**Novel Therapeutic Targets in ALL Therapy**

## Roman Crazzolara

*Department of Pediatrics, Medical University of Innsbruck Austria* 

#### **1. Introduction**

ALL is a malignant disorder of the blood system characterized by uncontrolled proliferation of bone marrow-derived B- and T-lymphocyte progenitors that are arrested in an early stage of development (Pui et al, 2008). This arrest is caused by aberrant gene fusions or inappropriate expression of oncogenes. The lymphoblasts replace the normal marrow elements, resulting in a substantial decrease in the production of normal blood cells. Consequently, anemia, thrombocytopenia, and neutropenia occur to varying degrees. Moreover, the expanding lymphoblasts can escape the bone marrow niche, and manifest as heptospenomegaly, enlargement of lymph nodes, thymus, and gonads, and infiltration of the meninges (Pui et al, 2008).

ALL has a peak incidence between the ages of 2 to 5 years, representing 25% of the malignant disorders in childhood (Pui et al, 2006). Currently, ALL is treated by administration of cyclic induction chemotherapy, which is intended to kill the majority of leukemic cells, followed by a consolidation chemotherapy, which should destroy any remaining leukemic cells (Freycon et al, 2008; Pui et al, 2008). For high risk patients, myeloablative regimens followed by allogeneic hematopoietic stem-cell transplantation are indicated to achieve more extensive eradication of leukemia and to induce graft versus leukemia effects against the disease. The implementation of risk-adapted strategies, which tailor the intensity of therapy to the risk of relapse, has resulted in a cure rate of more than 80% in children being long-term survivors (Pui et al, 2006). In contrast to the successes obtained with paediatric patients, treatment outcomes for adult patients remain poor with less than 40% being long-term survivors (Vitale et al, 2006). Due to relative nonspecific action and narrow therapeutic indices of antileukemic medications, serious short and longterm complications arise as a result of the intensification of many current therapies (Barr et al, 2008).

The identification of genetic and epigenetic changes that are associated with leukemogenesis and altered drug response provides insights into the molecular basis of ALL. The translation to the clinic might serve as a model for optimizing the treatment and generate innovative therapeutic agents. Finally, these could be implemented in more effective, and potentially less toxic, individually tailored treatment protocols, based on the underlying molecular abnormalities of a patient's leukemia. This review will concentrate on novel agents that show promising anti-leukemic activity in pre-clinical studies or clinical trials.

Novel Therapeutic Targets in ALL Therapy 167

monoclonal antibody (mAb), conjugated to a cytotoxic agent. After internalization of the antibody-drug complex, vital survival pathways for malignant cells can be blocked or dyregulated. Among these, CD22 antibody-targeted agents have more extensively been explored with significant preclinical success in models of acute leukemia (Dijoseph et al,

CMC-544 (inotuzumab ozogamicin) is a conjugate of a recombinant humanized antibody directed against the CD22 antigen and calicheamicin. After internalization into the target cell, calicheamicin binds to DNA in the minor groove in a sequence specific manner, causing double-strand DNA breaks followed by apoptotic death. *In vitro*, CMC-544 binds to CD22 with subnanomolar affinity and potently inhibits growth of ALL cell lines (Dijoseph et al, 2007). When administered to xenograft models, CMC-544 prevents engraftment, but also induces dose-dependent tumor regression in mice presenting with leukemia. Whereas the level of CD22 expression is significantly reduced after incubation with CMC-544, the CD20 level can be increased (Takeshita et al, 2009). Therefore, sequential administration of rituximab increases the cytotoxic effect and supports the rationale for a combination with

Currently, a phase I study is recruiting patients for the aminstration of CMC-544 with or without rituximab in relapsed or primary refractory ALL patients (NCT01134575, www.clinicaltrials.gov). Preliminary results indicate liver function abnormalities in 25% of patients, including periportal fibrosis and venoocclusive disease after allo SCT, among the most relevant side effects (Jabbour et al, 2011). Overall, complete response plus complete

Additional phase I studies are soon expected to open, including a single use of CMC-544 in refractory ALL patients (NCT01363297, www.clinicaltrials.gov) and a 2 dose level study in combination with cyclophosphamide, vincristine, dexamethasone, methotrexate, cytarabine

In several clinical studies alterations in the apoptotic threshold were proven to be predictive of poor response to treatment and adverse clinical outcome in patients with a variety of hematologic malignancies, including acute leukemia. Specifically, the up regulation of the BCL-2 family of proteins has been demonstrated to suppress caspase- and non-caspasemediated apoptosis mediated by several agents, including -irradiation and chemotherapy (Reed et al, 1995). As such, over expression of BCL-2 has been associated with an increased risk of relapse in childhood ALL (Hogarth et al, 1999), suggesting the use of factors that override the BCL-2 pathway might restore chemo sensitivity in chemo resistant leukemic

Since the discovery of BCL-2, a single antisense oligodeoxynucleotide - Genasense (G3139, oblimersen; Genta Inc.) - has been explored. *In vitro* studies have shown that when administered alone or in combination with chemotherapy, G3139 inhibits BCL2 expression, resulting in increased tumor cell apoptosis (Webb et al, 1997). Subsequently, its ability to effectively reduce BCL-2 protein levels has clearly been demonstrated in a mouse model transplanted with leukemia. This effect is correlated with enhanced induction of apoptosis, when cells are cultured with the anti-leukemic agents imatinib, daunorubicin, cytarabine and etoposide. Mice treated with G3139 have prolonged survival with some showing complete tumor regression. Of particular interest is the efficacy of G3139 in STI571 resistant

bone marrow response is achieved in more than 50% of the patients.

in elderly ALL patients (NCT01371630, www.clinicaltrials.gov).

2004).

other antibodies.

cells.

**4. BCL-2 Antisense therapy** 

## **2. CXCR4 inhibitors**

Several studies have been reported on the involvement of chemokines and adhesion molecules in the process of mobilization of ALL cells. Among the most studied molecules are the chemokine receptor CXCR4 and the chemokine stromal cell-derived factor-1 (SDF-1, CXCL12); in particular CXCR4, was found to be highly expressed in bone-marrow derived ALL cells enabling these cells to migrate across a gradient of CXCL12 concentrations (Crazzolara et al, 2001). Elevated levels of CXCL12 are not only found in the bone marrow environment, but also at extramedullary sites associated with ALL induced organ infiltration (Muller et al, 2001). Subsequently CXCL12 can act as survival and proliferation factor for CXCR4 positive cells and protect them from spontaneous and chemotherapy-induced apoptosis (Burger et al, 2000). In several studies, CXCL12 is also reported in the process of homing of CXCR4 positive cells in NOD/SCID xenograft mouse models (Nagasawa et al, 1996). Of note is the direct correlation between high CXCR4 expression on lymphoblasts and the extent of extramedullary infiltration in patients with ALL (Crazzolara et al, 2001).

Direct evidence for the involvement of CXCR4 and CXCL12 in the release of ALL cells in the blood is demonstrated from the finding that treatment of mice with small compounds that target CXCR4 and its ligand can disrupt the interaction between ALL cells and the stromal microenvironment. The polyphemusin II-derived inhibitors T140, TC140012, T134, and the bicyclam AMD3100 effectively inhibit CXCR4, and CXCL12-driven migration into bone marrow layers *in vitro*, thus enhancing the cytotoxic and anti-proliferative effects of the currently used agents vincristine and dexamethasone (Juarez et al, 2003). Disruption of the interaction of CXCR4 and its ligand *in vivo* mobilizes leukemic cells into the peripheral blood, potentially rendering them more susceptible to cytotoxic effects (Juarez et al, 2006). The importance of CXCR4 in the context of therapy is further supported by the observation, that CXCR4 expression is dynamically up regulated by chemotherapy exposure in ALL cells. Up regulation of surface CXCR4 may therefore be considered as a mechanism of chemo resistance in acute leukaemias that is potentially reversible with CXCR4-targeted therapy.

Concerns have been raised regarding potential side effects from CXCR4 inhibition, since CXCR4 knockout mice display severe defects in hematopoesis, vascular and cardiac development (Nagasawa et al, 1996). However, these defects are related to CXCR4 functions in early development, and short-term exposure to AMD3100 for stem cell mobilization does not result in any significant toxicity (Broxmeyer et al, 2005). Activity of different CXCR4 antagonists in animal models for solid tumors (Smith et al, 2004) generalizes the potential anti-neoplastic activity and suggests further clinical development of these agents in ALL. It is anticipated, that a phase I pediatric study has recently started to study the addition of the selective CXCR4 antagonist plerixafor to enhance the conditioning regimen cytotoxicity (NCT01068301, www.clinicaltrials.gov). Though the primary goal is to determine the maximum tolerated dose (MTD), additional trials are necessary to study the use of plerixafor as a complimentary agent with conditioning as well as other chemotherapeutic regimens for patients with relapsed or refractory hematologic malignancies.

## **3. CD22 immunoconjugates**

The approval of antibody-targeted agents for cancer treatment has provoked increased interest in the development of new and improved antibody-mediated therapies. This emerging approach centers on targeting cell clusters of differentiation on ALL cells with a

Several studies have been reported on the involvement of chemokines and adhesion molecules in the process of mobilization of ALL cells. Among the most studied molecules are the chemokine receptor CXCR4 and the chemokine stromal cell-derived factor-1 (SDF-1, CXCL12); in particular CXCR4, was found to be highly expressed in bone-marrow derived ALL cells enabling these cells to migrate across a gradient of CXCL12 concentrations (Crazzolara et al, 2001). Elevated levels of CXCL12 are not only found in the bone marrow environment, but also at extramedullary sites associated with ALL induced organ infiltration (Muller et al, 2001). Subsequently CXCL12 can act as survival and proliferation factor for CXCR4 positive cells and protect them from spontaneous and chemotherapy-induced apoptosis (Burger et al, 2000). In several studies, CXCL12 is also reported in the process of homing of CXCR4 positive cells in NOD/SCID xenograft mouse models (Nagasawa et al, 1996). Of note is the direct correlation between high CXCR4 expression on lymphoblasts and the extent of extramedullary infiltration

Direct evidence for the involvement of CXCR4 and CXCL12 in the release of ALL cells in the blood is demonstrated from the finding that treatment of mice with small compounds that target CXCR4 and its ligand can disrupt the interaction between ALL cells and the stromal microenvironment. The polyphemusin II-derived inhibitors T140, TC140012, T134, and the bicyclam AMD3100 effectively inhibit CXCR4, and CXCL12-driven migration into bone marrow layers *in vitro*, thus enhancing the cytotoxic and anti-proliferative effects of the currently used agents vincristine and dexamethasone (Juarez et al, 2003). Disruption of the interaction of CXCR4 and its ligand *in vivo* mobilizes leukemic cells into the peripheral blood, potentially rendering them more susceptible to cytotoxic effects (Juarez et al, 2006). The importance of CXCR4 in the context of therapy is further supported by the observation, that CXCR4 expression is dynamically up regulated by chemotherapy exposure in ALL cells. Up regulation of surface CXCR4 may therefore be considered as a mechanism of chemo resistance

Concerns have been raised regarding potential side effects from CXCR4 inhibition, since CXCR4 knockout mice display severe defects in hematopoesis, vascular and cardiac development (Nagasawa et al, 1996). However, these defects are related to CXCR4 functions in early development, and short-term exposure to AMD3100 for stem cell mobilization does not result in any significant toxicity (Broxmeyer et al, 2005). Activity of different CXCR4 antagonists in animal models for solid tumors (Smith et al, 2004) generalizes the potential anti-neoplastic activity and suggests further clinical development of these agents in ALL. It is anticipated, that a phase I pediatric study has recently started to study the addition of the selective CXCR4 antagonist plerixafor to enhance the conditioning regimen cytotoxicity (NCT01068301, www.clinicaltrials.gov). Though the primary goal is to determine the maximum tolerated dose (MTD), additional trials are necessary to study the use of plerixafor as a complimentary agent with conditioning as well as other chemotherapeutic

The approval of antibody-targeted agents for cancer treatment has provoked increased interest in the development of new and improved antibody-mediated therapies. This emerging approach centers on targeting cell clusters of differentiation on ALL cells with a

in acute leukaemias that is potentially reversible with CXCR4-targeted therapy.

regimens for patients with relapsed or refractory hematologic malignancies.

**2. CXCR4 inhibitors** 

in patients with ALL (Crazzolara et al, 2001).

**3. CD22 immunoconjugates** 

monoclonal antibody (mAb), conjugated to a cytotoxic agent. After internalization of the antibody-drug complex, vital survival pathways for malignant cells can be blocked or dyregulated. Among these, CD22 antibody-targeted agents have more extensively been explored with significant preclinical success in models of acute leukemia (Dijoseph et al, 2004).

CMC-544 (inotuzumab ozogamicin) is a conjugate of a recombinant humanized antibody directed against the CD22 antigen and calicheamicin. After internalization into the target cell, calicheamicin binds to DNA in the minor groove in a sequence specific manner, causing double-strand DNA breaks followed by apoptotic death. *In vitro*, CMC-544 binds to CD22 with subnanomolar affinity and potently inhibits growth of ALL cell lines (Dijoseph et al, 2007). When administered to xenograft models, CMC-544 prevents engraftment, but also induces dose-dependent tumor regression in mice presenting with leukemia. Whereas the level of CD22 expression is significantly reduced after incubation with CMC-544, the CD20 level can be increased (Takeshita et al, 2009). Therefore, sequential administration of rituximab increases the cytotoxic effect and supports the rationale for a combination with other antibodies.

Currently, a phase I study is recruiting patients for the aminstration of CMC-544 with or without rituximab in relapsed or primary refractory ALL patients (NCT01134575, www.clinicaltrials.gov). Preliminary results indicate liver function abnormalities in 25% of patients, including periportal fibrosis and venoocclusive disease after allo SCT, among the most relevant side effects (Jabbour et al, 2011). Overall, complete response plus complete bone marrow response is achieved in more than 50% of the patients.

Additional phase I studies are soon expected to open, including a single use of CMC-544 in refractory ALL patients (NCT01363297, www.clinicaltrials.gov) and a 2 dose level study in combination with cyclophosphamide, vincristine, dexamethasone, methotrexate, cytarabine in elderly ALL patients (NCT01371630, www.clinicaltrials.gov).

## **4. BCL-2 Antisense therapy**

In several clinical studies alterations in the apoptotic threshold were proven to be predictive of poor response to treatment and adverse clinical outcome in patients with a variety of hematologic malignancies, including acute leukemia. Specifically, the up regulation of the BCL-2 family of proteins has been demonstrated to suppress caspase- and non-caspasemediated apoptosis mediated by several agents, including -irradiation and chemotherapy (Reed et al, 1995). As such, over expression of BCL-2 has been associated with an increased risk of relapse in childhood ALL (Hogarth et al, 1999), suggesting the use of factors that override the BCL-2 pathway might restore chemo sensitivity in chemo resistant leukemic cells.

Since the discovery of BCL-2, a single antisense oligodeoxynucleotide - Genasense (G3139, oblimersen; Genta Inc.) - has been explored. *In vitro* studies have shown that when administered alone or in combination with chemotherapy, G3139 inhibits BCL2 expression, resulting in increased tumor cell apoptosis (Webb et al, 1997). Subsequently, its ability to effectively reduce BCL-2 protein levels has clearly been demonstrated in a mouse model transplanted with leukemia. This effect is correlated with enhanced induction of apoptosis, when cells are cultured with the anti-leukemic agents imatinib, daunorubicin, cytarabine and etoposide. Mice treated with G3139 have prolonged survival with some showing complete tumor regression. Of particular interest is the efficacy of G3139 in STI571 resistant

Novel Therapeutic Targets in ALL Therapy 169

The detection of NOTCH1 gain-of-function mutations in more than 50% of T-cell ALL patients has attracted much interest in the understanding of its role in the molecular pathogenesis of this leukemic subtype. Although expression of an activated NOTCH1 allele has been shown to cause T-cell leukemia in mice, the molecular mechanism for cellular transformation is largely unknown. However, the identification of c-myc as a direct and critical NOTCH1 target gene (Weng et al, 2004) has urged the screening and development of

The NOTCH1 inhibitor MK-0752 (Merck & Co.) has been developed for the treatment of Alzheimer's disease, since it cleaves amyloid precursor protein and prevents the formation of amyloid ß-peptides (Evin et al, 2006). Its activity in leukemia is demonstrated by the conservation of the NOTCH1 receptor within the transmembrane domain, preventing the release of the NOTCH intracellular domain, and thereby suppressing the transcription of target genes. This results in the arrest of cells in G0/1 , reduces viability, and increases apoptosis (DeAngelo et al, 2006). In a phase I trial of adult and pediatric patients with T-cell malignancies MK-0752 has been shown to be well-tolerated with diarrhoea being the doselimiting toxicity. Measurements of -secretase inhibition have shown a 24-69% decrease in plasma Abeta40 peptide levels compared to predose levels (DeAngelo et al, 2006). Only one patient with a NOTCH1 activating mutation has achieved a significant reduction of a

Currently, an additional phase I study is examining the safety of a newer NOTCH1 inhibitor with improved biological availability (PF-03084014, Pfizer; NCT01068301,

Heat shock protein 90 (Hsp90) is currently receiving considerable attention as a potential anticancer drug target. It is a molecular chaperone that regulates structural folding and active configuration of a variety of signal transduction and cell cycle regulatory proteins, including tyrosine and serine kinases such as c-src and Akt (Isaacs et al, 2003; Hawkins et al, 2005). Inhibition of Hsp90 disrupts the folding of these proteins, thus increasing their susceptibility to ubiquitination and proteosomal degradation. Although the exact mechanism by which Hsp90 inhibitors kill tumor cells remain to be defined, the ability to abrogate the AKT and BCR-ABL pathways makes these compounds particularly attractive

17-allylamino-17-demethoxygeldanamycin (17-AAG) is a toxic derivative of geldanamycin and is undergoing clinical examination as it has significant Hsp90-dependent antitumor activity and a favorable toxicity profile (Goetz et al, 2005). Following exposure to 17-AAG results in a rapid decrease of AKT phosphorylation and total AKT protein levels in pediatric ALL patients. This effect can be relevant when 17-AAG is combined with drugs that induce AKT activation, such as arsenic trioxide (Pelicano et al, 2006). Sequential administration causes rapid decline of phosphorylated AKT, increasing the number of cells accumulating in

Beyond the effect of 17-AAG on AKT function, BCR-ABL has also been shown to be a client protein for Hsp90 (Gorre et al, 2002). Treatment with 17-AAG results in significant down-

mediastinal mass at the end of the treatment, but has subsequently progressed.

www.clinicaltrials.gov) in adult patients with relapsed T-ALL.

**7. Heat-shock-protein antagonists** 

G0/1 phase as well as the rate of cleaved caspase-3.

for ALL therapy.

**6. Inhibitors of -secretase** 

NOTCH1 pathway therapeutics.

BCR-ABL transformed cells, suggesting its use in clinically acquired STI571 resistance (Tauchi et al, 2003). The very low toxicity observed in a phase I study of relapsed acute leukemia, demonstrates that G3139 can safely be administered with fludarabine and cytarabine salvage chemotherapy (FLAG) without dose limiting toxicity. Common side effects of this combination include fever, nausea; emesis, electrolyte imbalance, and fluid retention that are not dose limiting. Pharmacokinetics indicates steady-state concentrations within 24 hours and is associated with significant down-regulation of BCL-2 mRNA levels in 75% of patients (Marcucci et al, 2003). Despite an encouraging result of 45% overall survival is achieved, the specific role of G3139 cannot be discriminated from the antileukemic activity of FLAG. Based upon the results obtained so far, validation of the G3139 efficacy, still needs to be completed with phase II/III trials.

## **5. Proteasome inhibitors**

Malignant cells can harbour altered expression of proteasome subunits and their distribution between nucleus and cytoplasm can differ from normal cells. Subsequently, the ubiquitin-proteasome pathway is involved in malignant cellular hemostasis since it regulates the degradation of damaged, oxidized, or misfolded proteins and regulatory proteins that govern cell cycle, transcription factor activation, apoptosis, and cell trafficking. Key proteins degraded by this pathway include cyclins A, B and E, p21, p27, p53, cJun, cFos, IkB, Bcl2, BclX, and MAPK (Laney et al, 1999; Maki et al, 1996). Targeting proteasome subunits by proteasome inhibitors offers the possibility to sensitize surviving tumor cells to the effect of chemotherapy and to induce apoptosis in tumor cells.

Bortezomib (Velcade, PS-341; Millenium Pharmaceuticals) is the first proteasome inhibitor to be tested in humans and is currently approved for the treatment of relapsed multiple myeloma and mantle cell lymphoma. It is a potent and selective, reversible inhibitor of the 26S proteosome, a key regulatory multi-subunit protease, that controls cell cycle and apoptosis (Adams et al, 1998). As in a broad range of tumor cells, bortezomib has demonstrated significant activity in ALL cells, inducing apoptosis *in vitro* and *in vivo*. Additional studies have indicated that it may also potentiate the cytotoxic effects of chemotherapy. In particular, it enhances the *in vitro* cytotoxicity of dexamethasone, vincristine, asparaginase, cytarabine, doxorubicin, phenyl butyrate, trichostatin and HA14.1 (Horton et al, 2006; Sutheesophon et al, 2006).

A recent phase I clinical study in adults with refractory or relapsed acute leukemias indicates the dose of 1.25 mg/m2 to be safely administered on a twice weekly schedule for a 4 week period (Cortes et al, 2004). This is similar to the currently recommended dose of 1.3 mg/m2, given in multiple myeloma. Dose limiting toxicity includes orthostatic hypotension, nausea, diarrhoea and fluid retention. Evidence of biological activity is demonstrated by significant proteasome inhibition. It is anticipated, that few patients have achieved transient reduction of bone marrow blasts, with eventual recurrence of the initial blasts, usually during the time off therapy.

Unfortunately, experience in childhood is limited to a phase I trial of bortezomib in pediatric patients with solid tumors, showing minimal toxicity. Since it has non-overlapping toxicities with myelosuppressive agents used to treat ALL and promising *in vitro* activity, further investigation of bortezomib in this setting is warranted, particularly in studies with multimodal chemotherapy.

## **6. Inhibitors of -secretase**

168 Novel Aspects in Acute Lymphoblastic Leukemia

BCR-ABL transformed cells, suggesting its use in clinically acquired STI571 resistance (Tauchi et al, 2003). The very low toxicity observed in a phase I study of relapsed acute leukemia, demonstrates that G3139 can safely be administered with fludarabine and cytarabine salvage chemotherapy (FLAG) without dose limiting toxicity. Common side effects of this combination include fever, nausea; emesis, electrolyte imbalance, and fluid retention that are not dose limiting. Pharmacokinetics indicates steady-state concentrations within 24 hours and is associated with significant down-regulation of BCL-2 mRNA levels in 75% of patients (Marcucci et al, 2003). Despite an encouraging result of 45% overall survival is achieved, the specific role of G3139 cannot be discriminated from the antileukemic activity of FLAG. Based upon the results obtained so far, validation of the

Malignant cells can harbour altered expression of proteasome subunits and their distribution between nucleus and cytoplasm can differ from normal cells. Subsequently, the ubiquitin-proteasome pathway is involved in malignant cellular hemostasis since it regulates the degradation of damaged, oxidized, or misfolded proteins and regulatory proteins that govern cell cycle, transcription factor activation, apoptosis, and cell trafficking. Key proteins degraded by this pathway include cyclins A, B and E, p21, p27, p53, cJun, cFos, IkB, Bcl2, BclX, and MAPK (Laney et al, 1999; Maki et al, 1996). Targeting proteasome subunits by proteasome inhibitors offers the possibility to sensitize surviving tumor cells to

Bortezomib (Velcade, PS-341; Millenium Pharmaceuticals) is the first proteasome inhibitor to be tested in humans and is currently approved for the treatment of relapsed multiple myeloma and mantle cell lymphoma. It is a potent and selective, reversible inhibitor of the 26S proteosome, a key regulatory multi-subunit protease, that controls cell cycle and apoptosis (Adams et al, 1998). As in a broad range of tumor cells, bortezomib has demonstrated significant activity in ALL cells, inducing apoptosis *in vitro* and *in vivo*. Additional studies have indicated that it may also potentiate the cytotoxic effects of chemotherapy. In particular, it enhances the *in vitro* cytotoxicity of dexamethasone, vincristine, asparaginase, cytarabine, doxorubicin, phenyl butyrate, trichostatin and HA14.1

A recent phase I clinical study in adults with refractory or relapsed acute leukemias indicates the dose of 1.25 mg/m2 to be safely administered on a twice weekly schedule for a 4 week period (Cortes et al, 2004). This is similar to the currently recommended dose of 1.3 mg/m2, given in multiple myeloma. Dose limiting toxicity includes orthostatic hypotension, nausea, diarrhoea and fluid retention. Evidence of biological activity is demonstrated by significant proteasome inhibition. It is anticipated, that few patients have achieved transient reduction of bone marrow blasts, with eventual recurrence of the initial blasts, usually

Unfortunately, experience in childhood is limited to a phase I trial of bortezomib in pediatric patients with solid tumors, showing minimal toxicity. Since it has non-overlapping toxicities with myelosuppressive agents used to treat ALL and promising *in vitro* activity, further investigation of bortezomib in this setting is warranted, particularly in studies with

G3139 efficacy, still needs to be completed with phase II/III trials.

the effect of chemotherapy and to induce apoptosis in tumor cells.

(Horton et al, 2006; Sutheesophon et al, 2006).

during the time off therapy.

multimodal chemotherapy.

**5. Proteasome inhibitors** 

The detection of NOTCH1 gain-of-function mutations in more than 50% of T-cell ALL patients has attracted much interest in the understanding of its role in the molecular pathogenesis of this leukemic subtype. Although expression of an activated NOTCH1 allele has been shown to cause T-cell leukemia in mice, the molecular mechanism for cellular transformation is largely unknown. However, the identification of c-myc as a direct and critical NOTCH1 target gene (Weng et al, 2004) has urged the screening and development of NOTCH1 pathway therapeutics.

The NOTCH1 inhibitor MK-0752 (Merck & Co.) has been developed for the treatment of Alzheimer's disease, since it cleaves amyloid precursor protein and prevents the formation of amyloid ß-peptides (Evin et al, 2006). Its activity in leukemia is demonstrated by the conservation of the NOTCH1 receptor within the transmembrane domain, preventing the release of the NOTCH intracellular domain, and thereby suppressing the transcription of target genes. This results in the arrest of cells in G0/1 , reduces viability, and increases apoptosis (DeAngelo et al, 2006). In a phase I trial of adult and pediatric patients with T-cell malignancies MK-0752 has been shown to be well-tolerated with diarrhoea being the doselimiting toxicity. Measurements of -secretase inhibition have shown a 24-69% decrease in plasma Abeta40 peptide levels compared to predose levels (DeAngelo et al, 2006). Only one patient with a NOTCH1 activating mutation has achieved a significant reduction of a mediastinal mass at the end of the treatment, but has subsequently progressed.

Currently, an additional phase I study is examining the safety of a newer NOTCH1 inhibitor with improved biological availability (PF-03084014, Pfizer; NCT01068301, www.clinicaltrials.gov) in adult patients with relapsed T-ALL.

## **7. Heat-shock-protein antagonists**

Heat shock protein 90 (Hsp90) is currently receiving considerable attention as a potential anticancer drug target. It is a molecular chaperone that regulates structural folding and active configuration of a variety of signal transduction and cell cycle regulatory proteins, including tyrosine and serine kinases such as c-src and Akt (Isaacs et al, 2003; Hawkins et al, 2005). Inhibition of Hsp90 disrupts the folding of these proteins, thus increasing their susceptibility to ubiquitination and proteosomal degradation. Although the exact mechanism by which Hsp90 inhibitors kill tumor cells remain to be defined, the ability to abrogate the AKT and BCR-ABL pathways makes these compounds particularly attractive for ALL therapy.

17-allylamino-17-demethoxygeldanamycin (17-AAG) is a toxic derivative of geldanamycin and is undergoing clinical examination as it has significant Hsp90-dependent antitumor activity and a favorable toxicity profile (Goetz et al, 2005). Following exposure to 17-AAG results in a rapid decrease of AKT phosphorylation and total AKT protein levels in pediatric ALL patients. This effect can be relevant when 17-AAG is combined with drugs that induce AKT activation, such as arsenic trioxide (Pelicano et al, 2006). Sequential administration causes rapid decline of phosphorylated AKT, increasing the number of cells accumulating in G0/1 phase as well as the rate of cleaved caspase-3.

Beyond the effect of 17-AAG on AKT function, BCR-ABL has also been shown to be a client protein for Hsp90 (Gorre et al, 2002). Treatment with 17-AAG results in significant down-

Novel Therapeutic Targets in ALL Therapy 171

childhood ALL with rearrangement of the MLL gene and in ALL patients with hyperdiploidy (Armstrong et al, 2002, 2004); . *In vitro* studies have shown that coexpression of FLT3 ligand and activating mutations on the FLT3 gene can constitutively activate downstream targets. These include signal transducers and activators of transcription (STAT), MAPK, and AKT pathways that regulate proliferation, differentiation, and survival (Mizuki et al, 2000). Considering the worse prognosis of MLL patients and understanding the molecular mechanism of FLT3, has prompted the development of specific FLT3

Small-molecular FLT3 inhibitors have initially been developed for AML, in which FLT3 mutations represent the most common somatic genetic alteration. FLT3 inhibitors induce significant cytotoxic responses, and several of these agents have been tested in adult trials of AML (Fiedler et al, 2005; Smith et al, 2004). FLT3 inhibitors are well tolerated; toxicities include mild nausea, emesis and generalized weakness with the highest doses administered. In MLL-rearranged ALL, treatment with the FLT3 inhibitor PKC-412 (Midostaurin, Novartis Pharmaceuticals) has proved to be cytotoxic to Ba/F3 cells dependent upon activating mutations of FLT3 (Armstrong et al, 2003). Similarly, ALL with high hyperdiploidy and t (4;11), has shown pronounced apoptotic responses to treatment with CEP-701 (Lesaurtinib, Cephalon) (Brown et al, 2005). Synergistic effects have not been noted, when FLT3 inhibitors are used simultaneously or immediately following exposure to cytarabine, daunorubicin, mitoxantrone or etoposide. These effects might be of interest, if used to overcome rapid development of resistance to FLT3 inhibitors (Levis et al, 2004). Of note, pretreatment with CEP-701 in combination with cytarabine may act antagonistically, due to its cell-cycle

In MLL-rearranged ALL with wild type FLT3, FLT3 ligand induces quiescence and chemoresistance that can be overcome by FLT3 inhibition (Furuichi et al, 2007). It is possible that ligation of FLT3 in these ALL cases contributes to the poor response to chemotherapy by activating quiescence and self-renewal functions. It is therefore worth examining the ability of FLT3 inhibitors to reverse this response and increase chemosensitivity in this particularly difficult to treat group of patients. More specific FLT3 targeting has recently been developed by the application of anti-FLT3 antibodies (Piloto et al, 2006). IMC-NC7 consistently inhibits FLT3 phosphorylation, whereas IMC-EB10 stimulates its activation. Both treatments prolong survival and/or reduced engraftment of leukemic cells in a NOD/SCID mouse model, mainly through the recruitment of the host's immune system

A phase I/II clinical trial is currently recruiting patients that will evaluate the safety, tolerability, clinical response, pharmacokinetics and pharmacodynamics of PKC-412 in children who have relapsed or refractory acute leukemias, including MLL-rearranged ALL

Inhibitors of farnesyl transferase (FTI) have originally been developed to prevent attachment of intracellular Ras to the inner leaflet of the plasma membrane and, therefore, transduction of proliferative and survival signals (Gelb et al, 1997). Subsequent studies, however, have suggested that the cytotoxic actions of FTIs might also involve oncoproteins other than Ras, such as RhoB and members of phosphoinositide 3/OH kinase (PI3K)/AKT-2

against targeted cells, independently of receptor activation.

(NCT00866281, www.clinicaltrials.gov).

**10. Farnesyl transferase inhibitors** 

inhibitors.

inhibitory effect in AML.

regulation of intracellular levels of BCR-ABL, followed by a decrease in cell survival and the induction of apoptosis. Of interest is the effect on imatinib mesylate-resistant cells, in which sensitivity to imatinib can be restored.

Based on this selective toxicity, 17-AAG has been examined in phase I and II clinical trials in patients with acute myeloid and chronic lymphatic leukemia. These studies have shown that 17-AAG is reasonably well tolerated, with transient elevation of serum transaminases, nausea, vomiting, and diarrhoea being dose-limiting when this agent is administered as a 60-minute infusion on a weekly schedule. Further studies are required to determine tolerability and effectiveness in leukemic patients.

## **8. Resveratrol**

Recently, several natural or dietary substances have been shown to have antineoplastic activity. Much attention has been paid to the polyphenolic phytoalexin resveratrol (3,5,4' trihydroxy-trans-stilbene), since it inhibits events associated with tumor initiation, promotion and progress (Jang et al, 1997). Potentially, it inhibits free-radical formation and reduces oxidative and mutagenic stress (Aggarwal et al, 2004). Subsequently, resveratrol suppresses the growth of transformed cells and induces apoptosis through interaction with kinase pathways and activation of the caspase cascade (Bernhard et al, 2000).

In leukemic cells resveratrol arrests cells in the S-phase of the cell cycle. A mechanism, by which the replication machinery is arrested, is demonstrated by the inhibition of the ribonucleotide reductase in murine lymphoblastic leukemic cells. Additionally, both the NOTCH and the PI3K/AKT pathway are inhibited at higher concentrations of resveratrol. This is modulated by the activation of signalling systems, such as p53, p21waf-1 and Bax (Cecchinato et al, 2007). Also, resveratrol has been shown to induce mitochondrial depolarization and subsequent activation of downstream caspases associated with the intrinsic apoptotic pathway (Dorrie et al, 2001; Fontecave et al, 1998). Independence of Fasand TNF signalling in resveratrol-induced apoptosis might be a desirable property of a potential new therapeutic agent, since tumor cells develop strategies to escape Fas-mediated apoptosis (Bernhard et al, 2000).

Preliminary results of a phase II study of the synthetic resveratrol derivative SRT501 (GlaxoSmithKline) in multiple myeloma patients reveal, that administration of this formulation is limited by the development of the acute renal failure (NCT00920556, www.clinicaltrials.gov). Whereas this complication might be restricted to the underlying disease, tolerability and efficacy of resveratrol in leukemic patients still remain to be clarified in further clinical investigations.

## **9. Fms-like tyrosine kinase-3 inhibitors**

The fms-like tyrosine kinase-3 (FLT3) is a member of the class III receptor tyrosine kinase family and is largely expressed along with CD34 and CD117 in immature hematopoetic progenitor cells. Knock-out mice are associated with deficiencies in B-cell lymphopoesis and reconstitution of both T cells and myeloid cells after BMT, indicating a crucial role of FLT3 in the development of multipotent hematopoetic and lymphoid cells.

Similarly, FLT3 has been implicated in the pathogenesis of leukemia (Carow et al, 1996). In addition to the near-universal FLT3 expression in adult precursor B-ALL, gene expression analysis have shown, that the highest levels of FLT3 expression occur in infant and

regulation of intracellular levels of BCR-ABL, followed by a decrease in cell survival and the induction of apoptosis. Of interest is the effect on imatinib mesylate-resistant cells, in which

Based on this selective toxicity, 17-AAG has been examined in phase I and II clinical trials in patients with acute myeloid and chronic lymphatic leukemia. These studies have shown that 17-AAG is reasonably well tolerated, with transient elevation of serum transaminases, nausea, vomiting, and diarrhoea being dose-limiting when this agent is administered as a 60-minute infusion on a weekly schedule. Further studies are required to determine

Recently, several natural or dietary substances have been shown to have antineoplastic activity. Much attention has been paid to the polyphenolic phytoalexin resveratrol (3,5,4' trihydroxy-trans-stilbene), since it inhibits events associated with tumor initiation, promotion and progress (Jang et al, 1997). Potentially, it inhibits free-radical formation and reduces oxidative and mutagenic stress (Aggarwal et al, 2004). Subsequently, resveratrol suppresses the growth of transformed cells and induces apoptosis through interaction with

In leukemic cells resveratrol arrests cells in the S-phase of the cell cycle. A mechanism, by which the replication machinery is arrested, is demonstrated by the inhibition of the ribonucleotide reductase in murine lymphoblastic leukemic cells. Additionally, both the NOTCH and the PI3K/AKT pathway are inhibited at higher concentrations of resveratrol. This is modulated by the activation of signalling systems, such as p53, p21waf-1 and Bax (Cecchinato et al, 2007). Also, resveratrol has been shown to induce mitochondrial depolarization and subsequent activation of downstream caspases associated with the intrinsic apoptotic pathway (Dorrie et al, 2001; Fontecave et al, 1998). Independence of Fasand TNF signalling in resveratrol-induced apoptosis might be a desirable property of a potential new therapeutic agent, since tumor cells develop strategies to escape Fas-mediated

Preliminary results of a phase II study of the synthetic resveratrol derivative SRT501 (GlaxoSmithKline) in multiple myeloma patients reveal, that administration of this formulation is limited by the development of the acute renal failure (NCT00920556, www.clinicaltrials.gov). Whereas this complication might be restricted to the underlying disease, tolerability and efficacy of resveratrol in leukemic patients still remain to be

The fms-like tyrosine kinase-3 (FLT3) is a member of the class III receptor tyrosine kinase family and is largely expressed along with CD34 and CD117 in immature hematopoetic progenitor cells. Knock-out mice are associated with deficiencies in B-cell lymphopoesis and reconstitution of both T cells and myeloid cells after BMT, indicating a crucial role of FLT3

Similarly, FLT3 has been implicated in the pathogenesis of leukemia (Carow et al, 1996). In addition to the near-universal FLT3 expression in adult precursor B-ALL, gene expression analysis have shown, that the highest levels of FLT3 expression occur in infant and

in the development of multipotent hematopoetic and lymphoid cells.

kinase pathways and activation of the caspase cascade (Bernhard et al, 2000).

sensitivity to imatinib can be restored.

**8. Resveratrol** 

apoptosis (Bernhard et al, 2000).

clarified in further clinical investigations.

**9. Fms-like tyrosine kinase-3 inhibitors** 

tolerability and effectiveness in leukemic patients.

childhood ALL with rearrangement of the MLL gene and in ALL patients with hyperdiploidy (Armstrong et al, 2002, 2004); . *In vitro* studies have shown that coexpression of FLT3 ligand and activating mutations on the FLT3 gene can constitutively activate downstream targets. These include signal transducers and activators of transcription (STAT), MAPK, and AKT pathways that regulate proliferation, differentiation, and survival (Mizuki et al, 2000). Considering the worse prognosis of MLL patients and understanding the molecular mechanism of FLT3, has prompted the development of specific FLT3 inhibitors.

Small-molecular FLT3 inhibitors have initially been developed for AML, in which FLT3 mutations represent the most common somatic genetic alteration. FLT3 inhibitors induce significant cytotoxic responses, and several of these agents have been tested in adult trials of AML (Fiedler et al, 2005; Smith et al, 2004). FLT3 inhibitors are well tolerated; toxicities include mild nausea, emesis and generalized weakness with the highest doses administered. In MLL-rearranged ALL, treatment with the FLT3 inhibitor PKC-412 (Midostaurin, Novartis Pharmaceuticals) has proved to be cytotoxic to Ba/F3 cells dependent upon activating mutations of FLT3 (Armstrong et al, 2003). Similarly, ALL with high hyperdiploidy and t (4;11), has shown pronounced apoptotic responses to treatment with CEP-701 (Lesaurtinib, Cephalon) (Brown et al, 2005). Synergistic effects have not been noted, when FLT3 inhibitors are used simultaneously or immediately following exposure to cytarabine, daunorubicin, mitoxantrone or etoposide. These effects might be of interest, if used to overcome rapid development of resistance to FLT3 inhibitors (Levis et al, 2004). Of note, pretreatment with CEP-701 in combination with cytarabine may act antagonistically, due to its cell-cycle inhibitory effect in AML.

In MLL-rearranged ALL with wild type FLT3, FLT3 ligand induces quiescence and chemoresistance that can be overcome by FLT3 inhibition (Furuichi et al, 2007). It is possible that ligation of FLT3 in these ALL cases contributes to the poor response to chemotherapy by activating quiescence and self-renewal functions. It is therefore worth examining the ability of FLT3 inhibitors to reverse this response and increase chemosensitivity in this particularly difficult to treat group of patients. More specific FLT3 targeting has recently been developed by the application of anti-FLT3 antibodies (Piloto et al, 2006). IMC-NC7 consistently inhibits FLT3 phosphorylation, whereas IMC-EB10 stimulates its activation. Both treatments prolong survival and/or reduced engraftment of leukemic cells in a NOD/SCID mouse model, mainly through the recruitment of the host's immune system against targeted cells, independently of receptor activation.

A phase I/II clinical trial is currently recruiting patients that will evaluate the safety, tolerability, clinical response, pharmacokinetics and pharmacodynamics of PKC-412 in children who have relapsed or refractory acute leukemias, including MLL-rearranged ALL (NCT00866281, www.clinicaltrials.gov).

## **10. Farnesyl transferase inhibitors**

Inhibitors of farnesyl transferase (FTI) have originally been developed to prevent attachment of intracellular Ras to the inner leaflet of the plasma membrane and, therefore, transduction of proliferative and survival signals (Gelb et al, 1997). Subsequent studies, however, have suggested that the cytotoxic actions of FTIs might also involve oncoproteins other than Ras, such as RhoB and members of phosphoinositide 3/OH kinase (PI3K)/AKT-2

Novel Therapeutic Targets in ALL Therapy 173

alkylguanine DNA alkyltransferase levels, a potential mechanism of drug resistance, temozolomide should be administered in an extended low-dose schedule, as has been

Epigenetic changes to promoter regions have been identified in recent years as important factors in the pathogenesis of acute leukemia. They include DNA modifications that regulate chromatin structure and change gene expression without altering the nucleotide sequence. A promising new therapeutic strategy is aimed at removal of acetyl groups from histone proteins as well as other non-histone protein targets with histone deacetylation inhibitors (HDACIs), which results in chromatin remodeling that permits re-expression of silenced tumor suppressor genes in cancer cells (Brown et al, 2002). This in turn, can potentially

Several classes of HDACIs are currently under development for treatment of leukemia, including the short-chain fatty acids sodium phenylbutyrate and valproic acid, the hydroxamic acids suberoylanilide hydroxamic acid (SAHA, Vorinostat, Merck & Co.), FK-228 (Depsipeptide, Gloucester Pharmaceuticals) and LBH589 (Panobinostat, Novartis

*In vitro* studies have demonstrated that HDACIs trigger maturation, when administered at low concentrations, whereas at higher concentrations, apoptosis is induced. The factors that determine whether HDACIs engage apoptosis versus differentiation remain the subject of investigation, but they may involve the induction of death receptors such as Fas- and tumornecrosis-factor-related apoptosis-inducing ligand (TRAIL) (Bernhard et al, 2001; Inoue et al, 2004), the production of reactive oxygen species, which leads to mitochondrial disruption (Bernhard et al, 2001), independently of activation of caspase-8 or -3, followed by internucleosomal DNA fragmentation. Specifically in Philadelphia chromosome-positive (Ph+) ALL, HDACIs reduce viability and increase expression of the apoptosis associated proteins FANCG, FOXO3A, GADD45A, GADD45B and GADD45G (Scuto et al, 2008). If HDACIs are used at lower concentrations, they induce either G0/1 or G2-M arrest. These events are accompanied by induction of p53 and p21WAF1, and down-regulation of cell cycle-

*In vivo* FK-228 effectively inhibits HDAC in patients with chronic lymphatic leukemia (CLL) and acute myeloid leukemia (AML) (Byrd et al, 2005), but its use in the current schedule of administration is limited by progressive constitutional symptoms. Dose-limiting toxicities include anorexia, dehydration, diarrhoea, and fatigue. Several patients have evidence of anti-tumor activity following treatment, but no partial or complete response is noted. Both, SAHA and LBH589, have been reported to be tolerable for short- and long-term application in phase I clinical trials of adult refractory hematological malignancies (Garcia-Manero et al, 2008). Based on the safety and efficacy demonstrated in phase I/II trials, a single HDACI compound, SAHA, has been approved for the treatment of cutaneous T-cell lymphoma,

Regarding childhood ALL data, a recent phase I study of vorinostat has just been published. Drug disposition and tolerance in children is similar to that observed in adult patients, whereas the maximum tolerated dose (MTD) seems to be lower due to liver dysfunction. Furthermore, a phase II study is reported as currently recruiting participants (NCT00882206, www.clinicaltrials.gov). Specifically, this trial is investigating the use of decitabine and

suggested in a clinical trial of malignant gliomas (Khan et al, 2002).

result in cellular differentiation, inhibition of proliferation and/or apoptosis.

Pharmaceuticals), and the benzamides MS-275 and C1-994.

promoting proteins, including cyclin D1 and D2.

after demonstrating activity in heavily pre-treated patients.

**12. Histone deacetylase inhibitors** 

pathway (Lebowitz et al, 1998), suggesting that the mechanism of action of FTIs is significantly more complex than initially presumed.

Results of *in vitro* exposure of several leukemic cells provide evidence for the efficacy of FTIs in suppressing tumor cell proliferation. Particularly, the nonpeptidomimetic enzymespecific inhibitor R115777 (Tipifarnib, Johnson & Johnson) has been shown to decrease proliferation and sensitize apoptosis to chemotherapy-induced cell death. A phase I trial of R115777 in adults with poor-risk acute leukemia provides first evidence for *in vivo* efficacy. 29% of the 34 evaluable patients have shown a clinical response, including 2 patients with complete remission (Karp et al, 2001). Dose limiting toxicities on various schedules include central neurotoxicity and reversible nausea, renal insufficiency, polydipsia, paresthesia and myelosuppression.

Of note is the novel, orally active FTI SCH66336 (Lonafarnib, Schering-Plough), which competes with the enzyme for the CAAX portion of Ras and induces apoptosis in acute myeloid leukemia. It might be particularly attractive for a combination therapy with STI571, since it has been shown to inhibit the proliferation of STI571-resistant BCR-ABL-positive cell lines and synergize for the induction of apoptosis (Borthakur et al, 2006; Hoover et al, 2002).

## **11. DNA methylase inhibitors**

Aberrant DNA methylation of multiple promoter CpG islands is frequently observed in patients with ALL both at initial presentation and at the time of relapse. Indeed these methylation marks are stable in over 70% of patients with ALL at the time of relapse. Importantly, methylation of specific molecular pathways has been associated with an extremely poor prognosis in patients with ALL. This has been demonstrated in the aberrant methylation of the promotor region within tumor suppressor genes such as the fragile histidine triad (FHIT) of members of the cell cycle pathway such as p73, the cyclin dependent kinase inhibitors p57KIP2 and p15 (Bueso-Ramos et al, 2005). Pharmacologic modification of aberrant methylation can therefore be an attractive approach to regulate leukemic cell proliferation.

Recently, the pyrimidine nucleoside analog 5-Aza-2'-deoxycytidine (decitabine/Daco-gene, SuperGen) has received much interest as a strong hypomethylating agent with clinical activity in myelodysplastic syndrome, and acute and chronic myelogeous leukemia (Richel et al, 1991). *In vitro* exposure of decitabine results in hypomethylation and reactivation of putative tumor suppressor genes. As a result, low concentrations of decitabine stimulate cellular differentiation, whereas high concentrations directly interfere with DNA synthesis and mediate cytotoxicity in acute leukemias (Pinto et al, 1984). Combination therapy with cytarabine achieves complete remission in the majority of patients in a clinical trial of ALL (Richel et al, 1991). Analysis of cell membrane markers shows a loss of the early differentiation antigens CD34 and CD33 in leukemic bone marrow cells, which is suggestive of leukemic cell differentiation. Addition of HDACIs might synergize in controlling gene transcription as has successfully been shown in a clinical trial for t(8;14) AML (Klisovic et al, 2003).

Temozolomide (Temodal, Schering-Plough) is a second-generation oral alkylating agent with DNA methylating properties. Although the exact mechanism for methylation is not fully elucidated, it results in an active mismatch-repair pathway with consequent DNA strand breakage and apoptosis (D'Atri et al, 1998). Temozolomide is well tolerated when it is administered as a single agent (Seiter el al, 2002). Because of the chance to deplete O6-

pathway (Lebowitz et al, 1998), suggesting that the mechanism of action of FTIs is

Results of *in vitro* exposure of several leukemic cells provide evidence for the efficacy of FTIs in suppressing tumor cell proliferation. Particularly, the nonpeptidomimetic enzymespecific inhibitor R115777 (Tipifarnib, Johnson & Johnson) has been shown to decrease proliferation and sensitize apoptosis to chemotherapy-induced cell death. A phase I trial of R115777 in adults with poor-risk acute leukemia provides first evidence for *in vivo* efficacy. 29% of the 34 evaluable patients have shown a clinical response, including 2 patients with complete remission (Karp et al, 2001). Dose limiting toxicities on various schedules include central neurotoxicity and reversible nausea, renal insufficiency, polydipsia, paresthesia and

Of note is the novel, orally active FTI SCH66336 (Lonafarnib, Schering-Plough), which competes with the enzyme for the CAAX portion of Ras and induces apoptosis in acute myeloid leukemia. It might be particularly attractive for a combination therapy with STI571, since it has been shown to inhibit the proliferation of STI571-resistant BCR-ABL-positive cell lines and synergize for the induction of apoptosis (Borthakur et al, 2006; Hoover et al, 2002).

Aberrant DNA methylation of multiple promoter CpG islands is frequently observed in patients with ALL both at initial presentation and at the time of relapse. Indeed these methylation marks are stable in over 70% of patients with ALL at the time of relapse. Importantly, methylation of specific molecular pathways has been associated with an extremely poor prognosis in patients with ALL. This has been demonstrated in the aberrant methylation of the promotor region within tumor suppressor genes such as the fragile histidine triad (FHIT) of members of the cell cycle pathway such as p73, the cyclin dependent kinase inhibitors p57KIP2 and p15 (Bueso-Ramos et al, 2005). Pharmacologic modification of aberrant methylation can therefore be an attractive approach to regulate

Recently, the pyrimidine nucleoside analog 5-Aza-2'-deoxycytidine (decitabine/Daco-gene, SuperGen) has received much interest as a strong hypomethylating agent with clinical activity in myelodysplastic syndrome, and acute and chronic myelogeous leukemia (Richel et al, 1991). *In vitro* exposure of decitabine results in hypomethylation and reactivation of putative tumor suppressor genes. As a result, low concentrations of decitabine stimulate cellular differentiation, whereas high concentrations directly interfere with DNA synthesis and mediate cytotoxicity in acute leukemias (Pinto et al, 1984). Combination therapy with cytarabine achieves complete remission in the majority of patients in a clinical trial of ALL (Richel et al, 1991). Analysis of cell membrane markers shows a loss of the early differentiation antigens CD34 and CD33 in leukemic bone marrow cells, which is suggestive of leukemic cell differentiation. Addition of HDACIs might synergize in controlling gene transcription as has successfully been shown in a clinical trial for t(8;14) AML (Klisovic et al,

Temozolomide (Temodal, Schering-Plough) is a second-generation oral alkylating agent with DNA methylating properties. Although the exact mechanism for methylation is not fully elucidated, it results in an active mismatch-repair pathway with consequent DNA strand breakage and apoptosis (D'Atri et al, 1998). Temozolomide is well tolerated when it is administered as a single agent (Seiter el al, 2002). Because of the chance to deplete O6-

significantly more complex than initially presumed.

myelosuppression.

**11. DNA methylase inhibitors** 

leukemic cell proliferation.

2003).

alkylguanine DNA alkyltransferase levels, a potential mechanism of drug resistance, temozolomide should be administered in an extended low-dose schedule, as has been suggested in a clinical trial of malignant gliomas (Khan et al, 2002).

## **12. Histone deacetylase inhibitors**

Epigenetic changes to promoter regions have been identified in recent years as important factors in the pathogenesis of acute leukemia. They include DNA modifications that regulate chromatin structure and change gene expression without altering the nucleotide sequence. A promising new therapeutic strategy is aimed at removal of acetyl groups from histone proteins as well as other non-histone protein targets with histone deacetylation inhibitors (HDACIs), which results in chromatin remodeling that permits re-expression of silenced tumor suppressor genes in cancer cells (Brown et al, 2002). This in turn, can potentially result in cellular differentiation, inhibition of proliferation and/or apoptosis.

Several classes of HDACIs are currently under development for treatment of leukemia, including the short-chain fatty acids sodium phenylbutyrate and valproic acid, the hydroxamic acids suberoylanilide hydroxamic acid (SAHA, Vorinostat, Merck & Co.), FK-228 (Depsipeptide, Gloucester Pharmaceuticals) and LBH589 (Panobinostat, Novartis Pharmaceuticals), and the benzamides MS-275 and C1-994.

*In vitro* studies have demonstrated that HDACIs trigger maturation, when administered at low concentrations, whereas at higher concentrations, apoptosis is induced. The factors that determine whether HDACIs engage apoptosis versus differentiation remain the subject of investigation, but they may involve the induction of death receptors such as Fas- and tumornecrosis-factor-related apoptosis-inducing ligand (TRAIL) (Bernhard et al, 2001; Inoue et al, 2004), the production of reactive oxygen species, which leads to mitochondrial disruption (Bernhard et al, 2001), independently of activation of caspase-8 or -3, followed by internucleosomal DNA fragmentation. Specifically in Philadelphia chromosome-positive (Ph+) ALL, HDACIs reduce viability and increase expression of the apoptosis associated proteins FANCG, FOXO3A, GADD45A, GADD45B and GADD45G (Scuto et al, 2008). If HDACIs are used at lower concentrations, they induce either G0/1 or G2-M arrest. These events are accompanied by induction of p53 and p21WAF1, and down-regulation of cell cyclepromoting proteins, including cyclin D1 and D2.

*In vivo* FK-228 effectively inhibits HDAC in patients with chronic lymphatic leukemia (CLL) and acute myeloid leukemia (AML) (Byrd et al, 2005), but its use in the current schedule of administration is limited by progressive constitutional symptoms. Dose-limiting toxicities include anorexia, dehydration, diarrhoea, and fatigue. Several patients have evidence of anti-tumor activity following treatment, but no partial or complete response is noted. Both, SAHA and LBH589, have been reported to be tolerable for short- and long-term application in phase I clinical trials of adult refractory hematological malignancies (Garcia-Manero et al, 2008). Based on the safety and efficacy demonstrated in phase I/II trials, a single HDACI compound, SAHA, has been approved for the treatment of cutaneous T-cell lymphoma, after demonstrating activity in heavily pre-treated patients.

Regarding childhood ALL data, a recent phase I study of vorinostat has just been published. Drug disposition and tolerance in children is similar to that observed in adult patients, whereas the maximum tolerated dose (MTD) seems to be lower due to liver dysfunction. Furthermore, a phase II study is reported as currently recruiting participants (NCT00882206, www.clinicaltrials.gov). Specifically, this trial is investigating the use of decitabine and

Novel Therapeutic Targets in ALL Therapy 175

(R= relapse, eto= etoposide, ara-c= cytarabine, rit= rituximab, cpm= cyclophosphamide, vin= vincristine, dex= dexamethasone, flu= fludarabine, mit= mitoxantrone, asp= asparaginase, dau= daunorubicine, dox= doxorubicine, mer= mercaptopurine, vor= vorinostat, pre= predinison, mtx=

investigated for their antileukemic potential. Among them, the synthetic flavone derivative

Flavopiridol binds to the adenosine triphosphate (ATP) site of CDK, thereby reducing the activity of CDK1, CDK2, CDK4, CDK6, and CDK7, leading to cell cycle arrest in G0/1 and G2 (Sedlacek et al, 2001). In addition, flavopiridol disrupts the CDK9/cyclin T complex resulting in reduced phosphorylation of the carboxyl-terminal domain of RNA Pol-II and subsequent inhibition of mRNA synthesis (Chao et al, 2000). Consequently, short lived anti-

Table 1. Active and completed clinical trials with novel therapeutic targets for ALL.

flavopiridol (Alvocidib, L86-8275; Behringwerke) is the first to undergo human trials.

methotrexate, ima= imatinib)

vorinostat together with combination chemotherapy in treating patients with relapsed/refractory ALL or lymphoblastic lymphoma.

## **13. Mammalian target of rapamycin inhibitors**

The mammalian target of rapamycin (mTOR) is a critical effector in cell-signalling pathways, such as the PI3K/AKT transduction pathway, that are up regulated in malignant transformed cells (Dann et al, 2006). After activation of G-protein coupled receptors by various growth factors, mTOR mediates the phosphorylation of the 70 kDa S6 ribosomal protein kinase and the initiation factor 4E-binding protein 1 (Vignot et al, 2005). Subsequently, activated cyclin D1, CDK4, and Rb initiate the G0/1 to S phase progression. Accordingly, targeting the mTOR signalling pathway has extensively been analyzed for suppression of leukemic cell proliferation.

Inhibitors of mTOR include rapamycin (Sirolimus, Wyeth Pharmaceuticals) and the second generation analogs RAD001 (Everolimus, Novartis Pharmaceuticals), and CCI-779 (Temsirolimus, Wyeth Pharmaceuticals). Rapamycin was initially developed as immunosuppressive agent and was the first inhibitor to be used in a clinical setting for leukemic therapy. Rapamycin induces apoptosis in precursor B ALL lines *in vitro* and has *in vivo* activity in transgenic mice with pre-B leukemia/lymphoma (Brown et al, 2003). CCI-779 inhibits the growth of adult human ALL on bone marrow layers and reduces the number of blasts in the peripheral-blood and the degree of organ infiltration in human ALL engrafted NOD/SCID mice (Teachey et al, 2006). RAD001 reduces tumor mass *in vivo*, conferring prolonged survival of NOD/SCID mice engrafted with childhood ALL (Crazzolara et al, 2009). Mechanistic insight demonstrates the induction of autophagy in the absence of apoptosis, which is particular interesting in ALL, since resistance to current chemotherapies, such as dexamethasone, has been linked to certain defects in the apoptotic machinery.

Phase I trials in patients with various cancers show that mTOR inhibitors when used as monotherapy are well tolerated in humans, with little nephrotoxicity and neurotoxicity (Calne et al, 1989). They may further cause hyperlipidemia, mild myelosuppression, hypertension, skin rashes and mucositis. However, the toxicities of combining mTOR inhibitors with conventional cytotoxic agents have not been fully explored in both preclinical and clinical studies. Based on the preclinical work, a number of clinical trials evaluating the efficacy of mTOR inhibitors in ALL as single agents and in combination with other agents have been performed are still on-going (NCT01162551, www.clinicaltrials.gov). Two phase I/II trials of MTIs in patients with relapsed or refractory malignances, including one patient each with ALL, have shown that both patients have tolerated therapy, but neither have had an objective response. A recent interim result of an on-going phase 1 trial of sirolimus in children reveals that 3 of 7 patients relapsed/refractory ALL have stable disease.

## **14. Cyclin dependent kinase inhibitors**

Loss of p16 (INK4A) in hematopoietic stem cells is associated with enhanced self-renewal capacity and might facilitate progression of damaged stem cells into pre-cancerous cells that give rise to leukemia (Bhojwani et al, 2006). Based on higher frequency of p16 (INK4A) deletions in relapsed ALL, inhibitors of cyclin dependent kinases (CDKs) have been

vorinostat together with combination chemotherapy in treating patients with

The mammalian target of rapamycin (mTOR) is a critical effector in cell-signalling pathways, such as the PI3K/AKT transduction pathway, that are up regulated in malignant transformed cells (Dann et al, 2006). After activation of G-protein coupled receptors by various growth factors, mTOR mediates the phosphorylation of the 70 kDa S6 ribosomal protein kinase and the initiation factor 4E-binding protein 1 (Vignot et al, 2005). Subsequently, activated cyclin D1, CDK4, and Rb initiate the G0/1 to S phase progression. Accordingly, targeting the mTOR signalling pathway has extensively been analyzed for

Inhibitors of mTOR include rapamycin (Sirolimus, Wyeth Pharmaceuticals) and the second generation analogs RAD001 (Everolimus, Novartis Pharmaceuticals), and CCI-779 (Temsirolimus, Wyeth Pharmaceuticals). Rapamycin was initially developed as immunosuppressive agent and was the first inhibitor to be used in a clinical setting for leukemic therapy. Rapamycin induces apoptosis in precursor B ALL lines *in vitro* and has *in vivo* activity in transgenic mice with pre-B leukemia/lymphoma (Brown et al, 2003). CCI-779 inhibits the growth of adult human ALL on bone marrow layers and reduces the number of blasts in the peripheral-blood and the degree of organ infiltration in human ALL engrafted NOD/SCID mice (Teachey et al, 2006). RAD001 reduces tumor mass *in vivo*, conferring prolonged survival of NOD/SCID mice engrafted with childhood ALL (Crazzolara et al, 2009). Mechanistic insight demonstrates the induction of autophagy in the absence of apoptosis, which is particular interesting in ALL, since resistance to current chemotherapies, such as dexamethasone, has been linked to certain defects in the apoptotic machinery. Phase I trials in patients with various cancers show that mTOR inhibitors when used as monotherapy are well tolerated in humans, with little nephrotoxicity and neurotoxicity (Calne et al, 1989). They may further cause hyperlipidemia, mild myelosuppression, hypertension, skin rashes and mucositis. However, the toxicities of combining mTOR inhibitors with conventional cytotoxic agents have not been fully explored in both preclinical and clinical studies. Based on the preclinical work, a number of clinical trials evaluating the efficacy of mTOR inhibitors in ALL as single agents and in combination with other agents have been performed are still on-going (NCT01162551, www.clinicaltrials.gov). Two phase I/II trials of MTIs in patients with relapsed or refractory malignances, including one patient each with ALL, have shown that both patients have tolerated therapy, but neither have had an objective response. A recent interim result of an on-going phase 1 trial of sirolimus in children reveals that 3 of 7 patients relapsed/refractory ALL have stable

Loss of p16 (INK4A) in hematopoietic stem cells is associated with enhanced self-renewal capacity and might facilitate progression of damaged stem cells into pre-cancerous cells that give rise to leukemia (Bhojwani et al, 2006). Based on higher frequency of p16 (INK4A) deletions in relapsed ALL, inhibitors of cyclin dependent kinases (CDKs) have been

relapsed/refractory ALL or lymphoblastic lymphoma.

**13. Mammalian target of rapamycin inhibitors** 

suppression of leukemic cell proliferation.

**14. Cyclin dependent kinase inhibitors** 

disease.


(R= relapse, eto= etoposide, ara-c= cytarabine, rit= rituximab, cpm= cyclophosphamide, vin= vincristine, dex= dexamethasone, flu= fludarabine, mit= mitoxantrone, asp= asparaginase, dau= daunorubicine, dox= doxorubicine, mer= mercaptopurine, vor= vorinostat, pre= predinison, mtx= methotrexate, ima= imatinib)

Table 1. Active and completed clinical trials with novel therapeutic targets for ALL.

investigated for their antileukemic potential. Among them, the synthetic flavone derivative flavopiridol (Alvocidib, L86-8275; Behringwerke) is the first to undergo human trials.

Flavopiridol binds to the adenosine triphosphate (ATP) site of CDK, thereby reducing the activity of CDK1, CDK2, CDK4, CDK6, and CDK7, leading to cell cycle arrest in G0/1 and G2 (Sedlacek et al, 2001). In addition, flavopiridol disrupts the CDK9/cyclin T complex resulting in reduced phosphorylation of the carboxyl-terminal domain of RNA Pol-II and subsequent inhibition of mRNA synthesis (Chao et al, 2000). Consequently, short lived anti-

Novel Therapeutic Targets in ALL Therapy 177

Armstrong SA, Staunton JE, Silverman LB, Pieters R, de Boer ML, Minden MD, Sallan SE,

Armstrong SA, Kung AL, Mabon ME, Silverman LB, Stam RW, den Boer ML, Pieters R,

Armstrong SA, Mabon ME, Silverman LB, Li AH, Gribben JG, Fox EA, Sallan SE, Korsmeyer

Barr RD, Sala A. Osteonecrosis in children and adolescents with cancer. (2008). Pediatric

Bernhard D, Tinhofer I, Tonko M, Hubl H, Ausserlechner MJ, Greil R, Kofler R, Csordas A.

Bernhard D, Skvortsov S, Tinhofer I, Hubl H, Greil R, Csordas A, Kofler R. (2001). Inhibition

Borthakur G, Kantarjian H, Daley G, Talpaz M, O'Brien S, Garcia-Manero G, Giles F, Faderl

Broxmeyer HE, Orschell CM, Clapp DW, Hangoc G, Cooper S, Plett PA, Liles WC, Li XX,

Brown P, Levis M, Shurtleff S, Campana D, Downing J, Small D. (2005). FLT3 inhibition

Byrd JC, Marcucci G, Parthun MR, Xiao JJ, Klisovic RB, Moran M, Lin TS, Liu SJ, Sklenar AR,

Bueso-Ramos C, Xu YL, McDonnell TJ, Brisbay S, Pierce S, Kantarjian H, Rosner G, Garcia-

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pp.346-352

pp.1307-1318

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apoptotic proteins such as MCL-1 and BCL-2 and those that are cell cycle dependent such as cyclin D1 are depleted. This could provide an explanation for increased apoptosis observed following exposure to flavopiridol.

Pre-clinical studies have demonstrated the efficacy of flavopiridol in primary ALL samples. A phase I clinical trial of flavopiridol followed by cytarabine and mitoxantrone in patients with relapsed or refractory adult ALL has shown moderate biological and clinical benefits (Karp et al, 2005). Dose-limiting toxicities include sustained neutropenia, diarrhoea and mucositis. As suggested by a CLL clinical trial, a more sustained schedule of flavopiridol might be needed to improve the clinical response rate, without increasing neutropenia as the dose-limiting toxicity.

## **15. Conclusion**

The future of treatment optimization resides in exploring the molecular pathways involved in the pathogenesis of leukemia and in understanding the pharmacogenomic factors of the host. If successful, new protein products can be generated that are administered as targeted therapy with a narrow therapeutic window. A primary example of such therapy is given by the inhibitor of tyrosine kinase imatinib, which is the first anti-cancer agent that specifically targets the genetic defect underlying Philadelphia positive ALL. Although imatinib is currently implemented in the treatment of BCR/ABL positive ALL and has improved overall survival, its use as single agent should not be overestimated. In fact, imatinib is targeting a specific molecular pathway that regulates gene transcription, cell proliferation or survival, but not the principal genetic lesion. Accordingly, imatinib produces relatively short-lived remission in Ph+ ALL as single agent, but has the potential to improve survival, when combined with current chemotherapy. Similarly, it is likely that the key to the successful use of other molecular targeted therapies will be in the careful combination of novel agents with traditional chemotherapeutic regimes and/or with other molecularly targeted agents. Indeed many agents, including imatinib, flavopiridol and oblimersen are being tested in combination with standard chemotherapy, while imatinib is also being combined with inhibitors of farnesyl transferase, HDAC, cyclin dependent kinases, HSP90 and BCL-2. Ultimately, these combinations need to be based on a thorough understanding of the molecular pathways driving disease in each patient. Additionally, pharmacogenomic information will be essential for the development of optimal effective treatment regimes with minimal side effects.

## **16. Acknowledgment**

This work was supported by the Children's Cancer Aid Society Tirol and Vorarlberg.

## **17. References**


apoptotic proteins such as MCL-1 and BCL-2 and those that are cell cycle dependent such as cyclin D1 are depleted. This could provide an explanation for increased apoptosis observed

Pre-clinical studies have demonstrated the efficacy of flavopiridol in primary ALL samples. A phase I clinical trial of flavopiridol followed by cytarabine and mitoxantrone in patients with relapsed or refractory adult ALL has shown moderate biological and clinical benefits (Karp et al, 2005). Dose-limiting toxicities include sustained neutropenia, diarrhoea and mucositis. As suggested by a CLL clinical trial, a more sustained schedule of flavopiridol might be needed to improve the clinical response rate, without increasing neutropenia as the

The future of treatment optimization resides in exploring the molecular pathways involved in the pathogenesis of leukemia and in understanding the pharmacogenomic factors of the host. If successful, new protein products can be generated that are administered as targeted therapy with a narrow therapeutic window. A primary example of such therapy is given by the inhibitor of tyrosine kinase imatinib, which is the first anti-cancer agent that specifically targets the genetic defect underlying Philadelphia positive ALL. Although imatinib is currently implemented in the treatment of BCR/ABL positive ALL and has improved overall survival, its use as single agent should not be overestimated. In fact, imatinib is targeting a specific molecular pathway that regulates gene transcription, cell proliferation or survival, but not the principal genetic lesion. Accordingly, imatinib produces relatively short-lived remission in Ph+ ALL as single agent, but has the potential to improve survival, when combined with current chemotherapy. Similarly, it is likely that the key to the successful use of other molecular targeted therapies will be in the careful combination of novel agents with traditional chemotherapeutic regimes and/or with other molecularly targeted agents. Indeed many agents, including imatinib, flavopiridol and oblimersen are being tested in combination with standard chemotherapy, while imatinib is also being combined with inhibitors of farnesyl transferase, HDAC, cyclin dependent kinases, HSP90 and BCL-2. Ultimately, these combinations need to be based on a thorough understanding of the molecular pathways driving disease in each patient. Additionally, pharmacogenomic information will be essential for the development of optimal effective treatment regimes

This work was supported by the Children's Cancer Aid Society Tirol and Vorarlberg.

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**9** 

**Aberrant Proliferative and Apoptotic Pathways** 

Agostino Tafuri1, Michele Milella2, Stefano Iacovelli1, Fabiana De Cave1, Chiara Gregorj3, Paola Bergamo2, Andrea Miele1, Roberto Licchetta1, Marina Konopleva4, James A. McCubrey5, Alberto M. Martelli6,7, Robin Foà1, Maria Teresa Petrucci1 and Maria Rosaria Ricciardi1\*

Adult acute lymphoblastic leukemia (ALL) is characterized by a high relapse rate, with the majority of patients developing chemo-resistance and ultimately dying of the disease with a 5-year survival rate of 40% (Faderl et al., 2010). Significant advances, however, have been made in cases carrying the acquired genetic alteration BCR-ABL (ALL-Ph+) targeted by tyrosine-kinase inhibitors (Ottmann & Pfeifer, 2009). Therefore, several studies have recently been carried out to look for additional, therapeutically exploitable, genetic lesions. Aberrant activation of signal transduction pathways (STP) implicated in proliferation and survival mechanisms are generally involved in leukemogenesis and drug resistance (Zhao et al., 2010). Genes in the PI3K/PTEN/AKT/mTOR, RAS/RAF/MEK/ERK, and Jak/STAT pathways are frequently mutated and their expression is often altered in hematopoietic malignancies, including ALL (McCubrey et al., 2011; Steelman et al. 2008). In addition, deregulation of survival mechanisms may confer chemo-resistance to leukemic cells, particularly involving alterations of the Bcl-2 signaling cascade, which may represent one of the most important, potentially druggable, pathways for therapeutic intervention in ALL. Starting from our studies on chemo-resistance in ALL, particularly on multidrug resistance (MDR1) expression and prognostic significance, in this chapter we will illustrate the major pathways aberrantly activated in ALL - PI3K/PTEN/AKT/mTOR, RAF/RAS/MEK/ERK, and the Bcl-2 family of proteins - with the ultimate goal of summarizing novel targets for

*\* 1Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Italy 2Medical Oncology A, Regina Elena National Cancer Institute, Rome, Italy* 

*4Section of Molecular Hematology and Therapy, Department of Leukemia, University of Texas, M.D. Anderson* 

*5Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville,* 

*6Department of Human Anatomical Sciences, University of Bologna, Bologna, Italy & 7IGM-CNR, c/o IOR,* 

*3Hematology Unit, Campus Bio-Medico University Hospital, Rome, Italy* 

*Cancer Center, Houston, Texas, USA* 

**1. Introduction** 

*NC, USA* 

*Bologna, Italy* 

**in Acute Lymphoblastic Leukemia (ALL):** 

**Molecular Therapies to Overcome** 

**Chemo-Resistance** 

