gene expression studies have been employed to try and increase our understanding of the cytotoxic action of this compound in Multiple Myeloma. Mitsiades et al., (2002) performed gene expression profiling in a Multiple Myeloma cell line and demonstrated that bortezomib resulted in a downregulation of growth and survival signalling pathways and upregulation of molecules implicated in pro-apoptotic cascades, as well as upregulation of heat shock proteins and ubiquitin proteasome pathway members. Chen et al. (2010) performed a genome-wide siRNA screen in malignant cell lines to evaluate the genetic determinants that confer sensitivity to bortezomib. They found that bortezomib promotes apoptosis primarily by disregulating Myc and polyamines, interfering with protein translation and disrupting DNA damage repair pathways. More recently, Takeda and colleagues (2011) investigated genes affecting the toxicity of bortezomib in the fission yeast *S. pombe* and identified factors involved in the ubiquitin proteasome pathway, chromatin silencing, nuclear/cytoplasmic transportation, amino acid metabolism and vesicular trafficking. Gene expression profiling of Multiple Myeloma patients found that treatment with bortezomib resulted in an upregulation of proteasome genes and that high levels of the proteasome subunit PSMD4 was associated with an inferior prognosis (Shaughnessy et al., 2011). Further investigation into fully understanding the mechanism of action of bortezomib will help to identify therapeutic strategies to overcome resistance to bortezomib and to identify agents to enhance its efficacy.

#### **5. Clinical use of bortezomib in Multiple Myeloma**

#### **5.1 Bortezomib therapy for relapsed or refractory Multiple Myeloma**

Following encouraging preclinical results bortezomib was introduced into clinical trials to test for safety and efficacy in relapsed and refractory Multiple Myeloma. These studies established that bortezomib was effective and well-tolerated in Multiple Myeloma and led to approval of bortezomib in patients that had undergone at least two prior therapies. The incorporation of bortezomib into the treatment options for Myeloma represented a significant milestone as being the first proteasome inhibitor to be implemented into clinical use and also as the first novel therapy for Multiple Myeloma in over a decade. The main findings of the trials are outlined below.

Phase 1

8 Multiple Myeloma – An Overview

drug resistance. The success of bortezomib in Multiple Myeloma has been attributed not only to direct effects on Myeloma cells but also its effect on the bone marrow microenvironment. Vascular cell adhesion molecule-1 is a major ligand on bone marrow stromal cells that mediates binding of Multiple Myeloma cells via the cell surface molecule very late antigen-4. Early studies on proteasome inhibitors demonstrated that they downregulate cytokine-induced expression of vascular cell adhesion molecule-1 (Read et al., 1995). Hideshima et al., (2001) subsequently reported that treatment with bortezomib decreased binding of Myeloma cells to bone marrow stromal cells by 50% and consequently inhibited the related upregulation of interleukin-6 secretion and paracrine tumour growth. Bortezomib has also been demonstrated to have both direct and indirect effects on angiogenesis. Initial studies found that bortezomib treatment decreased the secretion of vascular endothelial growth factor from Myeloma cells, thereby decreasing vasculogenesis and angiogenesis (Nawrocki et al., 2002). More recent studies using functional assays including chemotaxis, adhesion to fibronectin and capillary formation demonstrated that bortezomib has direct anti-proliferative effects on vascular endothelial cells. Tamura et al. (2010) demonstrated that bortezomib potently inhibits cell growth of vascular endothelial cells by suppressing the G2/M transition of the cell cycle and increasing permeability, thus

A critical role of the bone marrow microenvironment in the efficacy of bortezomib in Multiple Myeloma was further established by Edwards et al. (2009). *In vivo* studies demonstrated that bortezomib had a greater effect on tumour burden when Myeloma cells were grown in the bone marrow of mice than when they were grown at sub-cutaneous sites.

Osteolytic lesions characterised with activated osteoclast activity accompanied with a reduction in osteoblast activity are a major feature of Multiple Myeloma. Bortezomib exhibits important effects on the development and progression of Myeloma-associated bone disease by reducing osteoclast activity and increasing osteoblast function, therefore reducing bone resorption and stimulating new bone formation. Both preclinical and clinical analysis have demonstrated that bortezomib exerts these effects in part by inhibiting dickkopf-1 and receptor activator of nuclear factor-kappa B ligand and increasing levels of alkaline phosphatase and osteocalcin (Terpos et al., 2006; Heider et al., 2006; Giuliani et al., 2007). However, a recent study by Lund and colleagues (2010) found that the combination of a glucocorticoid such as dexamethasone with bortezomib could inhibit the positive effects of bortezomib on osteoblast proliferation and differentiation, suggesting that bortezomib may

result in better healing of osteolytic lesions when used without a glucocorticoid.

While a number of mechanisms of action of bortezomib have been outlined above, the full mechanism of bortezomib-induced cytotoxicity remains to be elucidated. Gene expression studies have been employed to try and increase our understanding of the cytotoxic action of this compound in Multiple Myeloma. Mitsiades et al., (2002) performed gene expression profiling in a Multiple Myeloma cell line and demonstrated that bortezomib resulted in a downregulation of growth and survival signalling pathways and upregulation of molecules

acting as a vascular targeting drug.

**4.5 Bortezomib and bone formation** 

**4.6 Gene expression studies** 

Orlowski et al. (2002) conducted a Phase 1 trial evaluating the pharmacodynamics of bortezomib, along with toxicity and clinical responses in 27 patients with advanced refractory haematological malignancies. This study demonstrated that bortezomib could be safely administered, with a tolerable side-effect profile. There was significant evidence of anti-tumour activity in patients with Multiple Myeloma, with all 9 evaluable Multiple Myeloma patients showing some evidence of clinical benefit, including one complete response. Taken together with preclinical data this provided the rationale for Phase 2 clinical trials with bortezomib for the treatment of relapsed, refractory Myeloma.

#### Phase 2

The activity of bortezomib in relapsed and refractory Multiple Myeloma was confirmed with two Phase 2 trials, SUMMIT and CREST. SUMMIT (Study of Uncontrolled Multiple Myeloma managed with proteasome Inhibition Therapy) was a large multi-centre trial that enrolled 202 heavily pre-treated patients (Richardson et al., 2003). An overall response rate of 35% was achieved, including 10% of patients who achieved a complete or near complete response. Median time to progression was 7 months compared with 3 months on previous therapy. Grade 3 toxicities included cyclical thrombocytopenia, fatigue, peripheral

Proteasome Inhibitors in the Treatment of Multiple Myeloma 11

Bortezomib & alvocidib Phase 1 44% Holkova et al., 2011

Bortezomib & vorinostat Phase 1 42% Badros et al., 2009 Bortezomib & samarium lexidronam Phase 1 21% Berenson et al.,

Bortezomib & temsirolimus Phase 1/2 33% Ghobrial

Bortezomib, arsenic trioxide & ascorbic acid Phase 1/2 27% Berenson

Bortezomib & melphalan Phase 1/2 68% Berenson

Bortezomib, thalidomide & dexamethasone Phase 2 Pineda-Roman

Table 1. Bortezomib-based combination therapy for relapsed/refractory Multiple Myeloma. synergistic activity with bortezomib in preclinical studies and are under evaluation in early

Heat shock protein 90 is a chaperone that stabilizes numerous proteins that contribute to tumour cell survival and proliferation. Inhibition of heat shock protein 90 in Myeloma cells results in decreased expression of insulin-like growth factor-1 and interleukin-6 receptors, with a related decrease in the PI3K/Akt signalling pathway. Preclinical studies with the heat shock protein 90 inhibitor tanespimycin in combination with bortezomib demonstrated a synergistic effect and resulted in enhanced accumulation of ubiquitinated proteins (Mitsiades et al., 2006). A Phase 1 clinical trial of bortezomib in combination with tanespimycin demonstrated significant and durable responses (Richardson et al., 2011) and

a study of bortezomib in combination with KW-2478 is underway (NCT01063907).

Bortezomib & tanespimycin Phase 1 27% Richardson

**Response** 

Phase 1 Takamatsu

Phase 1 88% Kim et al., 2010

Phase 1/2 76% Popat et al., 2009

Phase 1/2 67% Palumbo et al., 2007

Phase 2 55% Chanan-Khan

Phase 2 66% Terpos et al., 2008

Phase 2 90% Kropff et al., 2007

**Reference** 

et al., 2011

et al., 2010

2009a

et al., 2011

et al., 2007

et al., 2006

et al., 2009

et al., 2008

**Combination Study Overall** 

Bortezomib, doxorubicin & intermediate

Bortezomib, low dose melphalan &

Bortezomib, melphalan, prednisone &

Bortezomib, melphalan, dexamethasone &

clinical trials in combination with bortezomib.

**5.3.1 Heat shock protein 90 inhibitors** 

Bortezomib, pegylated liposomal doxorubicin & thalidomide

intermittent thalidomide

cyclophosphamide

Bortezomib, dexamethasone &

Bortezomib, cyclophosphamide, thalidomide

dose dexamethasone

& dexamethasone

dexamethasone

thalidomide

neuropathy and neutropenia. Of these, the most clinically significant was peripheral neuropathy. The CREST (Clinical Response and Efficacy Study of PS-341 in the Treatment of relapsing Multiple Myeloma) trial was a smaller multicentre study that enrolled 54 patients with only one prior treatment (Jagannath et al., 2004). Patients were randomized to receive 1.0 or 1.3 mg/m2 bortezomib. The overall response rates were 30% for patients receiving 1.0 mg/m2 and 38% for patients receiving 1.3 mg/m2. Adverse effects were similar to those seen in SUMMIT, however, less peripheral neuropathy was seen with reduced dose used in CREST. These findings led to the approval of bortezomib by the Food and Drug Administration and European Medicines Agency for relapsed/refractory Multiple Myeloma patients that had at least 2 prior therapies (Kane et al., 2006). Bortezomib was the first new therapy approved for Multiple Myeloma for over a decade.

#### Phase 3

APEX (Assessment of Proteasome inhibition for EXtending remissions) was a Phase 3 study of 668 patients with relapsed and refractory Multiple Myeloma after one to three prior treatments, who were randomized to receive either bortezomib or high-dose dexamethasone (Richardson et al., 2005). Bortezomib induced a better overall response rate than dexamethasone (38% vs. 18%), including a 13% vs. 2% complete or near complete response. Median time to progression for bortezomib was 6.22 months vs. 3.49 months for dexamethasone and overall survival was 29.3 months vs. 23.7 months. The adverse events were similar to those observed previously, however the rates of adverse events were higher for bortezomib.

#### **5.2 Bortezomib-based combination therapy in relapsed or refractory Multiple Myeloma**

Early preclinical work demonstrated that bortezomib sensitized Myeloma cells to other chemotherapeutic agents and this prompted clinical investigation of bortezomib-based combination therapies in relapsed or refractory Multiple Myeloma. Dexamethasone was the first agent to be combined with bortezomib in the clinic and is the most common agent to be used in bortezomib-based combinations. Both preclinical data and clinical trials showed that the combination increased anti-Myeloma activity. Data from the SUMMIT and CREST trials demonstrated additional responses in 18% and 33% of patients who received both drugs, including patients who had previously been refractory to dexamethasone. Bortezomib has since been demonstrated to enhance the activity of many chemotherapeutic agents in Multiple Myeloma, demonstrating promising response rates in early clinical trials (summarized in Table 1). Larger Phase 3 trials will be required to confirm response and survival to these combinations.

#### **5.3 Bortezomib–based combinations with novel therapies**

The increased understanding of intracellular pathways that are involved in the proliferation and survival of Myeloma cells has led to the identification of novel targets for therapeutic intervention. Numerous small molecule inhibitors have been developed in recent years, targeted against key cellular proteins or signalling pathways that may enhance the antitumour effect of bortezomib, or overcome resistance to bortezomib. These novel small molecule compounds include heat shock protein 90 inhibitors, histone deacetylase inhibitors, farnesyltransferase inhibitors, Bcl-2 inhibitors, monoclonal antibodies and a number of different kinase inhibitors. Many of these novel agents have demonstrated

neuropathy and neutropenia. Of these, the most clinically significant was peripheral neuropathy. The CREST (Clinical Response and Efficacy Study of PS-341 in the Treatment of relapsing Multiple Myeloma) trial was a smaller multicentre study that enrolled 54 patients with only one prior treatment (Jagannath et al., 2004). Patients were randomized to receive 1.0 or 1.3 mg/m2 bortezomib. The overall response rates were 30% for patients receiving 1.0 mg/m2 and 38% for patients receiving 1.3 mg/m2. Adverse effects were similar to those seen in SUMMIT, however, less peripheral neuropathy was seen with reduced dose used in CREST. These findings led to the approval of bortezomib by the Food and Drug Administration and European Medicines Agency for relapsed/refractory Multiple Myeloma patients that had at least 2 prior therapies (Kane et al., 2006). Bortezomib was the first new

APEX (Assessment of Proteasome inhibition for EXtending remissions) was a Phase 3 study of 668 patients with relapsed and refractory Multiple Myeloma after one to three prior treatments, who were randomized to receive either bortezomib or high-dose dexamethasone (Richardson et al., 2005). Bortezomib induced a better overall response rate than dexamethasone (38% vs. 18%), including a 13% vs. 2% complete or near complete response. Median time to progression for bortezomib was 6.22 months vs. 3.49 months for dexamethasone and overall survival was 29.3 months vs. 23.7 months. The adverse events were similar to those observed previously, however the rates of adverse events were higher

**5.2 Bortezomib-based combination therapy in relapsed or refractory Multiple Myeloma**  Early preclinical work demonstrated that bortezomib sensitized Myeloma cells to other chemotherapeutic agents and this prompted clinical investigation of bortezomib-based combination therapies in relapsed or refractory Multiple Myeloma. Dexamethasone was the first agent to be combined with bortezomib in the clinic and is the most common agent to be used in bortezomib-based combinations. Both preclinical data and clinical trials showed that the combination increased anti-Myeloma activity. Data from the SUMMIT and CREST trials demonstrated additional responses in 18% and 33% of patients who received both drugs, including patients who had previously been refractory to dexamethasone. Bortezomib has since been demonstrated to enhance the activity of many chemotherapeutic agents in Multiple Myeloma, demonstrating promising response rates in early clinical trials (summarized in Table 1). Larger Phase 3 trials will be required to confirm response and

The increased understanding of intracellular pathways that are involved in the proliferation and survival of Myeloma cells has led to the identification of novel targets for therapeutic intervention. Numerous small molecule inhibitors have been developed in recent years, targeted against key cellular proteins or signalling pathways that may enhance the antitumour effect of bortezomib, or overcome resistance to bortezomib. These novel small molecule compounds include heat shock protein 90 inhibitors, histone deacetylase inhibitors, farnesyltransferase inhibitors, Bcl-2 inhibitors, monoclonal antibodies and a number of different kinase inhibitors. Many of these novel agents have demonstrated

therapy approved for Multiple Myeloma for over a decade.

Phase 3

for bortezomib.

survival to these combinations.

**5.3 Bortezomib–based combinations with novel therapies** 


Table 1. Bortezomib-based combination therapy for relapsed/refractory Multiple Myeloma.

synergistic activity with bortezomib in preclinical studies and are under evaluation in early clinical trials in combination with bortezomib.

#### **5.3.1 Heat shock protein 90 inhibitors**

Heat shock protein 90 is a chaperone that stabilizes numerous proteins that contribute to tumour cell survival and proliferation. Inhibition of heat shock protein 90 in Myeloma cells results in decreased expression of insulin-like growth factor-1 and interleukin-6 receptors, with a related decrease in the PI3K/Akt signalling pathway. Preclinical studies with the heat shock protein 90 inhibitor tanespimycin in combination with bortezomib demonstrated a synergistic effect and resulted in enhanced accumulation of ubiquitinated proteins (Mitsiades et al., 2006). A Phase 1 clinical trial of bortezomib in combination with tanespimycin demonstrated significant and durable responses (Richardson et al., 2011) and a study of bortezomib in combination with KW-2478 is underway (NCT01063907).

Proteasome Inhibitors in the Treatment of Multiple Myeloma 13

antibody siltuximab. Preclinical evaluation of siltuximab and bortezomib demonstrated enhanced cytotoxicity of bortezomib in Myeloma cell lines and primary cells in the presence of bone marrow stromal cells (Voorhees et al., 2007). A Phase 2 trial is evaluating this combination in relapsed Myeloma (NCT00401843). Elotuzumab is directed towards CS1, a cell surface glycoprotein expressed at high levels on Multiple Myeloma cells. This anti-CS1 antibody demonstrated significantly enhanced anti-tumour activity in combination with bortezomib in *in vitro* and *in vivo* models of Myeloma (van Rhee et al., 2009) and Phase 1/2 trials are underway (NCT00726869). AVE1642 is an anti-insulin-like growth factor 1 antibody that demonstrated synergistic apoptosis in combination with bortezomib in preclinical studies (Descamps et al., 2009), however, response rates from a Phase 1 study were insufficient to warrant further investigation (Moreau et al., 2011). Early phase clinical trials combining bortezomib with anti-chemokine receptor 4 and anti-CD40 antibodies are

Mammalian target of rapamycin (mTOR) inhibitors inhibit the mTOR kinase and related signalling pathways resulting in decreased expression of cyclins and c-Myc, increased expression of p27 and G1 arrest. *In vitro* studies have demonstrated synergistic action of the mTOR inhibitors NVP-BEZ235 and pp242 with bortezomib (Baumann et al., 2009; Hoang et al., 2010). A Phase 1/2 study of bortezomib in combination with mTOR inhibitor temsirolimus demonstrated a partial response rate of 33% in heavily pre-treated refractory

Cyclin-dependent kinase inhibitors are small molecule inhibitors that induce cell cycle arrest. Cyclin dependent kinase inhibitors (seliciclib and alvocidib) were shown to be synergistic with proteasome inhibitors in leukaemic cell lines (Dai et al., 2003, 2004). A subsequent study demonstrated that the cyclin dependent kinase inhibitor PD0332991 sensitizes an *in vivo* Multiple Myeloma model to bortezomib through enhanced induction of mitochondrial depolarization (Menu et al., 2008). A combination of bortezomib along with the cyclin dependent kinase inhibitor alvocidib (flavopiridol) was recently assessed in a Phase 1 trial for refractory B-cell malignancies and demonstrated an overall response rate of

Perifosine is an alkylphospholipid that inhibits Akt activation and associated growth and drug resistance in Multiple Myeloma. As a single agent, perifosine demonstrated significant toxicity both *in vivo* and *in vitro* and it has also been shown to inhibit bortezomib-induced upregulation of survivin resulting in enhanced bortezomib cytotoxicity (Hideshima et al., 2007). Perifosine is currently being evaluated in a Phase 1/2 study in combination with bortezomib with or without dexamethasone in relapsed Myeloma (NCT00401011) and a Phase 3 study of perifosine in combination with bortezomib and dexamethasone is currently

also underway (NCT01359657 and NCT00664898).

**5.3.6 Mammalian target of rapamycin inhibitor** 

Myeloma (Ghobrial et al., 2011).

**5.3.8 Akt inhibitors** 

recruiting (NCT01002248).

**5.3.7 Cyclin-dependent kinase inhibitors** 

44% with manageable toxicities (Holkova et al., 2011).

#### **5.3.2 Histone deactylase inhibitors**

Ubiquitinated and misfolded proteins are degraded not only by proteasomes but also by aggresomes. Aggresome formation, which is dependent on the histone deacetylase HDAC6, is increased in response to inhibition of proteasome function. Histone deacetylase inhibitors are a class of compounds that regulate gene expression by interfering with the function of histone deacetylases. Preclinical studies demonstrated that the combination of bortezomib with a HDAC inhibitor resulted in significant cytotoxicity and show a marked accumulation of polyubiquitinated proteins (Catley et al., 2006; Nawrocki et al., 2008). A Phase 1 trial of bortezomib and vorinostat in relapsed/refractory myeloma demonstrated encouraging results, with an overall response rate of 42%, including 3 responses among 9 bortezomib refractory patients (Badros et al., 2009). Further trials of bortezomib along with HDAC inhibitors vorinostat and panobinostat are currently being investigated.

#### **5.3.3 Farnesyltransferase inhibitors**

Farnesyltransferase inhibitors block activation of the Ras dependent MAPK signalling pathway to regulate signal transduction and proliferation. Combination of the farnesyltranserase inhibitors lonafarnib and tipifarnib with bortezomib induced synergistic cell death and overcame cell adhesion-mediated drug resistance in Multiple Myeloma cell lines and primary cells (David et al., 2005; Yanamandra et al., 2006). David and colleagues (2005) observed that this combination resulted in a down-regulation of Akt signalling, an effect which was absent when either drug was used alone. Early phase clinical trials evaluating bortezomib and tipifarnib combination therapy are ongoing (NCT00243035; NCT00972712).

#### **5.3.4 Bcl-2 inhibitors**

Bcl-2 family members play a critical role in mediating tumour cell survival and chemoresistance in Multiple Myeloma. There are a number of small molecule inhibitors available that interfere with the function of Bcl-2 proteins and induce apoptosis in Multiple Myeloma cells. In preclinical studies, three Bcl-2 inhibitors obatoclax, ABT-737 and ABT-263 have shown synergistic activity with bortezomib (Chauhan et al., 2007; Trudel et al., 2007; Ackler et al., 2010). The combination of bortezomib with a Bcl-2 inhibitor resulted in enhanced NOXA-mediated activation of Bak and increased activation of the mitochondrial apoptotic pathways. Obatoclax is being investigated in combination with bortezomib in early clinical trials (NCT00719901).

#### **5.3.5 Monoclonal antibodies**

Monoclonal antibody therapy can selectively target specific molecules, proteins or receptors involved in disease processes. There are a number of antigens currently under investigation as potential targets in Multiple Myeloma in combination with bortezomib. Bevacizumab is a monoclonal antibody that is targeted towards vascular endothelial growth factor to disrupt angiogenensis (Brekken et al., 2000). The combination of bevacizumab and bortezomib is being evaluated in Phase 2 studies for relapsed Myeloma (NCT00464178). Interleukin-6, a key intermediate in Multiple Myeloma signalling pathways, is targeted by the chimeric

Ubiquitinated and misfolded proteins are degraded not only by proteasomes but also by aggresomes. Aggresome formation, which is dependent on the histone deacetylase HDAC6, is increased in response to inhibition of proteasome function. Histone deacetylase inhibitors are a class of compounds that regulate gene expression by interfering with the function of histone deacetylases. Preclinical studies demonstrated that the combination of bortezomib with a HDAC inhibitor resulted in significant cytotoxicity and show a marked accumulation of polyubiquitinated proteins (Catley et al., 2006; Nawrocki et al., 2008). A Phase 1 trial of bortezomib and vorinostat in relapsed/refractory myeloma demonstrated encouraging results, with an overall response rate of 42%, including 3 responses among 9 bortezomib refractory patients (Badros et al., 2009). Further trials of bortezomib along with HDAC inhibitors vorinostat and panobinostat are

Farnesyltransferase inhibitors block activation of the Ras dependent MAPK signalling pathway to regulate signal transduction and proliferation. Combination of the farnesyltranserase inhibitors lonafarnib and tipifarnib with bortezomib induced synergistic cell death and overcame cell adhesion-mediated drug resistance in Multiple Myeloma cell lines and primary cells (David et al., 2005; Yanamandra et al., 2006). David and colleagues (2005) observed that this combination resulted in a down-regulation of Akt signalling, an effect which was absent when either drug was used alone. Early phase clinical trials evaluating bortezomib and tipifarnib combination therapy are ongoing (NCT00243035;

Bcl-2 family members play a critical role in mediating tumour cell survival and chemoresistance in Multiple Myeloma. There are a number of small molecule inhibitors available that interfere with the function of Bcl-2 proteins and induce apoptosis in Multiple Myeloma cells. In preclinical studies, three Bcl-2 inhibitors obatoclax, ABT-737 and ABT-263 have shown synergistic activity with bortezomib (Chauhan et al., 2007; Trudel et al., 2007; Ackler et al., 2010). The combination of bortezomib with a Bcl-2 inhibitor resulted in enhanced NOXA-mediated activation of Bak and increased activation of the mitochondrial apoptotic pathways. Obatoclax is being investigated in combination with bortezomib in

Monoclonal antibody therapy can selectively target specific molecules, proteins or receptors involved in disease processes. There are a number of antigens currently under investigation as potential targets in Multiple Myeloma in combination with bortezomib. Bevacizumab is a monoclonal antibody that is targeted towards vascular endothelial growth factor to disrupt angiogenensis (Brekken et al., 2000). The combination of bevacizumab and bortezomib is being evaluated in Phase 2 studies for relapsed Myeloma (NCT00464178). Interleukin-6, a key intermediate in Multiple Myeloma signalling pathways, is targeted by the chimeric

**5.3.2 Histone deactylase inhibitors** 

currently being investigated.

NCT00972712).

**5.3.4 Bcl-2 inhibitors** 

early clinical trials (NCT00719901).

**5.3.5 Monoclonal antibodies** 

**5.3.3 Farnesyltransferase inhibitors** 

antibody siltuximab. Preclinical evaluation of siltuximab and bortezomib demonstrated enhanced cytotoxicity of bortezomib in Myeloma cell lines and primary cells in the presence of bone marrow stromal cells (Voorhees et al., 2007). A Phase 2 trial is evaluating this combination in relapsed Myeloma (NCT00401843). Elotuzumab is directed towards CS1, a cell surface glycoprotein expressed at high levels on Multiple Myeloma cells. This anti-CS1 antibody demonstrated significantly enhanced anti-tumour activity in combination with bortezomib in *in vitro* and *in vivo* models of Myeloma (van Rhee et al., 2009) and Phase 1/2 trials are underway (NCT00726869). AVE1642 is an anti-insulin-like growth factor 1 antibody that demonstrated synergistic apoptosis in combination with bortezomib in preclinical studies (Descamps et al., 2009), however, response rates from a Phase 1 study were insufficient to warrant further investigation (Moreau et al., 2011). Early phase clinical trials combining bortezomib with anti-chemokine receptor 4 and anti-CD40 antibodies are also underway (NCT01359657 and NCT00664898).

#### **5.3.6 Mammalian target of rapamycin inhibitor**

Mammalian target of rapamycin (mTOR) inhibitors inhibit the mTOR kinase and related signalling pathways resulting in decreased expression of cyclins and c-Myc, increased expression of p27 and G1 arrest. *In vitro* studies have demonstrated synergistic action of the mTOR inhibitors NVP-BEZ235 and pp242 with bortezomib (Baumann et al., 2009; Hoang et al., 2010). A Phase 1/2 study of bortezomib in combination with mTOR inhibitor temsirolimus demonstrated a partial response rate of 33% in heavily pre-treated refractory Myeloma (Ghobrial et al., 2011).

#### **5.3.7 Cyclin-dependent kinase inhibitors**

Cyclin-dependent kinase inhibitors are small molecule inhibitors that induce cell cycle arrest. Cyclin dependent kinase inhibitors (seliciclib and alvocidib) were shown to be synergistic with proteasome inhibitors in leukaemic cell lines (Dai et al., 2003, 2004). A subsequent study demonstrated that the cyclin dependent kinase inhibitor PD0332991 sensitizes an *in vivo* Multiple Myeloma model to bortezomib through enhanced induction of mitochondrial depolarization (Menu et al., 2008). A combination of bortezomib along with the cyclin dependent kinase inhibitor alvocidib (flavopiridol) was recently assessed in a Phase 1 trial for refractory B-cell malignancies and demonstrated an overall response rate of 44% with manageable toxicities (Holkova et al., 2011).

#### **5.3.8 Akt inhibitors**

Perifosine is an alkylphospholipid that inhibits Akt activation and associated growth and drug resistance in Multiple Myeloma. As a single agent, perifosine demonstrated significant toxicity both *in vivo* and *in vitro* and it has also been shown to inhibit bortezomib-induced upregulation of survivin resulting in enhanced bortezomib cytotoxicity (Hideshima et al., 2007). Perifosine is currently being evaluated in a Phase 1/2 study in combination with bortezomib with or without dexamethasone in relapsed Myeloma (NCT00401011) and a Phase 3 study of perifosine in combination with bortezomib and dexamethasone is currently recruiting (NCT01002248).

Proteasome Inhibitors in the Treatment of Multiple Myeloma 15

and with thalidomide, dexamethasone and chemotherapy (Barlogie et al., 2007). The bortezomib-based combinations all demonstrated superior response rates than the regimens without bortezomib. A number of other combinations incorporating bortezomib for both transplant eligible and ineligible patients are in clinical trials and are achieving overall

Bortezomib, melphalan & prednisone Phase 1/2 95% Gasparetto et al., 2010 Bortezomib & melphelan Phase 1/2 87% Lonial et al., 2010

Bortezomib & dexamethasone Phase 2 66% Harousseau et al., 2006

Bortezomib & high dose melphalan Phase 2 70% Roussel et al., 2010

Bortezomib & dexamethasone Phase 2 86% Corso et al., 2010

Bortezomib & thalidomide Phase 2 82% Ghosh et al., 2011

Bortezomib & dexamethasone Phase 3 79% Harousseau et al., 2010

Table 2. Bortezomib-based combinations for induction and front-line therapy.

Response

Phase 1 83% Badros et al., 2006

Phase 1 96% Kumar et al., 2010

Phase 1/2 89/95% Popat et al., 2008

Phase 1/2 100% Richardson et al., 2010

Phase 1/2 96% Jakubowiak et al., 2011

Phase 2 68% Rosinol et al., 2007

Phase 2 88% Reeder et al., 2009

Phase 2 74% Berenson et al., 2009b

Phase 2 85% Jakubowaik et al., 2009

Phase 2 95% Besinger et al., 2010

Phase 2 75% Kim et al., 2011

Phase 2 78% Sher et al., 2011

Phase 3 31% Cavo et al., 2010

Reference

response rates of up to 100% (Table 2).

Bortezomib, thalidomide &

Bortezomib, dexamethasone, cyclophosphamide & lenalidomide

Bortezomib, doxorubicin &

Bortezomib, lenalidomide &

Bortezomib, lenanlidomide,

Alternating bortezomib &

Bortezomib, ascorbic acid &

Vincristine, adriamycin & dexamethasone followed by bortezomib, thalidomide &

pegylated liposomal doxorubicin &

Bortezomib, cyclophosphamide &

Bortezomib, pegylated liposomal doxorubicin & dexamethasone

Bortezomib, cyclophosphamide &

Bortezomib, pegylated liposomal doxorubicin & thalidomide

Bortezomib, thalidomide &

chemotherapy

dexamethasone

dexamethasone

dexamethasone

dexamethasone

dexamethasone

dexamethasone

dexamethasone

dexamethasone

melphalan

Combination Study Phase Overall

#### **5.3.9 Multi-kinase inhibitors**

Sorafenib and dasatinib are multi-kinase inhibitors that have been shown to enhance anti-Myeloma activity with bortezomib. Sorafenib inhibits RAF kinase, VEGF receptors, plateletderived growth factor β, Flt-3, c-Kit and RET receptor tyrosine kinases. The combination of sorafenib and bortezomib produced synergistic apoptosis in a number of malignant cell lines and was dependent on Akt inhibition (Yu et al., 2006). This combination is currently being investigated in a Phase 1/2 trial in relapsed Myeloma (NCT00536575). Dasatinib is an inhibitor of c-abl, src family proteins, EphA2 and btk. The triple combination of dasatinib along with bortezomib and dexamethasone produced greater synergistic effects compared to single agents or double combinations in Multiple Myeloma cell line models and primary cells (de Queiroz Crusoe et al., 2011). A Phase 1 study combining all three agents in relapsed or refractory Myeloma has recently been completed (NCT00560352).

#### **5.3.10 Other combinations**

The combination of bortezomib with second generation immunomodulatory drug pomalidomide (NCT01212952), telomerase inhibitor GRN163L (NCT00718601), aurora A kinase inhibitor MLN8237 (Gorgun et al., 2010; NCT01034553), p38 mitogen-activated kinase inhibitor SCIO-469 (Navas et al., 2006; NCT00095680) and protease inhibitor nelfinavir mesylate (NCT01164709) are all being evaluated in early clinical trials. In addition there are numerous more novel targeted therapies under preclinical assessment in combination with proteasome inhibitors.

#### **5.4 Bortezomib in front-line therapy**

For over 40 years melphalan and prednisone was the standard therapy for patients with newly diagnosed Multiple Myeloma that were ineligible for high-dose therapy and autologous stem cell transplantation. Following encouraging activity of bortezomib combined with melphalan in patients with relapsed or refractory Myeloma, bortezomib plus melphalan and prednisone was evaluated in a Phase 1/2 trial for newly diagnosed Myeloma patients who were at least 65 years of age. The combination gave a response rate of 89% and a median time to progression of 27 months. This led to the Phase 3 trial VISTA (Velcade as Initial Standard Therapy in Multiple Myeloma), which compared bortezomib, melphalan and prednisone with melphalan and prednisone in newly diagnosed Myeloma patients who were ineligible for high-dose therapy. Results of this trial demonstrated that when bortezomib was included in the regimen the overall response rate increased from 30% to 71% and the time to progression was 24 months compared with 16.6 months (San Miguel et al., 2008). There was also fewer bone disease events, improvement in bone remodelling and evidence of bone healing. These results suggested a benefit for bortezomib at earlier use and provided the framework for approval of bortezomib for use as front-line therapy.

In newly diagnosed patients who were candidates for high-dose therapy with autologous stem cell transplantation, the combination of vincristine, doxorubicin and dexamethasone was the standard induction therapy. Four randomized trials evaluated the role of bortezomib–based combinations for induction therapy in transplant candidates. Bortezomib was combined with dexamethasone (Harousseau et al., 2010), with adriamycin and dexamethasone (Popat et al., 2008), with thalidomide and dexamethasone (Cavo et al., 2010)

Sorafenib and dasatinib are multi-kinase inhibitors that have been shown to enhance anti-Myeloma activity with bortezomib. Sorafenib inhibits RAF kinase, VEGF receptors, plateletderived growth factor β, Flt-3, c-Kit and RET receptor tyrosine kinases. The combination of sorafenib and bortezomib produced synergistic apoptosis in a number of malignant cell lines and was dependent on Akt inhibition (Yu et al., 2006). This combination is currently being investigated in a Phase 1/2 trial in relapsed Myeloma (NCT00536575). Dasatinib is an inhibitor of c-abl, src family proteins, EphA2 and btk. The triple combination of dasatinib along with bortezomib and dexamethasone produced greater synergistic effects compared to single agents or double combinations in Multiple Myeloma cell line models and primary cells (de Queiroz Crusoe et al., 2011). A Phase 1 study combining all three agents in relapsed

The combination of bortezomib with second generation immunomodulatory drug pomalidomide (NCT01212952), telomerase inhibitor GRN163L (NCT00718601), aurora A kinase inhibitor MLN8237 (Gorgun et al., 2010; NCT01034553), p38 mitogen-activated kinase inhibitor SCIO-469 (Navas et al., 2006; NCT00095680) and protease inhibitor nelfinavir mesylate (NCT01164709) are all being evaluated in early clinical trials. In addition there are numerous more novel targeted therapies under preclinical assessment in combination with

For over 40 years melphalan and prednisone was the standard therapy for patients with newly diagnosed Multiple Myeloma that were ineligible for high-dose therapy and autologous stem cell transplantation. Following encouraging activity of bortezomib combined with melphalan in patients with relapsed or refractory Myeloma, bortezomib plus melphalan and prednisone was evaluated in a Phase 1/2 trial for newly diagnosed Myeloma patients who were at least 65 years of age. The combination gave a response rate of 89% and a median time to progression of 27 months. This led to the Phase 3 trial VISTA (Velcade as Initial Standard Therapy in Multiple Myeloma), which compared bortezomib, melphalan and prednisone with melphalan and prednisone in newly diagnosed Myeloma patients who were ineligible for high-dose therapy. Results of this trial demonstrated that when bortezomib was included in the regimen the overall response rate increased from 30% to 71% and the time to progression was 24 months compared with 16.6 months (San Miguel et al., 2008). There was also fewer bone disease events, improvement in bone remodelling and evidence of bone healing. These results suggested a benefit for bortezomib at earlier use and

provided the framework for approval of bortezomib for use as front-line therapy.

In newly diagnosed patients who were candidates for high-dose therapy with autologous stem cell transplantation, the combination of vincristine, doxorubicin and dexamethasone was the standard induction therapy. Four randomized trials evaluated the role of bortezomib–based combinations for induction therapy in transplant candidates. Bortezomib was combined with dexamethasone (Harousseau et al., 2010), with adriamycin and dexamethasone (Popat et al., 2008), with thalidomide and dexamethasone (Cavo et al., 2010)

or refractory Myeloma has recently been completed (NCT00560352).

**5.3.9 Multi-kinase inhibitors** 

**5.3.10 Other combinations** 

proteasome inhibitors.

**5.4 Bortezomib in front-line therapy** 

and with thalidomide, dexamethasone and chemotherapy (Barlogie et al., 2007). The bortezomib-based combinations all demonstrated superior response rates than the regimens without bortezomib. A number of other combinations incorporating bortezomib for both transplant eligible and ineligible patients are in clinical trials and are achieving overall response rates of up to 100% (Table 2).


Table 2. Bortezomib-based combinations for induction and front-line therapy.

Proteasome Inhibitors in the Treatment of Multiple Myeloma 17

chymotrypsin-like (β5 and LMP7) subunit, leading to more sustained proteasome inhibition. In preclinical studies carfilzomib was shown to exhibit equal potency but greater selectivity than bortezomib for the chymotrypsin-like activity. *In vitro* and *in vivo* studies demonstrated anti-tumour activity, tolerability and dosing flexibility in several xenograft models (Kuhn et al., 2007; Demo et al., 2007). Carfilzomib has also been shown to act synergistically with histone deacetylase inhibitors *in vitro* in lymphoma and leukaemia (Fuchs et al., 2009; Dasmahapatra et al., 2010; 2011). Results from Phase 1 studies in patients with haematological malignancies demonstrated that carfilzomib was well tolerated and may exhibit less peripheral neuropathy than bortezomib (O'Connor et al., 2009). Phase 2 trials of carfilzomib as a single agent in relapsed and refractory Multiple Myeloma demonstrated an overall response rate of 35.5% including patients with bortezomib-refractory disease. (Zangari et al., 2011) The main toxicities were fatigue and nausea, with limited peripheral neuropathy seen in less than 10% of patients. Carfilzomib is currently progressing in a number of trials for relapsed and newly diagnosed Multiple Myeloma and as both a single

Marizomib, also known as Salinosporamide A, is a β-lactone compound derived from the marine bacterium *Salinospora tropica* (Macherla et al., 2005) and is structurally related to the lactacystin-derived proteasome inhibitor Omuralide. In contrast to bortezomib which is a slowly reversible inhibitor of chymotrypsin-like activity, marizomib binds irreversibly to all three catalytic activities of the proteasome. While bortezomib is administered intravenously, marizomib has the advantage of being orally bioactive. Initial *in vitro* studies established the effectiveness of this compound in Multiple Myeloma cell lines, including those that were resistant to bortezomib (Chauhan et al., 2005). Animal tumour model studies demonstrated reduced tumour growth without significant toxicity (Chauhan et al., 2005; Singh et al., 2010). Preclinical studies demonstrated synergistic results when marizomib was combined with bortezomib or lenalidomide (Chauhan et al., 2008; 2010a). Phase 1 trials of marizomib in Myeloma are currently ongoing. Marizomib displays a broader, faster acting and more durable proteasome inhibition than bortezomib and treatment does not appear to induce the limiting toxicities associated with bortezomib, such as peripheral neuropathy and

MLN9708 like bortezomib is also a boron containing peptide proteasome inhibitor and was selected from a panel of inhibitors based on having a biochemical profile distinct from that of bortezomib. MLN9708 hydrolyses immediately in plasma to its biologically active form MLN2238. MLN2238 displays similar potency and selectivity for the chymotrypsin-like proteasome subunit, however, it has a substantially shorter half-life than bortezomib which may improve tissue distribution. Cell viability studies revealed a strong anti-proliferative effect on a variety of tumour cell lines and *in vivo* studies have demonstrated efficacy in human prostate xenograft, colon cancer and lymphoma models where both intravenous and oral dosing were effective (Kupperman et al., 2010). MLN2238 has been demonstrated to induce apoptosis in cells resistant to both conventional therapies and to bortezomib. Synergistic activity is seen by combining this compound with lenelidomide, HDAC

agent and in combination.

**7.2 Marizomib (NPI-0052)** 

thrombocytopenia.

**7.3 MLN9708/MLN2238** 

#### **6. Resistance to bortezomib**

Despite the clinical success of bortezomib, many patients with Multiple Myeloma are unresponsive and drug resistance can also develop (Dispenzieri et al., 2010). The mechanisms underlying this drug resistance, both intrinsic and acquired, are as yet poorly understood.

Resistance to proteasome inhibitors may occur either at the level of the proteasome complex or in the downstream signalling pathways. Several researchers have approached this problem by growing cell lines in increasing concentration of bortezomib. Ri et al. (2010) found a unique point mutation in the proteasome β5 subunit (PSMB5) in bortezomib resistant Multiple Myeloma cell lines. Using overexpression studies they demonstrated that the mutation may act by interfering with the Unfolded Protein Response pathway. Shaughnessy and colleagues have recently applied gene expression studies to a group of 142 Multiple Myeloma patients and identified PSMD4 as associated with adverse response to bortezomib; PSMD4 is one of the non-ATPase subunits of the proteasome 19S regulator (Shaughnessy et al., 2011).

The anti-tumour effects of bortezomib have been mainly attributed to its' actions on the NFκB and Bcl-2 regulatory protein pathways. It is not therefore surprising that polymorphisms of the NFκB family genes have been associated with treatment outcome in Multiple Myeloma patients. Studies with lymphoid cell lines have recently shown Noxa/Bcl-2 interactions contribute to bortezomib resistance (Smith et al., 2011) and there have been similar reports in Mantle Cell Lymphoma cell lines (Weniger et al., 2011); there is no supporting clinical evidence as yet. Overexpression of apoptosis regulators REDD1 and survivin have also been associated with bortezomib resistance in cell line models (Decaux et al., 2010; Ling et al., 2010).

In cases where drug resistance is directly associated with the proteasome enzymatic complex it may be possible to overcome resistance by using second generation inhibitors which act through a different mechanism to bortezomib (Ruschak et al., 2011; Arastu-Kapur et al., 2011; Chauhan et al., 2011). Knowledge of the resistance mechanism may also allow rational design of future combination therapies.

#### **7. Second generation inhibitors**

The success of bortezomib in the clinic prompted the development of a new generation of structurally distinct proteasome inhibitors. In addition to bortezomib, there are currently five proteasome inhibitors in clinical development, representing three different structural classes - peptide boronic acids, peptide epoxyketones and β-lactones (Figure 2). These inhibitors bind either reversibly or irreversibly to catalytic sites within the proteasome.

#### **7.1 Carfilzomib**

Epoxomicin, a member of the epoxyketone family of natural peptide proteasome inhibitors, inhibits proteasome activity through a unique mechanism, by binding to both the hydroxyl and amino groups of the catalytic site threonine residue (Groll & Huber, 2004). Carfilzomib (formerly PR-171) is an epoxomicin-based proteasome inhibitor, with improved pharmaceutical properties. Unlike bortezomib, carfilzomib binds irreversibly to the chymotrypsin-like (β5 and LMP7) subunit, leading to more sustained proteasome inhibition. In preclinical studies carfilzomib was shown to exhibit equal potency but greater selectivity than bortezomib for the chymotrypsin-like activity. *In vitro* and *in vivo* studies demonstrated anti-tumour activity, tolerability and dosing flexibility in several xenograft models (Kuhn et al., 2007; Demo et al., 2007). Carfilzomib has also been shown to act synergistically with histone deacetylase inhibitors *in vitro* in lymphoma and leukaemia (Fuchs et al., 2009; Dasmahapatra et al., 2010; 2011). Results from Phase 1 studies in patients with haematological malignancies demonstrated that carfilzomib was well tolerated and may exhibit less peripheral neuropathy than bortezomib (O'Connor et al., 2009). Phase 2 trials of carfilzomib as a single agent in relapsed and refractory Multiple Myeloma demonstrated an overall response rate of 35.5% including patients with bortezomib-refractory disease. (Zangari et al., 2011) The main toxicities were fatigue and nausea, with limited peripheral neuropathy seen in less than 10% of patients. Carfilzomib is currently progressing in a number of trials for relapsed and newly diagnosed Multiple Myeloma and as both a single agent and in combination.

#### **7.2 Marizomib (NPI-0052)**

16 Multiple Myeloma – An Overview

Despite the clinical success of bortezomib, many patients with Multiple Myeloma are unresponsive and drug resistance can also develop (Dispenzieri et al., 2010). The mechanisms underlying this drug resistance, both intrinsic and acquired, are as yet poorly

Resistance to proteasome inhibitors may occur either at the level of the proteasome complex or in the downstream signalling pathways. Several researchers have approached this problem by growing cell lines in increasing concentration of bortezomib. Ri et al. (2010) found a unique point mutation in the proteasome β5 subunit (PSMB5) in bortezomib resistant Multiple Myeloma cell lines. Using overexpression studies they demonstrated that the mutation may act by interfering with the Unfolded Protein Response pathway. Shaughnessy and colleagues have recently applied gene expression studies to a group of 142 Multiple Myeloma patients and identified PSMD4 as associated with adverse response to bortezomib; PSMD4 is one of the non-ATPase subunits of the proteasome 19S regulator

The anti-tumour effects of bortezomib have been mainly attributed to its' actions on the NFκB and Bcl-2 regulatory protein pathways. It is not therefore surprising that polymorphisms of the NFκB family genes have been associated with treatment outcome in Multiple Myeloma patients. Studies with lymphoid cell lines have recently shown Noxa/Bcl-2 interactions contribute to bortezomib resistance (Smith et al., 2011) and there have been similar reports in Mantle Cell Lymphoma cell lines (Weniger et al., 2011); there is no supporting clinical evidence as yet. Overexpression of apoptosis regulators REDD1 and survivin have also been associated with bortezomib resistance in cell line models (Decaux et

In cases where drug resistance is directly associated with the proteasome enzymatic complex it may be possible to overcome resistance by using second generation inhibitors which act through a different mechanism to bortezomib (Ruschak et al., 2011; Arastu-Kapur et al., 2011; Chauhan et al., 2011). Knowledge of the resistance mechanism may also allow

The success of bortezomib in the clinic prompted the development of a new generation of structurally distinct proteasome inhibitors. In addition to bortezomib, there are currently five proteasome inhibitors in clinical development, representing three different structural classes - peptide boronic acids, peptide epoxyketones and β-lactones (Figure 2). These inhibitors bind either reversibly or irreversibly to catalytic sites within the proteasome.

Epoxomicin, a member of the epoxyketone family of natural peptide proteasome inhibitors, inhibits proteasome activity through a unique mechanism, by binding to both the hydroxyl and amino groups of the catalytic site threonine residue (Groll & Huber, 2004). Carfilzomib (formerly PR-171) is an epoxomicin-based proteasome inhibitor, with improved pharmaceutical properties. Unlike bortezomib, carfilzomib binds irreversibly to the

**6. Resistance to bortezomib** 

(Shaughnessy et al., 2011).

al., 2010; Ling et al., 2010).

**7.1 Carfilzomib** 

rational design of future combination therapies.

**7. Second generation inhibitors** 

understood.

Marizomib, also known as Salinosporamide A, is a β-lactone compound derived from the marine bacterium *Salinospora tropica* (Macherla et al., 2005) and is structurally related to the lactacystin-derived proteasome inhibitor Omuralide. In contrast to bortezomib which is a slowly reversible inhibitor of chymotrypsin-like activity, marizomib binds irreversibly to all three catalytic activities of the proteasome. While bortezomib is administered intravenously, marizomib has the advantage of being orally bioactive. Initial *in vitro* studies established the effectiveness of this compound in Multiple Myeloma cell lines, including those that were resistant to bortezomib (Chauhan et al., 2005). Animal tumour model studies demonstrated reduced tumour growth without significant toxicity (Chauhan et al., 2005; Singh et al., 2010). Preclinical studies demonstrated synergistic results when marizomib was combined with bortezomib or lenalidomide (Chauhan et al., 2008; 2010a). Phase 1 trials of marizomib in Myeloma are currently ongoing. Marizomib displays a broader, faster acting and more durable proteasome inhibition than bortezomib and treatment does not appear to induce the limiting toxicities associated with bortezomib, such as peripheral neuropathy and thrombocytopenia.

#### **7.3 MLN9708/MLN2238**

MLN9708 like bortezomib is also a boron containing peptide proteasome inhibitor and was selected from a panel of inhibitors based on having a biochemical profile distinct from that of bortezomib. MLN9708 hydrolyses immediately in plasma to its biologically active form MLN2238. MLN2238 displays similar potency and selectivity for the chymotrypsin-like proteasome subunit, however, it has a substantially shorter half-life than bortezomib which may improve tissue distribution. Cell viability studies revealed a strong anti-proliferative effect on a variety of tumour cell lines and *in vivo* studies have demonstrated efficacy in human prostate xenograft, colon cancer and lymphoma models where both intravenous and oral dosing were effective (Kupperman et al., 2010). MLN2238 has been demonstrated to induce apoptosis in cells resistant to both conventional therapies and to bortezomib. Synergistic activity is seen by combining this compound with lenelidomide, HDAC

Proteasome Inhibitors in the Treatment of Multiple Myeloma 19

malignancies (Kuhn et al., 2009). At the time of writing this review there were no clinical trials of immunoproteasome inhibitors in progress, however, it is likely that the encouraging preclinical data on PR-924 and ISPS-101 will form the basis for future clinical evaluation of

Fig. 2. Structure and class of proteasome inhibitors in clinical trials.

novel inhibitors offers a powerful approach to Myeloma therapy.

Proteasome Inhibitors have provided a major new therapeutic strategy for the treatment of Multiple Myeloma. Bortezomib, the first-in-class of these inhibitors, has shown remarkable success since its introduction almost ten years ago. Second generation compounds are already demonstrating increased selectivity with a more acceptable therapeutic window. Researchers are turning to other parts of the Ubiquitin Proteasome Pathway to look for potential druggable targets which would confer greater specificity. The E3 ligases play a key role in substrate selection and the Pharma already have agents in their pipeline which show promise in modifying their action. Modulation of the Ubiquitin Proteasome Pathway with

Ackler, S., M. J. Mitten, K. Foster, A. Oleksijew, M. Refici, S. K. Tahir, Y. Xiao, et al. 2010. The

bcl-2 inhibitor ABT-263 enhances the response of multiple chemotherapeutic regimens in hematologic tumors in vivo. *Cancer Chemotherapy and Pharmacology* 66

these compounds.

**8. Conclusion** 

**9. References** 

(5) (Oct): 869-80.

inhibitors and dexamethasone *in vitro*. It is well tolerated in plasmacytoma xenograft mouse models and demonstrates significantly longer survival time than mice treated with bortezomib (Chauhan et al., 2011). This compound is currently being evaluated in Phase 1 studies in patients with lymphoma and non-haematological malignancies and in Phase 1/2 trials for Multiple Myeloma.

#### **7.4 CEP-18770**

CEP-18770 is a next-generation boronic acid-based proteasome inhibitor and in common with bortezomib it is a reversible inhibitor, primarily of the chymotrypsin-like activity. CEP-18770 was demonstrated to induce apoptosis in Multiple Myeloma cell lines and primary Myeloma cells, while displaying a favourable cytotoxicity profile towards normal cells (Piva et al., 2008; Dorsey et al., 2008). Its anti-tumour activity was demonstrated in several animal tumour models and it has been shown to demonstrate marked anti-Myeloma effects in combination with bortezomib and melphalan (Sanchez et al., 2010). CEP-18770 has completed early Phase 1 trials for solid tumours and non-Hodgkin's lymphoma and is currently being evaluated in Phase 1/2 trials for Multiple Myeloma.

#### **7.5 ONX0912**

ONX0912 (formerly PR-047) is a novel orally available analogue of the proteasome inhibitor carfilzomib. Carfilzomib, in common with bortezomib, is administered intravenously, however, proteasome inhibitor therapy requires twice weekly dosing and therefore an orally available inhibitor would be more advantageous. ONX0912 has demonstrated similar antitumour activity to carfilzomib *in vitro* in cell lines and primary cells and enhanced the anti-Myeloma activity of bortezomib, lenolidomide and histone deacetylase inhibitors; animal models of Multiple Myeloma, non-Hodgkin's lymphoma and colorectal cancer demonstrated reduced tumour progression and prolonged survival (Zhou et al., 2009; Roccaro et al., 2010; Chauhan et al., 2010b). A Phase 1 trial of ONX0912 in advanced solid tumours is currently recruiting.

#### **7.6 Immunoproteasome Inhibitors**

A novel approach that is looking promising is the use of proteasome inhibitors that specifically inhibit catalytic activities of the immunoproteasome. Immunoproteasomes are constitutively expressed in immune tissues and expressed at a much lower level in other cell types. Thus targeting immunoproteasomes confers a certain amount of specificity and provides an opportunity to overcome toxicities associated with proteasome inhibition, such as peripheral neuropathy and gastrointestinal effects. A number of immunoproteasome specific inhibitors have recently been described and exhibit encouraging preclinical activity in haematological malignancies. PR-924 is a tripeptide epoxyketone related to carfilzomib. It exhibits 100-fold greater selectivity for the LMP7 subunit than carfilzomib and was demonstrated to inhibit the growth of Multiple Myeloma cell lines and primary tumour cells and inhibited tumour growth in animal models without significant toxicity (Singh et al., 2010). The immunoproteasome inhibitor IPSI-101 is a peptide aldehyde which preferentially inhibits the LMP2 subunit. IPSI-101 induced accumulation of polyubiquitinated proteins and pro-apoptotic protein and inhibited proliferation in *in vitro* models of haematological malignancies (Kuhn et al., 2009). At the time of writing this review there were no clinical trials of immunoproteasome inhibitors in progress, however, it is likely that the encouraging preclinical data on PR-924 and ISPS-101 will form the basis for future clinical evaluation of these compounds.

Fig. 2. Structure and class of proteasome inhibitors in clinical trials.

#### **8. Conclusion**

18 Multiple Myeloma – An Overview

inhibitors and dexamethasone *in vitro*. It is well tolerated in plasmacytoma xenograft mouse models and demonstrates significantly longer survival time than mice treated with bortezomib (Chauhan et al., 2011). This compound is currently being evaluated in Phase 1 studies in patients with lymphoma and non-haematological malignancies and in Phase 1/2

CEP-18770 is a next-generation boronic acid-based proteasome inhibitor and in common with bortezomib it is a reversible inhibitor, primarily of the chymotrypsin-like activity. CEP-18770 was demonstrated to induce apoptosis in Multiple Myeloma cell lines and primary Myeloma cells, while displaying a favourable cytotoxicity profile towards normal cells (Piva et al., 2008; Dorsey et al., 2008). Its anti-tumour activity was demonstrated in several animal tumour models and it has been shown to demonstrate marked anti-Myeloma effects in combination with bortezomib and melphalan (Sanchez et al., 2010). CEP-18770 has completed early Phase 1 trials for solid tumours and non-Hodgkin's lymphoma and is

ONX0912 (formerly PR-047) is a novel orally available analogue of the proteasome inhibitor carfilzomib. Carfilzomib, in common with bortezomib, is administered intravenously, however, proteasome inhibitor therapy requires twice weekly dosing and therefore an orally available inhibitor would be more advantageous. ONX0912 has demonstrated similar antitumour activity to carfilzomib *in vitro* in cell lines and primary cells and enhanced the anti-Myeloma activity of bortezomib, lenolidomide and histone deacetylase inhibitors; animal models of Multiple Myeloma, non-Hodgkin's lymphoma and colorectal cancer demonstrated reduced tumour progression and prolonged survival (Zhou et al., 2009; Roccaro et al., 2010; Chauhan et al., 2010b). A Phase 1 trial of ONX0912 in advanced solid

A novel approach that is looking promising is the use of proteasome inhibitors that specifically inhibit catalytic activities of the immunoproteasome. Immunoproteasomes are constitutively expressed in immune tissues and expressed at a much lower level in other cell types. Thus targeting immunoproteasomes confers a certain amount of specificity and provides an opportunity to overcome toxicities associated with proteasome inhibition, such as peripheral neuropathy and gastrointestinal effects. A number of immunoproteasome specific inhibitors have recently been described and exhibit encouraging preclinical activity in haematological malignancies. PR-924 is a tripeptide epoxyketone related to carfilzomib. It exhibits 100-fold greater selectivity for the LMP7 subunit than carfilzomib and was demonstrated to inhibit the growth of Multiple Myeloma cell lines and primary tumour cells and inhibited tumour growth in animal models without significant toxicity (Singh et al., 2010). The immunoproteasome inhibitor IPSI-101 is a peptide aldehyde which preferentially inhibits the LMP2 subunit. IPSI-101 induced accumulation of polyubiquitinated proteins and pro-apoptotic protein and inhibited proliferation in *in vitro* models of haematological

currently being evaluated in Phase 1/2 trials for Multiple Myeloma.

trials for Multiple Myeloma.

**7.4 CEP-18770** 

**7.5 ONX0912** 

tumours is currently recruiting.

**7.6 Immunoproteasome Inhibitors** 

Proteasome Inhibitors have provided a major new therapeutic strategy for the treatment of Multiple Myeloma. Bortezomib, the first-in-class of these inhibitors, has shown remarkable success since its introduction almost ten years ago. Second generation compounds are already demonstrating increased selectivity with a more acceptable therapeutic window. Researchers are turning to other parts of the Ubiquitin Proteasome Pathway to look for potential druggable targets which would confer greater specificity. The E3 ligases play a key role in substrate selection and the Pharma already have agents in their pipeline which show promise in modifying their action. Modulation of the Ubiquitin Proteasome Pathway with novel inhibitors offers a powerful approach to Myeloma therapy.

#### **9. References**

Ackler, S., M. J. Mitten, K. Foster, A. Oleksijew, M. Refici, S. K. Tahir, Y. Xiao, et al. 2010. The bcl-2 inhibitor ABT-263 enhances the response of multiple chemotherapeutic regimens in hematologic tumors in vivo. *Cancer Chemotherapy and Pharmacology* 66 (5) (Oct): 869-80.

Proteasome Inhibitors in the Treatment of Multiple Myeloma 21

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Proteasome Inhibitors in the Treatment of Multiple Myeloma 31

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**2** 

**Regulatory Cells and Multiple Myeloma** 

The concept of immune surveillance posits innate and adaptive immune cell mediated recognition and elimination of tumor cells, which express either tumor specific antigens or molecules by cellular stress. Both innate and adaptive immunity are important to inhibit tumor formation and rejection of transplanted tumors (Dunn et al., 2004; Pardoll, 2003). Despite immune surveillance, tumors do progress. Therefore, new concept immunoediting provides complete explanation of immune system in cancers. Immunoediting has three phases against tumors including elimination, equilibrium and escape (Swann & Smyth, 2007). Elimination phase is similar to immune surveillance, where the immune system detects tumor cells and kills them. In the equilibrium phase, tumor cells become dormant, and the immune system selectively destroys susceptible tumor clones and prevents tumor progression. In the escape phase, immune responses fail to suppress the tumor growth which leads to development of tumor variants that are resistant to anti-tumor responses. The major immune cells involved in targeting the tumor mass are CD8 T cells (MHC I dependent) and NK cells (MHC I independent/deficient) (Pardoll, 2003; Smyth et al., 2000, 2001). Perforin and Fas/FasL pathways constitute for contact-mediated cytotoxicity represented by NK and CD8 T cells (Lieberman, 2003; Russell & Ley, 2002). Also, other pathways play an important role in tumor elimination, such as IFN-γ and IFN-α/β. Regulatory T cells have been initially described in studies by Sakaguchi et al who proved the role of (CD4+CD25+) regulatory T (Treg) cells in maintaining the tolerance against self-antigens (Sakaguchi et al., 1995, 2004, 2008). Treg cells have been shown to contain distinct populations (Table 1) which are able to actively suppress the function of other immune cells, including CD4+CD25- T cells, CD8 T cells, dendritic cells, macrophages, B cells, NK cells and NKT cells (Azuma et al., 2003; Chen, 2006; Lim et al., 2005; Murakami et al., 2002; Romagnani et al., 2005; Trzonkowski et al., 2004). Several studies recently proved that Treg cells could induce tolerance against tumors (Nagai et al., 2004; von Boehmer, 2005; Yamaguchi & Sakaguchi, 2006). Also, studies addressed the expansion of Treg cells in various non-hematological and hematological malignancies (Beyer & Schultze, 2006b). Treg cells were also proved to

**1. Introduction**

Karthick Raja Muthu Raja1,2 and Roman Hajek1,2,3 *1Babak Myeloma Group, Department of Pathological Physiology,* 

> *Faculty of Medicine, Masaryk University, Brno, 2Department of Molecular and Cellular Biology, Faculty of Science, Masaryk University, Brno, 3Department of Internal Medicine- Hematooncology,*

> > *Faculty Hospital, Brno,*

*Czech Republic* 


### **Regulatory Cells and Multiple Myeloma**

Karthick Raja Muthu Raja1,2 and Roman Hajek1,2,3

*1Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, 2Department of Molecular and Cellular Biology, Faculty of Science, Masaryk University, Brno, 3Department of Internal Medicine- Hematooncology, Faculty Hospital, Brno, Czech Republic* 

#### **1. Introduction**

32 Multiple Myeloma – An Overview

Yu, C., B. B. Friday, J. P. Lai, L. Yang, J. Sarkaria, N. E. Kay, C. A. Carter, L. R. Roberts, S. H.

Zangari, M., M. Aujay, F. Zhan, K. L. Hetherington, T. Berno, R. Vij, S. Jagannath, et al. 2011.

Zhou, H. J., M. A. Aujay, M. K. Bennett, M. Dajee, S. D. Demo, Y. Fang, M. N. Ho, et al. 2009.

*Therapeutics* 5 (9) (Sep): 2378-87.

(Jun): 484-7.

38.

Kaufmann, and A. A. Adjei. 2006. Cytotoxic synergy between the multikinase inhibitor sorafenib and the proteasome inhibitor bortezomib in vitro: Induction of apoptosis through akt and c-jun NH2-terminal kinase pathways. *Molecular Cancer* 

Alkaline phosphatase variation during carfilzomib treatment is associated with best response in multiple myeloma patients. *European Journal of Haematology* 86 (6)

Design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (PR-047). *Journal of Medicinal Chemistry* 52 (9) (May 14): 3028-

> The concept of immune surveillance posits innate and adaptive immune cell mediated recognition and elimination of tumor cells, which express either tumor specific antigens or molecules by cellular stress. Both innate and adaptive immunity are important to inhibit tumor formation and rejection of transplanted tumors (Dunn et al., 2004; Pardoll, 2003). Despite immune surveillance, tumors do progress. Therefore, new concept immunoediting provides complete explanation of immune system in cancers. Immunoediting has three phases against tumors including elimination, equilibrium and escape (Swann & Smyth, 2007). Elimination phase is similar to immune surveillance, where the immune system detects tumor cells and kills them. In the equilibrium phase, tumor cells become dormant, and the immune system selectively destroys susceptible tumor clones and prevents tumor progression. In the escape phase, immune responses fail to suppress the tumor growth which leads to development of tumor variants that are resistant to anti-tumor responses. The major immune cells involved in targeting the tumor mass are CD8 T cells (MHC I dependent) and NK cells (MHC I independent/deficient) (Pardoll, 2003; Smyth et al., 2000, 2001). Perforin and Fas/FasL pathways constitute for contact-mediated cytotoxicity represented by NK and CD8 T cells (Lieberman, 2003; Russell & Ley, 2002). Also, other pathways play an important role in tumor elimination, such as IFN-γ and IFN-α/β. Regulatory T cells have been initially described in studies by Sakaguchi et al who proved the role of (CD4+CD25+) regulatory T (Treg) cells in maintaining the tolerance against self-antigens (Sakaguchi et al., 1995, 2004, 2008). Treg cells have been shown to contain distinct populations (Table 1) which are able to actively suppress the function of other immune cells, including CD4+CD25- T cells, CD8 T cells, dendritic cells, macrophages, B cells, NK cells and NKT cells (Azuma et al., 2003; Chen, 2006; Lim et al., 2005; Murakami et al., 2002; Romagnani et al., 2005; Trzonkowski et al., 2004). Several studies recently proved that Treg cells could induce tolerance against tumors (Nagai et al., 2004; von Boehmer, 2005; Yamaguchi & Sakaguchi, 2006). Also, studies addressed the expansion of Treg cells in various non-hematological and hematological malignancies (Beyer & Schultze, 2006b). Treg cells were also proved to

Regulatory Cells and Multiple Myeloma 35

does not exclude the non-regulatory Foxp3 expressing T cells. These cells express CD45RO, lack suppressive ability and secrete pro-inflammatory cytokines IL-2, IFN-γ and IL-17 (Miyara et al., 2009). In conclusion, heterogenic expression of FoxP3 by nonregulatory and Treg cells precludes the inclusion of FoxP3 as a sole marker in humans to

The existence of Treg cells was uncovered more than four decades ago in studies showing that neonatally thymectomized mice developed autoimmunity, which could be prevented by reconstitution with CD4 T cells (Nishizuka & Sakakura, 1969; Sakaguchi et al., 1982). Further work characterized these Treg cells as CD4+ T cells expressing high levels of IL-2 receptor α chain (Sakuguchi et al., 1995). Fontenot et al determined that in mice, forkhead transcription factor FoxP3 is a specific marker of Treg cells and a master regulator in development and function of Treg cells (Fontenot et al., 2003). Types and functions of

**Types Origin Functions References** 

In mice, depletion of these cells leads to autoimmunity. In humans, mutation in FoxP3 gene located on X chromosome leads to fatal immune disorder IPEX syndrome (Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked syndrome)

Induced in the periphery from naïve T cells in the presence of IL-10. These cells lack FoxP3 expression but secrete IL-10 and TGF-β.

Induced in the periphery from naïve T cells in the presence of TGF-β. Suppression is mediated by TGF-β. Rare Th3 cells express FoxP3 molecule due to induction by TGF-β.

In mice and humans these cells constitute about 1%-3% and 1%, respectively. Double negative Treg cells inhibit T cell activation and proliferation in an antigen specific manner.

Suppress naïve and effector T cell responses and inhibit maturation and function of dendritic cells.

Gambineri et al., 2003.

Groux et al., 1997; Vieira et al., 2004

Apostolou & von Boehmer, 2004; Chen et al., 2003.

> Fischer et al., 2005.

> Peng et al., 2007.

characterize the Treg cells.

CD4+ natural regulatory T cells

> CD4+ Tr1 regulatory cells

CD4+ Th3

Double negative Treg cells

**3. Types and functions of T regulatory cells** 

various Treg cells are summarized in Table 1 and Table 2.

Arise in thymus and disseminate to periphery; constitute about 10%-15% of CD4 cells

Periphery

Periphery

site (breast cancer)

cells Periphery

γδ T cells Infiltrate into tumor

inversely correlate with outcome of various cancers, including gastric malignancies, ovarian and breast cancers (Curiel et al., 2004; Merlo et al., 2009; Sasada et al., 2003). However, it appears that in certain diseases, such as follicular lymphoma and Hodgkin's lymphoma, higher number of FoxP3+ cells correlate with better survival (Alvaro et al., 2005; Carreras et al., 2006).

Multiple myeloma is a plasma cell proliferative disorder and the second most common hematological malignancy standing next to lymphoma (Kyle & Rajkumar, 2008). Multiple myeloma is clinically characterized by ≥ 10% of plasma cell infiltration in the bone marrow, ≥ 30g/L of monoclonal protein and presence of CRAB symptoms (hypercalcemia, renal insufficiency, anemia and bone lytic lesions) (Raja et al., 2010). It has been proved that B and T lymphocyte populations in multiple myeloma significantly associate with survival (Kay et al., 2001). Multiple myeloma patients commonly present with defects in numbers and function of various immune cells including dendritic cells, B cells, T cells and NK cells (Pratt et al., 2007). Concept of immunoediting also fits in multiple myeloma because of its several stages, starting from premalignant stage known as monoclonal gammopathy of undetermined significance to symptomatic stage (Swann & Smyth, 2007). Elimination phase of immunoediting has been explained in premalignant stage of myeloma, where strong T cell response was observed against the tumor clone compared to malignant stage (Dhodapkar, 2005). Followed by surveillance, T cells patrol the premalignant clone (equilibrium) and finally lose responses against malignant clones which lead to symptomatic myeloma (escape) (Dhodapkar et al., 2003). Recently, in multiple myeloma several studies showed elevated level of Treg cells; these cells were functionally active in suppressing the function of naïve T cells. In this chapter, we focus on describing general aspects of Treg cells, including subtypes, functions, migration and induction of tolerance at tumor microenvironment. Then, we discuss the role of CD4 Treg cells in multiple myeloma and their association with stages and survival, and influence of immunomodulatory drugs on multiple myeloma patient's Treg cells. Additionally, we discuss the role of Th17 cells, CD8 Treg cells and myeloid-derived suppressor cells (MDSCs) in multiple myeloma.

#### **2. Phenotypic characterizations of regulatory T cells in humans**

In mice, CD4 Treg cells can be easily characterized as CD4+FoxP3+ and high expression of CD25 (Sakaguchi et al., 1995). However, identification of human Treg cells is ambiguous because approximately 1-2 % of CD25 expressing T cells are functional Treg cells (Allan et al., 2005; Baecher-Allan et al., 2001). Isolation of CD25hi T cells excludes FoxP3 low and CD25 low/intermediate Treg cells which are found to be naïve Treg cell population (CD45RA+/CD45RO-). Unclear identification of CD25 (intermediate and high) expression on CD4 Treg cells influence the reproducibility of results in various disorders. Several groups suggested that negative expression of CD127 in Treg cells might help in characterization. But it will not ensure the accurate identification of Treg cells because activated CD4 T cells are also CD127- (Liu et al., 2006; Seddiki et al., 2006). Alternatively, CD62L expression could differentiate the recently activated CD4 T cells (CD62L low) and Treg cells (CD25hi+CD62L+CD127low) but CD62L expression is not solely restricted to Treg cells (Hamann et al., 2000). Characterization of Treg cells using the CD127 and CD25

inversely correlate with outcome of various cancers, including gastric malignancies, ovarian and breast cancers (Curiel et al., 2004; Merlo et al., 2009; Sasada et al., 2003). However, it appears that in certain diseases, such as follicular lymphoma and Hodgkin's lymphoma, higher number of FoxP3+ cells correlate with better survival (Alvaro et al.,

Multiple myeloma is a plasma cell proliferative disorder and the second most common hematological malignancy standing next to lymphoma (Kyle & Rajkumar, 2008). Multiple myeloma is clinically characterized by ≥ 10% of plasma cell infiltration in the bone marrow, ≥ 30g/L of monoclonal protein and presence of CRAB symptoms (hypercalcemia, renal insufficiency, anemia and bone lytic lesions) (Raja et al., 2010). It has been proved that B and T lymphocyte populations in multiple myeloma significantly associate with survival (Kay et al., 2001). Multiple myeloma patients commonly present with defects in numbers and function of various immune cells including dendritic cells, B cells, T cells and NK cells (Pratt et al., 2007). Concept of immunoediting also fits in multiple myeloma because of its several stages, starting from premalignant stage known as monoclonal gammopathy of undetermined significance to symptomatic stage (Swann & Smyth, 2007). Elimination phase of immunoediting has been explained in premalignant stage of myeloma, where strong T cell response was observed against the tumor clone compared to malignant stage (Dhodapkar, 2005). Followed by surveillance, T cells patrol the premalignant clone (equilibrium) and finally lose responses against malignant clones which lead to symptomatic myeloma (escape) (Dhodapkar et al., 2003). Recently, in multiple myeloma several studies showed elevated level of Treg cells; these cells were functionally active in suppressing the function of naïve T cells. In this chapter, we focus on describing general aspects of Treg cells, including subtypes, functions, migration and induction of tolerance at tumor microenvironment. Then, we discuss the role of CD4 Treg cells in multiple myeloma and their association with stages and survival, and influence of immunomodulatory drugs on multiple myeloma patient's Treg cells. Additionally, we discuss the role of Th17 cells, CD8 Treg cells and myeloid-derived suppressor cells

2005; Carreras et al., 2006).

(MDSCs) in multiple myeloma.

**2. Phenotypic characterizations of regulatory T cells in humans** 

In mice, CD4 Treg cells can be easily characterized as CD4+FoxP3+ and high expression of CD25 (Sakaguchi et al., 1995). However, identification of human Treg cells is ambiguous because approximately 1-2 % of CD25 expressing T cells are functional Treg cells (Allan et al., 2005; Baecher-Allan et al., 2001). Isolation of CD25hi T cells excludes FoxP3 low and CD25 low/intermediate Treg cells which are found to be naïve Treg cell population (CD45RA+/CD45RO-). Unclear identification of CD25 (intermediate and high) expression on CD4 Treg cells influence the reproducibility of results in various disorders. Several groups suggested that negative expression of CD127 in Treg cells might help in characterization. But it will not ensure the accurate identification of Treg cells because activated CD4 T cells are also CD127- (Liu et al., 2006; Seddiki et al., 2006). Alternatively, CD62L expression could differentiate the recently activated CD4 T cells (CD62L low) and Treg cells (CD25hi+CD62L+CD127low) but CD62L expression is not solely restricted to Treg cells (Hamann et al., 2000). Characterization of Treg cells using the CD127 and CD25 does not exclude the non-regulatory Foxp3 expressing T cells. These cells express CD45RO, lack suppressive ability and secrete pro-inflammatory cytokines IL-2, IFN-γ and IL-17 (Miyara et al., 2009). In conclusion, heterogenic expression of FoxP3 by nonregulatory and Treg cells precludes the inclusion of FoxP3 as a sole marker in humans to characterize the Treg cells.

#### **3. Types and functions of T regulatory cells**

The existence of Treg cells was uncovered more than four decades ago in studies showing that neonatally thymectomized mice developed autoimmunity, which could be prevented by reconstitution with CD4 T cells (Nishizuka & Sakakura, 1969; Sakaguchi et al., 1982). Further work characterized these Treg cells as CD4+ T cells expressing high levels of IL-2 receptor α chain (Sakuguchi et al., 1995). Fontenot et al determined that in mice, forkhead transcription factor FoxP3 is a specific marker of Treg cells and a master regulator in development and function of Treg cells (Fontenot et al., 2003). Types and functions of various Treg cells are summarized in Table 1 and Table 2.


Regulatory Cells and Multiple Myeloma 37

**Treg cells Mechanism of Suppression References** 

Expression of CTLA-4 under the control of FoxP3 facilitates Treg cells interaction with CD80 and CD86 on APCs and induces the suppression of T cell activation. CTLA-4 mediated interaction between Treg cells and APCs leads to upregulation of indoleamine 2, 3 dioxygenase (IDO) production by the APCs which leads to degradation of the essential amino acid tryptophan. Recently, it was proposed that IDO mediated depletion of tryptophan inhibited T cell proliferation and response against tumors.

**4. Mechanism of migration and induction of tolerance in the tumor bed by** 

There are several chemokine receptors and ligands are involved in the migration of Treg cells. Before going into detail, we would like to summarize the migration process of Treg cells from thymus to secondary lymphoid organ in normal physiological conditions. Preliminary switch occurs in chemokine receptors CCR8/CCR9 and CXCR4 to CCR7 on thymic precursors of Treg cells. These precursor Treg cells are a homogeneous population and express CD62L, CCR7 and CXCR4low (Lee et al., 2007). After migration from thymus to the periphery, these Treg cells acquire complete expression of CXCR4. Thymic emigrant Treg cells most exclusively migrate to secondary lymphoid organ for antigen contact. After the antigen contact, the second switch occurs in the receptors where they downregulate the CCR7 and CXCR4 expression. Consequently, upregulation of effector/memory chemokine receptors (CCR2, CCR4, CCR6, CCR8, and CCR9) occurs to enhance the heterogenic property of Treg cell population (Lee et al., 2007). It was shown that CCR7 mediated migration of Treg cells into secondary lymphoid organ is an important process for encountering mature dendritic cells and consequent differentiation and proliferation (Schneider et al., 2007). All peripheral blood Treg cells express CCR4 receptor and show chemotactic response to ligands CCL22 and CCL17. In ovarian cancer, Treg cell migration and accumulation at the tumor site is attributed by CCL22 chemokine (Curiel et al., 2004). Secretion of CCL22 by lymphoma cells largely recruits the intratumoral Treg cells that express CCR4 but not CCR8 (Ishida et al., 2006; Yang et al., 2006b). In non-hematological malignancies, it was observed that increased frequency of CCR4 expressing Treg cells associated with CCL22 and CCL17 chemokines. An *in vitro* study in gastric cancer showed that migratory activity of Treg cells was induced by CCL22 and CCL17 chemokines

Several molecules and receptors contribute the suppressive function of Treg cells in the tumor microenvironment. Suppression mechanism by Treg cells is not solely dependent on cell-cell contact rather it is also supported by soluble IL-10 and TGF-β molecules (Strauss et al., 2007). Treg cells do not depend always on direct inhibition of effector T cells; rather, it impedes function of dendritic cells via IL-10 and TGF-β. This mechanism downregulates NF-kB and subsequently changes downstream molecules (CD80, CD86 and CD40) as well as

Mellor & Munn, 2004; Munn et al., 1999; Oderup et al., 2006; Read et al., 2000.

**Functions of** 

Inhibition of antigen presenting cells (APCs)

**regulatory cells** 

(Mizukami et al., 2008).

Table 2. Functions of T regulatory cells


Table 1. Subsets of T regulatory cells


**Types Origin Functions References** 

**Treg cells Mechanism of Suppression References** 

Mainly cytokines such as IL-10, TGF- β and IL-35 secreted by Treg cells are involved in inhibitory function. Chen et al proved in murine colon carcinoma IL-10 induced suppression of tumor specific CD8 T cell immunity. Peptide inhibitor targeted against the surface TGF-β on Treg cells abrogated their function and enhanced antitumor response. In mouse model of inflammatory bowel disease, it was shown that IL-35 played a role in severity of inflammatory bowel disease. In humans IL-35 is not expressed constitutively by Treg cells.

Perforin/granzyme pathway is well known to be associated with CD8 T cells and NK cells for destruction of intracellular pathogens and tumor cells. Recent studies have shown Treg cells also use the perforin/granzyme pathway. An *in vitro* study demonstrated Treg cells activated with anti CD3 and anti CD46 antibodies expressed granzyme A and B. In murine Treg cells, it was shown that perforin lacking Treg cells also exhibit suppressive function. Cao et al reported in a tumor inoculation system the adoptively transferred Treg cells induced suppression of tumor immunity (CD8 T cells and NK cells) specifically by granzyme B pathway. Fas ligand utilizing Treg cells presence was reported in head and neck squamous cell carcinoma patients and found to suppress CD8 T cells.

There are several subsets of CD8 Treg cells: Qa-1 specific CD8 Treg cells suppress autoreactive T cells expressing Qa-1 molecule associated with self peptide. CD8+CD28- Treg cells express FoxP3α molecule and suppress other cells via contact dependent mechanism. CD8+CD25+ Treg cells suppress both naïve CD4 and CD8 T cells by contact dependent or independent (IL-10) mechanisms. CD8+CD25+ Treg cells mostly accumulate in the tumor bed rather than peripheral tissues.

Filaci et al., 2007; Kiniwa et al., 2007; Sarantopoulos et al., 2004; Wang, 2008.

> Bardel et al., 2008; Chen et al., 2005; Collison et al., 2007; Gil-Guerrero et al., 2008; Loser et al., 2007.

Cao et al., 2007; Gondek et al., 2005; Grossman et al, 2004; Lieberman, 2003; Russell & Ley, 2002; Strauss et al., 2009.

CD8 regulatory T cells

**Functions of** 

Inhibitory cytokines

Cytotoxicity

Most of CD8 Treg cells are induced by antigen- specific manner

Table 1. Subsets of T regulatory cells


Table 2. Functions of T regulatory cells

#### **4. Mechanism of migration and induction of tolerance in the tumor bed by regulatory cells**

There are several chemokine receptors and ligands are involved in the migration of Treg cells. Before going into detail, we would like to summarize the migration process of Treg cells from thymus to secondary lymphoid organ in normal physiological conditions. Preliminary switch occurs in chemokine receptors CCR8/CCR9 and CXCR4 to CCR7 on thymic precursors of Treg cells. These precursor Treg cells are a homogeneous population and express CD62L, CCR7 and CXCR4low (Lee et al., 2007). After migration from thymus to the periphery, these Treg cells acquire complete expression of CXCR4. Thymic emigrant Treg cells most exclusively migrate to secondary lymphoid organ for antigen contact. After the antigen contact, the second switch occurs in the receptors where they downregulate the CCR7 and CXCR4 expression. Consequently, upregulation of effector/memory chemokine receptors (CCR2, CCR4, CCR6, CCR8, and CCR9) occurs to enhance the heterogenic property of Treg cell population (Lee et al., 2007). It was shown that CCR7 mediated migration of Treg cells into secondary lymphoid organ is an important process for encountering mature dendritic cells and consequent differentiation and proliferation (Schneider et al., 2007). All peripheral blood Treg cells express CCR4 receptor and show chemotactic response to ligands CCL22 and CCL17. In ovarian cancer, Treg cell migration and accumulation at the tumor site is attributed by CCL22 chemokine (Curiel et al., 2004). Secretion of CCL22 by lymphoma cells largely recruits the intratumoral Treg cells that express CCR4 but not CCR8 (Ishida et al., 2006; Yang et al., 2006b). In non-hematological malignancies, it was observed that increased frequency of CCR4 expressing Treg cells associated with CCL22 and CCL17 chemokines. An *in vitro* study in gastric cancer showed that migratory activity of Treg cells was induced by CCL22 and CCL17 chemokines (Mizukami et al., 2008).

Several molecules and receptors contribute the suppressive function of Treg cells in the tumor microenvironment. Suppression mechanism by Treg cells is not solely dependent on cell-cell contact rather it is also supported by soluble IL-10 and TGF-β molecules (Strauss et al., 2007). Treg cells do not depend always on direct inhibition of effector T cells; rather, it impedes function of dendritic cells via IL-10 and TGF-β. This mechanism downregulates NF-kB and subsequently changes downstream molecules (CD80, CD86 and CD40) as well as

Regulatory Cells and Multiple Myeloma 39

mechanisms of suppression by Treg cells were revealed. Expression of CD39 in conjunction with CD73 generates the adenosine molecule and its interaction with adenosine A2A receptor on activated T cells creates strong immunosuppressive loops; this suggests one of the important suppressive mechanisms of Treg cells (Deaglio et al., 2007). Transfer of cyclic adenosine monophosphate by Treg cells to effector T cells via cell-cell interaction induces suppression and impedes IL-2 secretion (Bopp et al., 2007). Also, recently it was proven that effector T cells are suppressed via apoptosis induced by deprivation of γ cytokines by Treg cells. Strong association was observed between Treg cell induced apoptosis and increased level of pro-apoptotic proteins Bim and Bad as well as decreased level of pro-survival protein kinase Akt (Pandiyan et al., 2007). Taking all these observations into consideration, the significance of chemokines and their receptors in migration of Treg cells could act as a suitable target for deprivation of Treg cells in the tumor microenvironment; this might also enhance anti-tumor responses. To add more flavors in impeding Treg cell migration, a chimeric monoclonal antibody targeting CCR4 receptor is already under clinical trial. All of the above mentioned mechanisms of suppression collectively work together to induce suppression of immune cells and tolerance in the tumor bed. In some circumstances, these mechanisms might

Recently, several research groups analyzed Treg cells in multiple myeloma. So far, Treg cells data in multiple myeloma are conflicting. Study from Prabhala et al and Gupta et al reported decreased frequency of peripheral blood Treg cells in multiple myeloma when compared to control group (Gupta et al., 2011; Prabhala et al., 2006). Both studies confirmed that FoxP3 expression was reduced in myeloma patients. In contrast to these studies, Feyler et al and Beyer et al reported increased frequencies of peripheral blood Treg cells in multiple myeloma patients (Feyler et al., 2009; Beyer et al., 2006a). Most of the studies confirmed that peripheral blood and bone marrow Treg cells frequencies were comparable. According to Beyer et al, Treg cells associated markers, such as CTLA-4, GITR, CD62L and OX40 were elevated in myeloma patients compared to healthy subjects (Beyer et al., 2006a). Contrasting to this observation, Prabhala et al showed decreased frequency of CTLA-4 expression on Treg cells of monoclonal gammopathy of undetermined significance and myeloma cohorts than healthy subjects (Prabhala et al., 2006). These opposing results may be due to Treg cells identification strategy. For instance, Prabhala et al identified Treg cells as CD4+FoxP3+, Gupta et al characterized Treg cells with the inclusion of CD127 in their gating, Feyler et al identified Treg cells as CD4+CD25hi+FoxP3+ and Beyer et al identified Treg cells using only CD4 and CD25 markers (Beyer et al., 2006a; Feyler et al., 2009; Gupta et al., 2011; Prabhala et al., 2006). Most of the studies in other cancers including hematological and nonhematological malignancies showed elevated level of Treg cells and these cells are associated with worse prognosis. To support the concept of tumor based expansion of Treg cells, studies clearly showed that established Treg cell clones recognized tumor antigen in MHC class II restricted manner (Wang et al., 2004). In multiple myeloma, no strong conclusions could be made due to the existence of equal number of conflicting results with

independently act to enforce the suppression of anti-tumor responses.

**5. Regulatory T cells and multiple myeloma** 

regard to Treg cells frequency.

soluble factors, such as TNF-α, IL-12 and CCL5 (Larmonier et al., 2007). Moreover, Treg cells in tumor bed prevent CD4 T cell mediated generation of CD8 T cell cytotoxic responses (Chaput et al., 2007). Prostaglandin E2 is the effector molecule released by Treg cells; it is also important for activation of Treg cells. This molecule efficiently suppresses the effector T cell responses via COX2 induction. Prostaglandin E2 mediated suppression by Treg cells was observed in colorectal cancer patients (Mahic et al., 2006). Cell-cell contact dependent suppression is partly attributed to CTLA-4 expression by Treg cells (Read et al., 2000). Expression of CTLA-4 also facilitates the TGF-β mediated suppression via intensifying the TGF-β signals at the interaction point of Treg cell and target cell (Oida et al., 2006). Tumor infiltrating Treg cells express ICOS molecule but peripheral Treg cells do not express ICOS. ICOS receptor and its ligand are involved in Treg cell mediated suppression. Treg cells with ICOS low expression did not show strong suppressive function as compared to Treg cells with ICOS high expression (Strauss et al., 2008). Very recently, other novel

Fig. 1. Mechanism of accumulation and expansion of T regulatory cells in the tumor bed This schematic diagram represents a mechanism of immune tolerance induced by Treg cells in the tumor microenvironment. CCL22 as well H-ferritin secretion by tumor cells and tumor infiltrating macrophages recruit naïve Treg cells (CCR4+) in the tumor bed. These accumulated naïve Treg cells differentiate and proliferate into memory Treg cells via interaction with prostaglandin E2 (PGE2) induced tolerogenic dendritic cells. These expanded memory Treg cells along with tolerogenic dendritic cells impede the functions of tumor effector T cells.

soluble factors, such as TNF-α, IL-12 and CCL5 (Larmonier et al., 2007). Moreover, Treg cells in tumor bed prevent CD4 T cell mediated generation of CD8 T cell cytotoxic responses (Chaput et al., 2007). Prostaglandin E2 is the effector molecule released by Treg cells; it is also important for activation of Treg cells. This molecule efficiently suppresses the effector T cell responses via COX2 induction. Prostaglandin E2 mediated suppression by Treg cells was observed in colorectal cancer patients (Mahic et al., 2006). Cell-cell contact dependent suppression is partly attributed to CTLA-4 expression by Treg cells (Read et al., 2000). Expression of CTLA-4 also facilitates the TGF-β mediated suppression via intensifying the TGF-β signals at the interaction point of Treg cell and target cell (Oida et al., 2006). Tumor infiltrating Treg cells express ICOS molecule but peripheral Treg cells do not express ICOS. ICOS receptor and its ligand are involved in Treg cell mediated suppression. Treg cells with ICOS low expression did not show strong suppressive function as compared to Treg cells with ICOS high expression (Strauss et al., 2008). Very recently, other novel

Fig. 1. Mechanism of accumulation and expansion of T regulatory cells in the tumor bed This schematic diagram represents a mechanism of immune tolerance induced by Treg cells in the tumor microenvironment. CCL22 as well H-ferritin secretion by tumor cells and tumor infiltrating macrophages recruit naïve Treg cells (CCR4+) in the tumor bed. These accumulated naïve Treg cells differentiate and proliferate into memory Treg cells via interaction with prostaglandin E2 (PGE2) induced tolerogenic dendritic cells. These

expanded memory Treg cells along with tolerogenic dendritic cells impede the functions of

tumor effector T cells.

mechanisms of suppression by Treg cells were revealed. Expression of CD39 in conjunction with CD73 generates the adenosine molecule and its interaction with adenosine A2A receptor on activated T cells creates strong immunosuppressive loops; this suggests one of the important suppressive mechanisms of Treg cells (Deaglio et al., 2007). Transfer of cyclic adenosine monophosphate by Treg cells to effector T cells via cell-cell interaction induces suppression and impedes IL-2 secretion (Bopp et al., 2007). Also, recently it was proven that effector T cells are suppressed via apoptosis induced by deprivation of γ cytokines by Treg cells. Strong association was observed between Treg cell induced apoptosis and increased level of pro-apoptotic proteins Bim and Bad as well as decreased level of pro-survival protein kinase Akt (Pandiyan et al., 2007). Taking all these observations into consideration, the significance of chemokines and their receptors in migration of Treg cells could act as a suitable target for deprivation of Treg cells in the tumor microenvironment; this might also enhance anti-tumor responses. To add more flavors in impeding Treg cell migration, a chimeric monoclonal antibody targeting CCR4 receptor is already under clinical trial. All of the above mentioned mechanisms of suppression collectively work together to induce suppression of immune cells and tolerance in the tumor bed. In some circumstances, these mechanisms might independently act to enforce the suppression of anti-tumor responses.

#### **5. Regulatory T cells and multiple myeloma**

Recently, several research groups analyzed Treg cells in multiple myeloma. So far, Treg cells data in multiple myeloma are conflicting. Study from Prabhala et al and Gupta et al reported decreased frequency of peripheral blood Treg cells in multiple myeloma when compared to control group (Gupta et al., 2011; Prabhala et al., 2006). Both studies confirmed that FoxP3 expression was reduced in myeloma patients. In contrast to these studies, Feyler et al and Beyer et al reported increased frequencies of peripheral blood Treg cells in multiple myeloma patients (Feyler et al., 2009; Beyer et al., 2006a). Most of the studies confirmed that peripheral blood and bone marrow Treg cells frequencies were comparable. According to Beyer et al, Treg cells associated markers, such as CTLA-4, GITR, CD62L and OX40 were elevated in myeloma patients compared to healthy subjects (Beyer et al., 2006a). Contrasting to this observation, Prabhala et al showed decreased frequency of CTLA-4 expression on Treg cells of monoclonal gammopathy of undetermined significance and myeloma cohorts than healthy subjects (Prabhala et al., 2006). These opposing results may be due to Treg cells identification strategy. For instance, Prabhala et al identified Treg cells as CD4+FoxP3+, Gupta et al characterized Treg cells with the inclusion of CD127 in their gating, Feyler et al identified Treg cells as CD4+CD25hi+FoxP3+ and Beyer et al identified Treg cells using only CD4 and CD25 markers (Beyer et al., 2006a; Feyler et al., 2009; Gupta et al., 2011; Prabhala et al., 2006). Most of the studies in other cancers including hematological and nonhematological malignancies showed elevated level of Treg cells and these cells are associated with worse prognosis. To support the concept of tumor based expansion of Treg cells, studies clearly showed that established Treg cell clones recognized tumor antigen in MHC class II restricted manner (Wang et al., 2004). In multiple myeloma, no strong conclusions could be made due to the existence of equal number of conflicting results with regard to Treg cells frequency.

Regulatory Cells and Multiple Myeloma 41

correlated unfavorably with progression free survival and overall survival of myeloma patients, so Treg cells could be targeted along with the tumor cells in multiple myeloma.

Immunomodulatory drugs are orally bioavailable agents, derived from thalidomide (first generation immunomodulatory drug). The second generation immunomodulatory drugs are lenalidomide and pomalidomide which share similar chemical structure with thalidomide (Galustian et al., 2004). Quach et al recently reviewed the functions of

Co-stimulation of CD4 and CD8 T cells, activation of NK and NKT cells, production of Th1 cytokines, enhancement of antibody dependent cellular cytotoxicity and Treg cell

Anti-angiogenesis, inhibition of inflammatory effects, anti-osteoclastogenesis and

Induction of cyclin dependent kinase inhibitors, such as p15, p21 and p27 which results in cell cycle arrest (G0/G1 phase), increases expression of early growth genes (1, 2), downregulation of NF-kB with subsequent reduction in anti-apoptotic proteins FLIP and

Minnema et al showed that in relapsed myeloma patients (after allogeneic stem cell transplantation), lenalidomide increased frequencies of Treg cells during treatment (Minnema et al., 2009). In contrast, CD4 and CD8 T cells were decreased. This study also showed that ratio of FoxP3+ T cells to IFN-γ secreting T cells was significantly increased during treatment (Minnema et al., 2009). Increased Treg cells always favour allogeneic stem cell transplanted patients because these cells inhibit graft versus host disease (Rezvani et al., 2006). However, Minnema et al study did not show the advantage of increased Treg cells with relevance to graft versus host disease, probably due to small patient numbers (Minnema et al., 2009). Contrasting to this study, an *in vitro* observation demonstrated that lenalidomide and pomalidomide are able to inhibit the proliferation of Treg cells at very low concentrations (10 μM and 1 μM) but thalidomide failed to inhibit the proliferation even at maximum concentration of 200 μM. This study reveals lenalidomide and pomalidomide inhibited the Treg cells mediated suppression of CD4+CD25- cells and also proposes that FoxP3 molecule was targeted efficiently by lenalidomide and pomalidomide but no alterations to GITR were observed (Galustian et al., 2009). Lenalidomide and pomalidomide also inhibit the function of Treg cells by hindering the expression of OX-40 (CD134) molecule (Galustian et al., 2009; Valzasina et al., 2005). Carcinoma animal model study showed Treg cells were depleted after cyclophosphamide treatment; tumor growth was also

**6. Influence of immunomodulatory drugs on T regulatory cells of multiple** 

immunomodulatory drugs (Quach et al., 2010). The functions are:

**6.2 Hampering tumor microenvironment interactions** 

downregulation of adhesion molecules on plasma cells.

**myeloma patients** 

**6.1 Immune modulation** 

**6.3 Direct anti-tumor effects** 

suppression.

clAP2.

#### **5.1 Immunosuppressive function of T regulatory cells in multiple myeloma**

Most studies in myeloma agree that Treg cells efficiently suppress both autologous and allogeneic responder cells (CD4+CD25-) similarly to healthy subjects (Beyer et al., 2006a; Brimnes et al., 2010; Feyler et al., 2009; Gupta et al., 2011). Exclusively, Prabhala et al showed that multiple myeloma patients Treg cells lack suppressive function (Prabhala et al., 2006). This contrasting result by Prabhala et al might be due to the use of whole peripheral blood mononuclear cells depleted with CD25+ cells as responder cells (Prabhala et al., 2006). The suppressive nature of Treg cells could be well appreciated by the presence of intracellular cytokines TGF-β and IL-10. Beyer et al confirmed that myeloma patients Treg cells express increased level of TGF-β and IL-10 when compared to healthy subjects (Beyer et al., 2006a). *In vitro* matured dendritic cells using inflammatory cytokines generate functionally active FoxP3+ Treg cells from CD25- T cells. Treg cells derived from dendritic cells are functionally similar in between healthy subjects and myeloma patients. Also, an *in vivo* study showed administration of cytokine matured dendritic cells in myeloma subjects enhanced increase of Treg cell numbers (Banerjee et al., 2006). This study also proposed that use of human dendritic cell vaccination may affect the balance of effector T cells generation because of Treg cell enhancement (Banerjee et al., 2006). In allogeneic stem cell transplanted multiple myeloma patients, donor derived Treg cells reconstituted largely in the bone marrow compartment and prevented graft versus host disease (Atanackovic et al., 2008). This data suggest that *in vivo* inhibitory function of Treg cells and also *in vitro* assay showed reconstituted Treg cells possess complete inhibitory function. Moreover, Atanackovic et al proved that reconstitution of Treg cells in bone marrow positively correlates with time passed since transplantation. Donor derived Treg cells reconstituted in the bone marrow were found to be memory Treg cells, which indicates that Treg cells indeed expanded outside the thymus (Atanackovic et al., 2008). Most of the studies strongly suggest that myeloma patients Treg cells are functional in suppressing the conventional T cell proliferation, and this suppressive function encourages the immune impairments and dysfunctions. However, Treg cells suppressive function could be appreciated in the case of graft versus host disease where donor cells require engraftment to ensure the anti-tumor effects.

#### **5.2 Association of international staging system and myeloma survival status with T regulatory cells**

Based on the international staging system (ISS), Treg cells were increased in newly diagnosed multiple myeloma patients. This observation was noticed only in ISS 1 and ISS 2 (Feyler et al., 2009). On the other hand, Gupta et al found a trend of decrease in Treg cells in stages ISS 2 and ISS 3 (Gupta et al., 2011). Apart from ISS, paraprotein level of myeloma patients was positively correlated with frequencies of Treg cells (Feyler et al., 2009).

Giannopoulos et al demonstrated patients with higher level of Treg cells have significantly reduced survival time compared to patients with lower level of Treg cells (21 months vs. median not reached) (Giannopoulos et al., 2010). Our observation also showed that patients with increased peripheral blood Treg cells (≥ 5%) have shorter progression free survival compared to patients with reduced Treg cells (< 5%) cohort (13 months vs. median not reached) (Muthu Raja et al., 2011). These data showing that elevated level of Treg cells

Most studies in myeloma agree that Treg cells efficiently suppress both autologous and allogeneic responder cells (CD4+CD25-) similarly to healthy subjects (Beyer et al., 2006a; Brimnes et al., 2010; Feyler et al., 2009; Gupta et al., 2011). Exclusively, Prabhala et al showed that multiple myeloma patients Treg cells lack suppressive function (Prabhala et al., 2006). This contrasting result by Prabhala et al might be due to the use of whole peripheral blood mononuclear cells depleted with CD25+ cells as responder cells (Prabhala et al., 2006). The suppressive nature of Treg cells could be well appreciated by the presence of intracellular cytokines TGF-β and IL-10. Beyer et al confirmed that myeloma patients Treg cells express increased level of TGF-β and IL-10 when compared to healthy subjects (Beyer et al., 2006a). *In vitro* matured dendritic cells using inflammatory cytokines generate functionally active FoxP3+ Treg cells from CD25- T cells. Treg cells derived from dendritic cells are functionally similar in between healthy subjects and myeloma patients. Also, an *in vivo* study showed administration of cytokine matured dendritic cells in myeloma subjects enhanced increase of Treg cell numbers (Banerjee et al., 2006). This study also proposed that use of human dendritic cell vaccination may affect the balance of effector T cells generation because of Treg cell enhancement (Banerjee et al., 2006). In allogeneic stem cell transplanted multiple myeloma patients, donor derived Treg cells reconstituted largely in the bone marrow compartment and prevented graft versus host disease (Atanackovic et al., 2008). This data suggest that *in vivo* inhibitory function of Treg cells and also *in vitro* assay showed reconstituted Treg cells possess complete inhibitory function. Moreover, Atanackovic et al proved that reconstitution of Treg cells in bone marrow positively correlates with time passed since transplantation. Donor derived Treg cells reconstituted in the bone marrow were found to be memory Treg cells, which indicates that Treg cells indeed expanded outside the thymus (Atanackovic et al., 2008). Most of the studies strongly suggest that myeloma patients Treg cells are functional in suppressing the conventional T cell proliferation, and this suppressive function encourages the immune impairments and dysfunctions. However, Treg cells suppressive function could be appreciated in the case of graft versus host disease where donor cells require engraftment to ensure the anti-tumor

**5.2 Association of international staging system and myeloma survival status with** 

patients was positively correlated with frequencies of Treg cells (Feyler et al., 2009).

Based on the international staging system (ISS), Treg cells were increased in newly diagnosed multiple myeloma patients. This observation was noticed only in ISS 1 and ISS 2 (Feyler et al., 2009). On the other hand, Gupta et al found a trend of decrease in Treg cells in stages ISS 2 and ISS 3 (Gupta et al., 2011). Apart from ISS, paraprotein level of myeloma

Giannopoulos et al demonstrated patients with higher level of Treg cells have significantly reduced survival time compared to patients with lower level of Treg cells (21 months vs. median not reached) (Giannopoulos et al., 2010). Our observation also showed that patients with increased peripheral blood Treg cells (≥ 5%) have shorter progression free survival compared to patients with reduced Treg cells (< 5%) cohort (13 months vs. median not reached) (Muthu Raja et al., 2011). These data showing that elevated level of Treg cells

**5.1 Immunosuppressive function of T regulatory cells in multiple myeloma** 

effects.

**T regulatory cells** 

correlated unfavorably with progression free survival and overall survival of myeloma patients, so Treg cells could be targeted along with the tumor cells in multiple myeloma.

#### **6. Influence of immunomodulatory drugs on T regulatory cells of multiple myeloma patients**

Immunomodulatory drugs are orally bioavailable agents, derived from thalidomide (first generation immunomodulatory drug). The second generation immunomodulatory drugs are lenalidomide and pomalidomide which share similar chemical structure with thalidomide (Galustian et al., 2004). Quach et al recently reviewed the functions of immunomodulatory drugs (Quach et al., 2010). The functions are:

#### **6.1 Immune modulation**

Co-stimulation of CD4 and CD8 T cells, activation of NK and NKT cells, production of Th1 cytokines, enhancement of antibody dependent cellular cytotoxicity and Treg cell suppression.

#### **6.2 Hampering tumor microenvironment interactions**

Anti-angiogenesis, inhibition of inflammatory effects, anti-osteoclastogenesis and downregulation of adhesion molecules on plasma cells.

#### **6.3 Direct anti-tumor effects**

Induction of cyclin dependent kinase inhibitors, such as p15, p21 and p27 which results in cell cycle arrest (G0/G1 phase), increases expression of early growth genes (1, 2), downregulation of NF-kB with subsequent reduction in anti-apoptotic proteins FLIP and clAP2.

Minnema et al showed that in relapsed myeloma patients (after allogeneic stem cell transplantation), lenalidomide increased frequencies of Treg cells during treatment (Minnema et al., 2009). In contrast, CD4 and CD8 T cells were decreased. This study also showed that ratio of FoxP3+ T cells to IFN-γ secreting T cells was significantly increased during treatment (Minnema et al., 2009). Increased Treg cells always favour allogeneic stem cell transplanted patients because these cells inhibit graft versus host disease (Rezvani et al., 2006). However, Minnema et al study did not show the advantage of increased Treg cells with relevance to graft versus host disease, probably due to small patient numbers (Minnema et al., 2009). Contrasting to this study, an *in vitro* observation demonstrated that lenalidomide and pomalidomide are able to inhibit the proliferation of Treg cells at very low concentrations (10 μM and 1 μM) but thalidomide failed to inhibit the proliferation even at maximum concentration of 200 μM. This study reveals lenalidomide and pomalidomide inhibited the Treg cells mediated suppression of CD4+CD25- cells and also proposes that FoxP3 molecule was targeted efficiently by lenalidomide and pomalidomide but no alterations to GITR were observed (Galustian et al., 2009). Lenalidomide and pomalidomide also inhibit the function of Treg cells by hindering the expression of OX-40 (CD134) molecule (Galustian et al., 2009; Valzasina et al., 2005). Carcinoma animal model study showed Treg cells were depleted after cyclophosphamide treatment; tumor growth was also

Regulatory Cells and Multiple Myeloma 43

cells, especially mature dendritic cells. Dendritic cells induced Th17 cells were multifunctional and secreted IL-17 and IFN-γ (Dhodapkar et al., 2008). In myeloma, tumor cells are infiltrated by dendritic cells; Dhodapkar et al demonstrated that Th17 cells were enriched in the tumor microenvironment (Dhodapkar et al., 2008). These IL-17 producing cells were found to enhance the activation of osteoclasts (Noonan et al., 2010). This study also reported significant association between extent of bone lytic lesions and IL-17 cytokine producing Th17 cells (Noonan et al., 2010). These findings suggest that Th17 cells enhance

T cells play an important role in immunosurveillance against cancers. Eventually, these T cells may become CD4/CD8 regulatory T cells due to stimulation by and interaction with tumor cells. Thus, these generated regulatory cells promote the growth of tumor rather than inhibition of tumor (Wang, 2008). Mechanisms of suppression and expansion are well documented for CD4 Treg cells but recent research has been directed to screen the presence

These CD8 Treg cells downregulate autoimmune T cell responses. They are Qa-1 (MHC class 1b molecule) restricted and specifically target self-reactive activated T cells which express Qa-1

These cells are induced by MHC class I peptide antigens. CD8+CD28- Treg cells were found to express FoxP3 α molecule and mediate suppression by cell-cell contact mechanism. CD8+CD28- Treg cells also indirectly target CD4 T cells to become tolerogenic via dendritic cells and non-professional antigen presenting cells. These cells were identified in tumors as well as in the context of transplantation (Cortesini et al., 2001; Filaci & Suciu-Foca, 2002;

These cells express CD122, Foxp3 and GITR molecules typically associated with CD4 Treg cells (Cosmi et al., 2003; Kiniwa et al., 2007; Lee et al., 2008). They suppress naive CD4 and CD8 T cells via contact dependent mechanism or soluble IL-10. These Treg cells are different from Qa-1 specific CD8 Treg cells but similar to CD4 Treg cells. CD8+CD25hi+ Treg cells are induced by the tumor environment and require antigen stimulation to suppress naïve T cells

So far, only a few studies have documented the role of CD8 Treg cells (CD8+CD25hi+FoxP3+) in cancers. In prostate and colorectal cancers, elevated levels of CD8

molecules associated with self peptide (Jiang & Chess, 2004; Sarantopoulos et al., 2004).

myeloma cell growth and development of bone lytic lesions.

of CD8 Treg cells in various tumors and inflammatory conditions.

**8. CD8 T regulatory cells in multiple myeloma** 

**8.1 Subtypes of CD8 T regulatory cells 8.1.1 Qa-1 restricted CD8 Treg cells** 

Filaci et al., 2007; Suciu-Foca et al., 2005).

**8.1.3 CD8+CD25hi+ Treg cells** 

**8.2 Role of CD8 T regulatory cells** 

(Wang, 2008).

**8.1.2 CD8+CD28- Treg cells** 

repressed due to depletion of Treg cells, and tumor clones were cleared followed by immunotherapy. Without administration of cyclophosphamide, no tumor regression or clearance was noticed in the tumor bearing animal (Ghiringhelli et al., 2004). Apart from sensitivity of Treg cells to imunomodulatory drugs and cyclophosphamide, a recent study showed that naturally occurring Treg cells were resistant to pro-apoptotic effect of proteasome inhibitor bortezomib. Long-term culturing of CD4 T cells in the presence of bortezomib promoted the emergence of Treg cells and significantly inhibited proliferation, IFN-γ production and CD40L expression by effector T cells (Blanco et al., 2009). Druginduced apoptosis resistance by Treg cells is due to the increased expression of BCL-2 and inhibitor of apoptosis protein 1 (IAP1); the expression of IAP1 is in response to TNF induced apoptosis. These BCL-2 and IAP1 protein expression was significantly elevated in Treg cells of B cell leukemic patients than healthy volunteers (Jak et al., 2009). Gupta et al confirmed that multiple myeloma patients treated with immunomodulatory drug combination showed increased Treg cells in relation to irrespective of the response achieved (Gupta et al., 2011). They also showed patients with stable and progressive disease had decreased Treg cells. Taking all these observations into consideration, no strong conclusion can be forwarded because of contrasting results between *in vitro* and *in vivo* studies. However, *in vitro* studies showed promising effects of immunomodulatory drugs on Treg cells when compared to proteasome inhibitor but immunomodulatory drugs effects might be diluted by inclusion of corticosteroid during treatment.

#### **7. Th17 cells in multiple myeloma**

Th17 cells are one of the subsets of CD4 T cells. These cells are differentiated in the presence of IL-6, IL-1β, IL-21 and IL-23 with or without TGF-β. Th17 cells secrete IL-17, IL-21, IL-22 and IL-26 cytokines. These cytokines are involved in anti-fungal and anti-parasite responses and participate in inflammatory and autoimmune reactions (Acosta-Rodriguez et al., 2007; Aujla et al., 2008; Bettelli et al., 2006; Ivanov et al., 2006; Veldhoen et al., 2008; Wilson et al., 2007; Zheng et al., 2008). Both Treg cells and Th17 cells originate from naïve CD4 T cells. For Treg cell differentiation, TGF-β is required whereas for Th17 cell differentiation, IL-6 and TGF-β are required (the role of TGF-β in human is unclear) (Bettelli et al., 2006; Veldhoen et al., 2006). Xu et al observed that mature Treg cells can be converted into Th17 cells in the presence of IL-6 (Xu et al., 2007). IL-6 and IL-21 might be involved in transition of Th17/Treg cells to Th17 cells. Some reports show switching of Th17 cells to Th1 cells, but reverse switching is not possible (Annunziato & Romagnani, 2009; Bending et al., 2009; Lee et al., 2009; Shi et al., 2008). Upregulated T-bet expression in Th17 cells resulted in a switch to Th1 cells in the presence of IL-12 or/and IL-23 (Annunziato et al., 2007; Lee et al., 2009).

Recently, a study reported increased Th17 cells and IL-17 in freshly isolated mononuclear cells and sera of myeloma patients compared to healthy subjects. In vitro polarization of CD4 T cells to Th17 cells showed increased Th17 cells in multiple myeloma patients than healthy subjects (Prabhala et al., 2010). Moreover, this study showed expression of IL-17 receptor on myeloma cells which promotes the growth of myeloma cells. IL-21 is a proinflammatory cytokine associated with Th17 cells, which is also capable of inducing STAT-3 mediated myeloma growth-promoting effects in synergism with insulin like growth factor-1 (Brenne et al., 2002). Most of the myeloma patients present with bone lytic disorder. In cell culture experiments, Dhodapkar et al showed dendritic cells were efficient inducers of Th17

repressed due to depletion of Treg cells, and tumor clones were cleared followed by immunotherapy. Without administration of cyclophosphamide, no tumor regression or clearance was noticed in the tumor bearing animal (Ghiringhelli et al., 2004). Apart from sensitivity of Treg cells to imunomodulatory drugs and cyclophosphamide, a recent study showed that naturally occurring Treg cells were resistant to pro-apoptotic effect of proteasome inhibitor bortezomib. Long-term culturing of CD4 T cells in the presence of bortezomib promoted the emergence of Treg cells and significantly inhibited proliferation, IFN-γ production and CD40L expression by effector T cells (Blanco et al., 2009). Druginduced apoptosis resistance by Treg cells is due to the increased expression of BCL-2 and inhibitor of apoptosis protein 1 (IAP1); the expression of IAP1 is in response to TNF induced apoptosis. These BCL-2 and IAP1 protein expression was significantly elevated in Treg cells of B cell leukemic patients than healthy volunteers (Jak et al., 2009). Gupta et al confirmed that multiple myeloma patients treated with immunomodulatory drug combination showed increased Treg cells in relation to irrespective of the response achieved (Gupta et al., 2011). They also showed patients with stable and progressive disease had decreased Treg cells. Taking all these observations into consideration, no strong conclusion can be forwarded because of contrasting results between *in vitro* and *in vivo* studies. However, *in vitro* studies showed promising effects of immunomodulatory drugs on Treg cells when compared to proteasome inhibitor but immunomodulatory drugs effects might be diluted by inclusion of

Th17 cells are one of the subsets of CD4 T cells. These cells are differentiated in the presence of IL-6, IL-1β, IL-21 and IL-23 with or without TGF-β. Th17 cells secrete IL-17, IL-21, IL-22 and IL-26 cytokines. These cytokines are involved in anti-fungal and anti-parasite responses and participate in inflammatory and autoimmune reactions (Acosta-Rodriguez et al., 2007; Aujla et al., 2008; Bettelli et al., 2006; Ivanov et al., 2006; Veldhoen et al., 2008; Wilson et al., 2007; Zheng et al., 2008). Both Treg cells and Th17 cells originate from naïve CD4 T cells. For Treg cell differentiation, TGF-β is required whereas for Th17 cell differentiation, IL-6 and TGF-β are required (the role of TGF-β in human is unclear) (Bettelli et al., 2006; Veldhoen et al., 2006). Xu et al observed that mature Treg cells can be converted into Th17 cells in the presence of IL-6 (Xu et al., 2007). IL-6 and IL-21 might be involved in transition of Th17/Treg cells to Th17 cells. Some reports show switching of Th17 cells to Th1 cells, but reverse switching is not possible (Annunziato & Romagnani, 2009; Bending et al., 2009; Lee et al., 2009; Shi et al., 2008). Upregulated T-bet expression in Th17 cells resulted in a switch to Th1 cells in the presence of IL-12 or/and IL-23 (Annunziato et al., 2007; Lee et al., 2009). Recently, a study reported increased Th17 cells and IL-17 in freshly isolated mononuclear cells and sera of myeloma patients compared to healthy subjects. In vitro polarization of CD4 T cells to Th17 cells showed increased Th17 cells in multiple myeloma patients than healthy subjects (Prabhala et al., 2010). Moreover, this study showed expression of IL-17 receptor on myeloma cells which promotes the growth of myeloma cells. IL-21 is a proinflammatory cytokine associated with Th17 cells, which is also capable of inducing STAT-3 mediated myeloma growth-promoting effects in synergism with insulin like growth factor-1 (Brenne et al., 2002). Most of the myeloma patients present with bone lytic disorder. In cell culture experiments, Dhodapkar et al showed dendritic cells were efficient inducers of Th17

corticosteroid during treatment.

**7. Th17 cells in multiple myeloma** 

cells, especially mature dendritic cells. Dendritic cells induced Th17 cells were multifunctional and secreted IL-17 and IFN-γ (Dhodapkar et al., 2008). In myeloma, tumor cells are infiltrated by dendritic cells; Dhodapkar et al demonstrated that Th17 cells were enriched in the tumor microenvironment (Dhodapkar et al., 2008). These IL-17 producing cells were found to enhance the activation of osteoclasts (Noonan et al., 2010). This study also reported significant association between extent of bone lytic lesions and IL-17 cytokine producing Th17 cells (Noonan et al., 2010). These findings suggest that Th17 cells enhance myeloma cell growth and development of bone lytic lesions.

#### **8. CD8 T regulatory cells in multiple myeloma**

T cells play an important role in immunosurveillance against cancers. Eventually, these T cells may become CD4/CD8 regulatory T cells due to stimulation by and interaction with tumor cells. Thus, these generated regulatory cells promote the growth of tumor rather than inhibition of tumor (Wang, 2008). Mechanisms of suppression and expansion are well documented for CD4 Treg cells but recent research has been directed to screen the presence of CD8 Treg cells in various tumors and inflammatory conditions.

#### **8.1 Subtypes of CD8 T regulatory cells**

#### **8.1.1 Qa-1 restricted CD8 Treg cells**

These CD8 Treg cells downregulate autoimmune T cell responses. They are Qa-1 (MHC class 1b molecule) restricted and specifically target self-reactive activated T cells which express Qa-1 molecules associated with self peptide (Jiang & Chess, 2004; Sarantopoulos et al., 2004).

#### **8.1.2 CD8+CD28- Treg cells**

These cells are induced by MHC class I peptide antigens. CD8+CD28- Treg cells were found to express FoxP3 α molecule and mediate suppression by cell-cell contact mechanism. CD8+CD28- Treg cells also indirectly target CD4 T cells to become tolerogenic via dendritic cells and non-professional antigen presenting cells. These cells were identified in tumors as well as in the context of transplantation (Cortesini et al., 2001; Filaci & Suciu-Foca, 2002; Filaci et al., 2007; Suciu-Foca et al., 2005).

#### **8.1.3 CD8+CD25hi+ Treg cells**

These cells express CD122, Foxp3 and GITR molecules typically associated with CD4 Treg cells (Cosmi et al., 2003; Kiniwa et al., 2007; Lee et al., 2008). They suppress naive CD4 and CD8 T cells via contact dependent mechanism or soluble IL-10. These Treg cells are different from Qa-1 specific CD8 Treg cells but similar to CD4 Treg cells. CD8+CD25hi+ Treg cells are induced by the tumor environment and require antigen stimulation to suppress naïve T cells (Wang, 2008).

#### **8.2 Role of CD8 T regulatory cells**

So far, only a few studies have documented the role of CD8 Treg cells (CD8+CD25hi+FoxP3+) in cancers. In prostate and colorectal cancers, elevated levels of CD8

Regulatory Cells and Multiple Myeloma 45

immunological reactions. In addition, increased peroxynitrite levels associated with tumor progression in several cancers (Gabrilovich & Nagaraj, 2009). Interaction of peroxynitrite producing MDSCs and T cells leads to nitration of T cell receptors and alters specific peptide

*In vivo* studies showed that MDSCs can induce *de novo* generation of Treg cells (Huang et al., 2006; Yang et al., 2006a). Induction of Treg cells by MDSCs requires tumor specific T cells together with IFN-γ and IL-10, but independent of nitric oxide production (Huang et al., 2006). In murine models, MDSCs induce generation of Treg cells by CTLA-4 (ovarian tumor) and arginase 1 (lymphoma) molecules (Serafini et al., 2008; Yang et al., 2006a). However, in contrast, other studies report no association of Treg cell generation with MDSCs (Dugast et

Accumulation of MDSCs in cancer patients is an immune evasion mechanism. MDSCs were found to be elevated in peripheral blood of solid tumors including breast, colon, prostrate, hepatocellular and esophageal carcinomas. It was also shown that increase of MDSCs in solid tumors is stage-dependent. Stage IV solid tumor patients showed increased level of MDSCs which correlated with metastatic tumor burden (Diaz-Montero et al., 2009). Early stage breast cancer patients who received cyclophosphamide plus doxorubicin also had increased level of MDSCs. In multiple myeloma, information about MDSCs is lacking. However, a recent study showed significant increase of MDSCs in multiple myeloma patients (Brimnes et al., 2010). This study identified MDSCs as CD14+HLADR-/low, which is contradictory to other studies. In our study, we identified MDSCs as CD33+CD11b+CD14-HLADR-. Cells with this phenotype were elevated in multiple myeloma patients and also an increasing trend was showed in monoclonal gammopathy of undetermined significance patients compared to healthy subjects (Muthu Raja et al., 2011). Due to limited studies on MDSCs of myeloma patients, no strong conclusion could be forwarded. However, studies have shown increased level of MDSCs. Further studies are

**10. Therapeutic targeting of regulatory and suppressor cells to enhance** 

Treg cells favored as a potential target in various cancers to enhance the anti-tumor responses. Chemotherapy agents such as fludarabine and cyclophosphamide were reported to reduce Treg cell numbers in animal models. Cyclophosphamide plus fludarabine and high dose IL-2 treatment was given to metastatic melanoma patients where transient decrease in Treg cells was observed (Powell et al., 2007). Use of chemotherapeutic agents to target Treg cells is relatively unspecific approach but targeting CD25 was found to be more selective in hitting Treg cells than chemotherapies. Various preclinical trials have shown that depletion of Treg cells via specific monoclonal antibodies targeting CD25 in combination with adoptive T cell transfer, denileukin diftitox (a fusion protein of diphtheria toxin and IL-2) and LMB-2 (a fusion protein of a single-chain Fv fragment of an anti-CD25

binding of T cells. This process leads to T cell unresponsiveness (Nagaraj et al., 2007).

**9.1.4 Induction of Treg cells** 

al., 2008, Movahedi et al., 2008).

**9.2 Role of myeloid-derived suppressor cells** 

required to prove their functional activity.

**anti-tumor responses** 

Treg cells have been reported (Chaput et al., 2009; Kiniwa et al., 2007). Both these studies showed that CD8 Treg cells are capable of suppressing naïve T cell proliferation. Additionally, Chaput et al demonstrated suppression of Th1 cytokine production. CD8 Treg cells from colorectal cancer patients were found to correlate with disease stage and microinvasive status (Chaput et al., 2009). In multiple myeloma, data on CD8 Treg cells are lacking. Our recent observation showed that CD8 Treg cells were significantly elevated in monoclonal gammopathy of undetermined significance and multiple myeloma when compared to healthy subjects (Muthu Raja et al., 2010). However, functional data of CD8 Treg cells are still lacking.

#### **9. Myeloid-derived suppressor cells (MDSCs) in multiple myeloma**

Myeloid-derived suppressor cells (MDSCs) are activated immature myeloid cells that have been prevented from differentiation to mature cells. These cells are expanded in pathological conditions (Gabrilovich & Nagaraj, 2009). MDSCs lack the expression of cell surface markers that are specifically expressed by monocytes, macrophages or dendritic cells and comprise a mixture of myeloid cells that have the morphology of granulocytes or monocytes (Youn et al., 2008). They are potent suppressors of T cells. Human MDSCs can be characterized phenotypically as CD14-CD11b+ or by CD33 expression which is a common marker for myeloid cells. Moreover, MDSCs lack the expression of mature lymphoid and myeloid markers as well HLA-DR (MHC class II). Healthy individuals were found to have approximately 0.5% of immature myeloid cells from total peripheral blood mononuclear cells (Gabrilovich & Nagaraj, 2009). In cancer patients and tumor models, accumulation of MDSCs occurs due to release of soluble factors by tumor cells or cells in tumor environment (Almand et al., 2001, Diaz-Montero et al., 2009).

#### **9.1 Mode of suppression**

#### **9.1.1 Arginase and inducible nitric oxide synthase**

Arginase and inducible nitric oxide synthase enzymes are released by MDSCs. Arginase depletes the non-essential amino acid L-arginine and leads to inhibition of T cell proliferation (Rodriguez et al., 2002). Inducible nitric oxide synthase mediates nitric oxide production. Nitric oxide suppresses the T cell function via induction of apoptosis, inhibition of MHC II expression and inhibition of STAT-5 and JAK3 function in T cells (Gabrilovich & Nagaraj, 2009).

#### **9.1.2 Reactive oxygen species**

This is also an important factor from MDSCs that contributes to suppressive activity. Reactive oxygen species release was noticed in tumor bearing mice and cancer patients. Several tumor derived factors including TGF-β, IL-6, IL-3, IL-10 and granulocyte macrophage colony-stimulating factor induce reactive oxygen species synthesis by MDSCs (Gabrilovich & Nagaraj, 2009).

#### **9.1.3 Peroxynitrite**

Peroxynitrite induces MDSCs mediated suppression of T cell function. Accumulation of peroxynitrite is noticed where recruitment of MDSCs occurs at the site of inflammation or

Treg cells have been reported (Chaput et al., 2009; Kiniwa et al., 2007). Both these studies showed that CD8 Treg cells are capable of suppressing naïve T cell proliferation. Additionally, Chaput et al demonstrated suppression of Th1 cytokine production. CD8 Treg cells from colorectal cancer patients were found to correlate with disease stage and microinvasive status (Chaput et al., 2009). In multiple myeloma, data on CD8 Treg cells are lacking. Our recent observation showed that CD8 Treg cells were significantly elevated in monoclonal gammopathy of undetermined significance and multiple myeloma when compared to healthy subjects (Muthu Raja et al., 2010). However, functional data of CD8

Myeloid-derived suppressor cells (MDSCs) are activated immature myeloid cells that have been prevented from differentiation to mature cells. These cells are expanded in pathological conditions (Gabrilovich & Nagaraj, 2009). MDSCs lack the expression of cell surface markers that are specifically expressed by monocytes, macrophages or dendritic cells and comprise a mixture of myeloid cells that have the morphology of granulocytes or monocytes (Youn et al., 2008). They are potent suppressors of T cells. Human MDSCs can be characterized phenotypically as CD14-CD11b+ or by CD33 expression which is a common marker for myeloid cells. Moreover, MDSCs lack the expression of mature lymphoid and myeloid markers as well HLA-DR (MHC class II). Healthy individuals were found to have approximately 0.5% of immature myeloid cells from total peripheral blood mononuclear cells (Gabrilovich & Nagaraj, 2009). In cancer patients and tumor models, accumulation of MDSCs occurs due to release of soluble factors by tumor cells or cells in tumor environment

Arginase and inducible nitric oxide synthase enzymes are released by MDSCs. Arginase depletes the non-essential amino acid L-arginine and leads to inhibition of T cell proliferation (Rodriguez et al., 2002). Inducible nitric oxide synthase mediates nitric oxide production. Nitric oxide suppresses the T cell function via induction of apoptosis, inhibition of MHC II expression

This is also an important factor from MDSCs that contributes to suppressive activity. Reactive oxygen species release was noticed in tumor bearing mice and cancer patients. Several tumor derived factors including TGF-β, IL-6, IL-3, IL-10 and granulocyte macrophage colony-stimulating factor induce reactive oxygen species synthesis by MDSCs

Peroxynitrite induces MDSCs mediated suppression of T cell function. Accumulation of peroxynitrite is noticed where recruitment of MDSCs occurs at the site of inflammation or

and inhibition of STAT-5 and JAK3 function in T cells (Gabrilovich & Nagaraj, 2009).

**9. Myeloid-derived suppressor cells (MDSCs) in multiple myeloma** 

Treg cells are still lacking.

**9.1 Mode of suppression** 

**9.1.2 Reactive oxygen species** 

(Gabrilovich & Nagaraj, 2009).

**9.1.3 Peroxynitrite** 

(Almand et al., 2001, Diaz-Montero et al., 2009).

**9.1.1 Arginase and inducible nitric oxide synthase** 

immunological reactions. In addition, increased peroxynitrite levels associated with tumor progression in several cancers (Gabrilovich & Nagaraj, 2009). Interaction of peroxynitrite producing MDSCs and T cells leads to nitration of T cell receptors and alters specific peptide binding of T cells. This process leads to T cell unresponsiveness (Nagaraj et al., 2007).

#### **9.1.4 Induction of Treg cells**

*In vivo* studies showed that MDSCs can induce *de novo* generation of Treg cells (Huang et al., 2006; Yang et al., 2006a). Induction of Treg cells by MDSCs requires tumor specific T cells together with IFN-γ and IL-10, but independent of nitric oxide production (Huang et al., 2006). In murine models, MDSCs induce generation of Treg cells by CTLA-4 (ovarian tumor) and arginase 1 (lymphoma) molecules (Serafini et al., 2008; Yang et al., 2006a). However, in contrast, other studies report no association of Treg cell generation with MDSCs (Dugast et al., 2008, Movahedi et al., 2008).

#### **9.2 Role of myeloid-derived suppressor cells**

Accumulation of MDSCs in cancer patients is an immune evasion mechanism. MDSCs were found to be elevated in peripheral blood of solid tumors including breast, colon, prostrate, hepatocellular and esophageal carcinomas. It was also shown that increase of MDSCs in solid tumors is stage-dependent. Stage IV solid tumor patients showed increased level of MDSCs which correlated with metastatic tumor burden (Diaz-Montero et al., 2009). Early stage breast cancer patients who received cyclophosphamide plus doxorubicin also had increased level of MDSCs. In multiple myeloma, information about MDSCs is lacking. However, a recent study showed significant increase of MDSCs in multiple myeloma patients (Brimnes et al., 2010). This study identified MDSCs as CD14+HLADR-/low, which is contradictory to other studies. In our study, we identified MDSCs as CD33+CD11b+CD14-HLADR-. Cells with this phenotype were elevated in multiple myeloma patients and also an increasing trend was showed in monoclonal gammopathy of undetermined significance patients compared to healthy subjects (Muthu Raja et al., 2011). Due to limited studies on MDSCs of myeloma patients, no strong conclusion could be forwarded. However, studies have shown increased level of MDSCs. Further studies are required to prove their functional activity.

#### **10. Therapeutic targeting of regulatory and suppressor cells to enhance anti-tumor responses**

Treg cells favored as a potential target in various cancers to enhance the anti-tumor responses. Chemotherapy agents such as fludarabine and cyclophosphamide were reported to reduce Treg cell numbers in animal models. Cyclophosphamide plus fludarabine and high dose IL-2 treatment was given to metastatic melanoma patients where transient decrease in Treg cells was observed (Powell et al., 2007). Use of chemotherapeutic agents to target Treg cells is relatively unspecific approach but targeting CD25 was found to be more selective in hitting Treg cells than chemotherapies. Various preclinical trials have shown that depletion of Treg cells via specific monoclonal antibodies targeting CD25 in combination with adoptive T cell transfer, denileukin diftitox (a fusion protein of diphtheria toxin and IL-2) and LMB-2 (a fusion protein of a single-chain Fv fragment of an anti-CD25

Regulatory Cells and Multiple Myeloma 47

Moreover, characterization of regulatory cells in humans is an ambiguous aspect due to lack of precise markers. Studies are needed to disclose specific characterization marker for human Treg cells, so that results do not vary between groups. In multiple myeloma, immunotherapeutic targeting of tumor cells at the pre-clinical and clinical studies showed remarkable immunological as well clinical responses in some cohort of patients. When compared to non-hematological malignancies, there are no clinical trials performed to target regulatory cells in myeloma patients. Investigations are required with the inclusion of preclinical and clinical studies in myeloma via combinational approach of targeting tumor cells as well regulatory cells. This approach might overcome tumor induced immunosuppression

We kindly thank Dr. Pavel Chrobak for his merit suggestions in preparing this chapter. We also acknowledge the support provided by the research grants MSM0021622434, LC06027,

Acosta-Rodriguez, E.V., Rivino, L., Geginat, J., Jarrossay, D., Gattorno, M., Lanzavecchia, A.,

Allan, S.E., Passerini, L., Bacchetta, R., Crellin, N., Dai, M., Orban, P.C., Ziegler, S.F.,

Almand, B., Clark, J.I., Nikitina, E., van Beynen, J., English, N.R., Knight, S.C., Carbone, D.P.

Annunziato, F., Cosmi, L., Santarlasci, V., Maggi, L., Liotta, F., Mazzinghi, B., Parente, E.,

Annunziato, F. & Romagnani, S. (2009). Do studies in humans better depict Th17 cells? *Blood*, Vol.114, No.11, (September 2009), pp. 2213-2219, ISSN 0006-4971 Apostolou, I. & von Boehmer, H. (2004). In vivo instruction of suppressor commitment in

*Immunology*, Vol.166, No.1, (January 2001), pp. 678-689, ISSN 0022-1767 Alvaro, T., Lejeune, M., Salvadó, M.T., Bosch, R., García, J.F., Jaén, J., Banham, A.H.,

Sallusto, F. & Napolitani, G. (2007). Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. *Nature Immunology*, Vol.8,

Roncarolo, M.G. & Levings, M.K. (2005). The role of 2 FOXP3 isoforms in the generation of human CD4+ Tregs. *Journal of Clinical Investigation*, vol.115, No.11,

& Gabrilovich, D.I. (2001). Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer. *Journal of* 

Roncador, G., Montalbán, C. & Piris, M.A. (2005). Outcome in Hodgkin's lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells. *Clinical Cancer Research*, Vol.11, No.4, (February 2005), pp. 1467-

Filì, L., Ferri, S., Frosali, F., Giudici, F., Romagnani, P., Parronchi, P., Tonelli, F., Maggi, E. & Romagnani, S. (2007). Phenotypic and functional features of human Th17 cells. *Journal of Experimental Medicine*, Vol.204, No.8, (August 2007), pp. 1849-

naive T cells. *Journal of Experimental Medicine*, Vol.199, No.10, (May 2004), pp. 1401-

in myeloma patients.

**13. References** 

**12. Acknowledgement** 

IGA NS10406, IGA NS10408, and GACR P304/10/1395.

No.6, (June 2007), pp. 639-646, ISSN 1529-2908

(November 2005), pp. 3276-3284, ISSN 0021-9738

1473, ISSN 1078-0432

1861, ISSN 0022-1007

1408, ISSN 0022-1007

antibody and the bacterial pseudomonas exotoxin A or 3) enhanced the anti-tumor immunity (Attia et al., 1997; Knutson et al., 2006; Litzinger et al., 2007; Shimizu et al., 1999). Translation of denileukin diftitox and LMB-2 into clinical studies along with vaccination showed efficient improvement in anti-tumor response plus reduced frequency of Treg cells in various cancers, including metastatic renal cell carcinoma, melanoma and colorectal carcinoma. Targeting CTLA-4 molecule is not a precise approach because both effector T cells and Treg cells express CTLA-4. However, a recent animal model study showed CTLA-4 deficient mice lacked the Treg cell mediated immune suppression (Wing et al., 2008). Disadvantage in blockade or depletion of Treg cells is autoimmune toxicity, which was observed in murine models and cancer patients (Dougan & Dranoff, 2009). Recent understanding of MDSCs in cancers provokes to target these suppressor cells. All-trans retinoic acid (ATRA) administration in animal models and *in vitro* study showed decreased number of MDSCs, activated CD4 and CD8 T cells and delayed tumor progression (Kusmartsev et al., 2008). ATRA administration with granulocyte macrophage colonystimulating factor helped in differentiation of MDSCs into myeloid dendritic cells in tumorbearing mice (Gabrilovich et al., 2001). In the clinical study ATRA plus IL-2 combination did not have impact on MDSCs of renal cell carcinoma patients. Chemotherapeutic agents, such as gemicitabine and 5-fluorouracil, were reported to reduce the peripheral blood MDSCs in animal models as well as *in vitro* (Le et al., 2009; Vincent et al., 2010). Cyclophosphamide and doxorubicin have negative impact on MDSCs of breast cancer patients (Diaz-Montero et al., 2009). A recent study proposed combination of cyclophosphamide with IL-2 enhanced the clearance of intra-tumoral Treg cells and MDSCs, and enhanced the generation of myeloid inflammatory cells which lack the suppressive function (Medina-Echeverz et al., 2011). STAT-3 is a key regulatory molecule in MDSCs, and this molecule is constitutively expressed by malignant cells. There are several STAT-3 inhibitory molecules under investigation. Sunitinib is one of the STAT-3 inhibitors which influence the phosphorylation of STAT-3 via tyrosine kinase; additionally, it has anti-angiogenic property. Currently, Sunitinib is under investigation for its efficiency on MDSCs (Ko et al., 2009). These observations are showing the efficiency of various inhibitors and chemotherapy agents to hinder the regulatory and suppressor cells *in vitro* and in pre-clinical trials. Unfortunately, clinical trials did not show flourishing impact in all cancers. This might be due to autoimmune toxicities caused by depletion or targeting of Treg cells. Approach of hitting the Treg cells needs further investigation; it is essential to target specifically the tumor associated Treg cells not the global Treg cells which might cause imbalance in the immune homeostasis.

#### **11. Conclusion**

Large evidence is available in hematological malignancies and solid tumors for elevated level of various regulatory and suppressor cells which impede anti-tumor responses. Therefore, targeting the regulatory cells could be a useful strategy to enhance the anti-tumor immunity. Approaches of depletion or inhibition of regulatory cells showed countable benefits in pre-clinical and clinical studies of some cancers including renal cell carcinoma, metastatic melanoma and colorectal carcinoma. Targeting regulatory T cells in a non-specific approach might cause detrimental autoimmune toxicities which is the key issue. Further studies are necessary to identify tumor associated regulatory cells which will enhance the depletion of specific regulatory cells but not the global population of regulatory cells.

antibody and the bacterial pseudomonas exotoxin A or 3) enhanced the anti-tumor immunity (Attia et al., 1997; Knutson et al., 2006; Litzinger et al., 2007; Shimizu et al., 1999). Translation of denileukin diftitox and LMB-2 into clinical studies along with vaccination showed efficient improvement in anti-tumor response plus reduced frequency of Treg cells in various cancers, including metastatic renal cell carcinoma, melanoma and colorectal carcinoma. Targeting CTLA-4 molecule is not a precise approach because both effector T cells and Treg cells express CTLA-4. However, a recent animal model study showed CTLA-4 deficient mice lacked the Treg cell mediated immune suppression (Wing et al., 2008). Disadvantage in blockade or depletion of Treg cells is autoimmune toxicity, which was observed in murine models and cancer patients (Dougan & Dranoff, 2009). Recent understanding of MDSCs in cancers provokes to target these suppressor cells. All-trans retinoic acid (ATRA) administration in animal models and *in vitro* study showed decreased number of MDSCs, activated CD4 and CD8 T cells and delayed tumor progression (Kusmartsev et al., 2008). ATRA administration with granulocyte macrophage colonystimulating factor helped in differentiation of MDSCs into myeloid dendritic cells in tumorbearing mice (Gabrilovich et al., 2001). In the clinical study ATRA plus IL-2 combination did not have impact on MDSCs of renal cell carcinoma patients. Chemotherapeutic agents, such as gemicitabine and 5-fluorouracil, were reported to reduce the peripheral blood MDSCs in animal models as well as *in vitro* (Le et al., 2009; Vincent et al., 2010). Cyclophosphamide and doxorubicin have negative impact on MDSCs of breast cancer patients (Diaz-Montero et al., 2009). A recent study proposed combination of cyclophosphamide with IL-2 enhanced the clearance of intra-tumoral Treg cells and MDSCs, and enhanced the generation of myeloid inflammatory cells which lack the suppressive function (Medina-Echeverz et al., 2011). STAT-3 is a key regulatory molecule in MDSCs, and this molecule is constitutively expressed by malignant cells. There are several STAT-3 inhibitory molecules under investigation. Sunitinib is one of the STAT-3 inhibitors which influence the phosphorylation of STAT-3 via tyrosine kinase; additionally, it has anti-angiogenic property. Currently, Sunitinib is under investigation for its efficiency on MDSCs (Ko et al., 2009). These observations are showing the efficiency of various inhibitors and chemotherapy agents to hinder the regulatory and suppressor cells *in vitro* and in pre-clinical trials. Unfortunately, clinical trials did not show flourishing impact in all cancers. This might be due to autoimmune toxicities caused by depletion or targeting of Treg cells. Approach of hitting the Treg cells needs further investigation; it is essential to target specifically the tumor associated Treg cells not the global Treg cells which might cause imbalance in the immune

Large evidence is available in hematological malignancies and solid tumors for elevated level of various regulatory and suppressor cells which impede anti-tumor responses. Therefore, targeting the regulatory cells could be a useful strategy to enhance the anti-tumor immunity. Approaches of depletion or inhibition of regulatory cells showed countable benefits in pre-clinical and clinical studies of some cancers including renal cell carcinoma, metastatic melanoma and colorectal carcinoma. Targeting regulatory T cells in a non-specific approach might cause detrimental autoimmune toxicities which is the key issue. Further studies are necessary to identify tumor associated regulatory cells which will enhance the depletion of specific regulatory cells but not the global population of regulatory cells.

homeostasis.

**11. Conclusion** 

Moreover, characterization of regulatory cells in humans is an ambiguous aspect due to lack of precise markers. Studies are needed to disclose specific characterization marker for human Treg cells, so that results do not vary between groups. In multiple myeloma, immunotherapeutic targeting of tumor cells at the pre-clinical and clinical studies showed remarkable immunological as well clinical responses in some cohort of patients. When compared to non-hematological malignancies, there are no clinical trials performed to target regulatory cells in myeloma patients. Investigations are required with the inclusion of preclinical and clinical studies in myeloma via combinational approach of targeting tumor cells as well regulatory cells. This approach might overcome tumor induced immunosuppression in myeloma patients.

#### **12. Acknowledgement**

We kindly thank Dr. Pavel Chrobak for his merit suggestions in preparing this chapter. We also acknowledge the support provided by the research grants MSM0021622434, LC06027, IGA NS10406, IGA NS10408, and GACR P304/10/1395.

#### **13. References**


Regulatory Cells and Multiple Myeloma 49

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Brenne, A.T., Ro, T.B., Waage, A., Sundan, A., Borset, M. & Hjorth-Hansen, H. (2002).

Brimnes, M.K., Vangsted, A.J., Knudsen, L.M., Gimsing, P., Gang, A.O., Johnsen, H.E. &

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Carreras, J., Lopez-Guillermo, A., Fox, B.C., Colomo, L., Martinez, A., Roncador, G.,

Chaput, N., Darrasse-Jèze, G., Bergot, A.S., Cordier, C., Ngo-Abdalla, S., Klatzmann, D. &

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**3** 

*Slovenia* 

**Effects of Recombinant Human Tumor Necrosis** 

Andrej Plesničar1, Gaj Vidmar2, Borut Štabuc3 and Blanka Kores Plesničar4

Multiple myeloma (MM) is a malignant B-cell disease, characterized by uncontrolled proliferation of differentiated plasma cells in bone marrow (BM), osteolytic bone lesions, monoclonal protein peaks in serum or urine and suppression of normal antibody production. Patients with MM usually present with a number of clinical signs and symptoms, including fatigue, infection, severe bone pain, bone fractures, hypercalcaemia, and renal disease (Bommert et al., 2006; Raman et al., 2007; Redzepovic et al., 2008). Despite clinical responses produced by conventional chemotherapy, radiotherapy, and an increasing number of new compounds and improvements in supportive therapy, MM remains largely

Tumor necrosis factor-α (TNF-α) is a known survival and proliferation factor for myeloma cell lines. It is produced by tumor and stromal cells in BM of patients with MM and induces tumor cell proliferation, migration, survival, drug resistance, and blood vessel proliferation (Harrison et al., 2006; Jourdan et al., 1999). Although TNF-α secreted by MM cells does not induce significant growth and drug resistance in tumor cells, it stimulates interleukin-6 (IL-6) secretion in bone marrow stromal cells more potently than vascular endothelial growth factor (VEGF) or transforming growth factor-β (TGF-β) (Yasui et al., 2005). Out of BM environment, circulating TNF-α levels are increased in MM patients with manifest bone disease, whose osteoblasts constitutively overexpress receptors for TNF-related apoptosisinducing ligand, intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic

In our previous study, treatment with native human leukocyte interferon-α (nhIFN-α), recombinant human interferon-α2a (rhIFN-α2a) and recombinant human interferon-α2b (rhIFN-α2b) in doses of 500 IU/ml, 1000/ml and 2000 IU/ml resulted in differential effects on P3-X63-Ag8.653 mouse myeloma cells. A statistically significant dose-dependent decrease in

incurable (Katzel et al., 2007; Ozdemir et al., 2004; Redzepovic et al., 2008).

protein-1 (MCP-1) (Silvestris et al., 2004).

**1. Introduction** 

**Factor-α and Its Combination with Native** 

**Ag8.653 Mouse Myeloma Cell Growth** 

*1University of Ljubljana, Faculty of Health Sciences, Ljubljana,* 

*3University of Ljubljana, Faculty of Medicine, Ljubljana, 4University of Maribor, Faculty of Medicine, Maribor,* 

*2Institute for Rehabilitation, Ljubljana,* 

**Human Leukocyte Interferon-α on P3-X63-**

