**1. Chronic lymphocytic leukemia**

One of the most prevalent types of leukemia is chronic lymphocytic leukemia (CLL). Leukemia is a type of cancer. Cancer means too little apoptosis of body cells. In the case of cancer, cells have mutations that prevent them from undergoing apoptosis. It is a general belief that CLL is an indolent disease associated with a prolonged (i.e., 10–20 years) clinical course, and unrelated causes to CLL lead to death. But it is true only for less than 30% of cases [1]. By convention, the history of chronic lymphocytic leukemia begins in 1845, but it could be said to have started when the first white cells, "the globuli albicanates," were noted by Joseph Lieutaud in

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1749. During the intervening years, many events have aided in our understanding of the etiology and treatment of CLL. In his discussion of the history of CLL, Rai [2] found it informative to define three eras: (1) the recognition of CLL as a clinical entity, 1845–1924; (2) initial clinical investigations, 1924–1973; and (3) the modern era, 1973–2002.

Overexpression of Bcl-2<sup>1</sup> and Fas-inhibitory molecules such as TOSO is the principle mechanism of apoptosis resistance in CLL cells. CLL lymphocytes are clonal B-cells arrested in the B-cell differentiation pathway at some intermediate stage between the pre-B-cell and mature B-cell, perhaps in the "activated, antigen-experienced" B-cell subset. Phenotypic features of B-cell CLL (B-CLL) lymphocytes are [3–5] (1) extremely low levels of surface membrane immunoglobulin (often abbreviated as SmIg or sIg), (2) expression of one or more B-cellassociated antigens (like CD19, CD20, CD21, CD23, and CD24) [6, 7], and (3) expression of CD5, a T-cell-associated antigen.

Until the early 1980s, it was not possible to study chromosomal abnormalities in CLL because of the inadequate number of metaphases induced by available techniques. Certain genetic abnormalities have been associated with patient outcomes. Patients with complex genomic changes appear to have more aggressive disease [8]. The most frequently observed abnormalities were trisomy 12 and 14q+. The cytogenetic abnormalities appear to be restricted to B-cells in B-CLL [9]. In two studies of patients with CLL using fluorescence in situ hybridization (FISH) techniques, chromosomal abnormalities were noted in 69–82% of the patients, with abnormalities of chromosomes 11, 12, and 13 being most commonly seen.

For establishment of BCR-signaling pathways independent of antigen ligation, CD19 is an important surface marker. It has an important role in regulation and amplification of signal transduction via lyn [13]. ZaP-70 is another tyrosine kinase that has important role in BCR

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Apoptosis is a kind of cell death. Extrinsic pathway of apoptosis triggers by death receptors. In CLL, they are CD95/Fas and trail (tumor-necrosis factor-related apoptosis-inducing ligand). After ligation by ligands (like CD40L), these receptors directly feed into a caspase cascade and lead to cell death [16]. The intrinsic pathway, or mitochondrial pathway, is regulated by the balance between antiapoptotic and proapoptotic members of the Bcl-2 family [15]. "BH3-only" proteins (e.g., Bim, Bid, Bmf, Puma, Bad, and noxa) are another class of Bcl-2

Non-death-transmitted signals drive from developmental cues or sensor platforms. Developmental cues like Bim-dependent B-cell killing upon BCR cross-linking [17] and sensor platforms like the DNA damage sensor network involving the ATM (ataxia telangiectasia mutated) and p53 tumor suppressors, which prominently determine survival and treatment outcomes in CLL [15]. Currently, one of the therapeutic strategies that kill CLL cells is the DNA damage response via p53 that leads to a dominant cell-death signal via Puma [18, 19]. A major problem encountered with this strategy is that a number of patients with CLL harbor defects in the DNA damage machinery that leads to deactivation of the pathway. The challenge thus seems to be

Another therapeutic strategy is the exploitation of CD95 signaling. But it seems to be restricted, as systemic CD95 triggering leads to fulminant liver toxicity [20]. The role of trail receptor

signaling. When syk is not expressed, it can partially restore BCR signaling [14].

*1.1.2. Aberrant apoptotic signaling pathway*

**Figure 1.** The role of BCR signaling in the biology of CLL [15].

family proteins which can modify this balance (**Figure 2**).

to bypass such resistance and produce p53-independent cell death [15].

### **1.1. Pathophysiology**

Chronic lymphocytic leukemia is a monoclonal disease of mature-appearing lymphocytes that accumulate in blood, lymph nodes, spleen, liver, and bone marrow. Most cases (>95%) are characterized by monoclonal lymphocytes expressing normal B-cell surface proteins including immunoglobulin (Ig), CD19, and CD20 and aberrantly expressing CD5, a protein normally found on T-cells. A small minority (<5%) of cases are of T-cell origin, expressing T-cell surface markers such as CD3 and CD4 or CD8. These T-cell leukemias are not uncommon in individuals with ataxia telangiectasia. The molecular biology of T-cell lymphocytic leukemia is distinct from that of B-cell CLL [10].

Molecular and cellular mechanisms of CLL can be divided into two parts.

### *1.1.1. B-cell receptor-signaling pathways*

B-cell receptor (BCR)-signaling pathways are triggered with or without antigen ligation in CLL. After antigenic BCR triggering downstream signaling of the BCR is dominated by the kinases lyn and syk, which transduce survival and antiapoptotic signals [11]. In CLL, the elevated expression of antiapoptotic Mcl-1, which leads to increased survival of malignant cells, occurred by prolonged activation of the MEK/ERK2 and Pi3K/AKT<sup>3</sup> pathways and with AKT after BCR signaling (**Figure 1**) [12].

<sup>1</sup> B-cell CLL Lymphoma 2.

<sup>2</sup> Mitogen-activated protein kinase/extracellular signal-regulated kinase.

<sup>3</sup> Phosphatidylinositol-3-kinase and protein kinase B.

**Figure 1.** The role of BCR signaling in the biology of CLL [15].

1749. During the intervening years, many events have aided in our understanding of the etiology and treatment of CLL. In his discussion of the history of CLL, Rai [2] found it informative to define three eras: (1) the recognition of CLL as a clinical entity, 1845–1924; (2) initial clinical

nism of apoptosis resistance in CLL cells. CLL lymphocytes are clonal B-cells arrested in the B-cell differentiation pathway at some intermediate stage between the pre-B-cell and mature B-cell, perhaps in the "activated, antigen-experienced" B-cell subset. Phenotypic features of B-cell CLL (B-CLL) lymphocytes are [3–5] (1) extremely low levels of surface membrane immunoglobulin (often abbreviated as SmIg or sIg), (2) expression of one or more B-cellassociated antigens (like CD19, CD20, CD21, CD23, and CD24) [6, 7], and (3) expression of

Until the early 1980s, it was not possible to study chromosomal abnormalities in CLL because of the inadequate number of metaphases induced by available techniques. Certain genetic abnormalities have been associated with patient outcomes. Patients with complex genomic changes appear to have more aggressive disease [8]. The most frequently observed abnormalities were trisomy 12 and 14q+. The cytogenetic abnormalities appear to be restricted to B-cells in B-CLL [9]. In two studies of patients with CLL using fluorescence in situ hybridization (FISH) techniques, chromosomal abnormalities were noted in 69–82% of the patients,

Chronic lymphocytic leukemia is a monoclonal disease of mature-appearing lymphocytes that accumulate in blood, lymph nodes, spleen, liver, and bone marrow. Most cases (>95%) are characterized by monoclonal lymphocytes expressing normal B-cell surface proteins including immunoglobulin (Ig), CD19, and CD20 and aberrantly expressing CD5, a protein normally found on T-cells. A small minority (<5%) of cases are of T-cell origin, expressing T-cell surface markers such as CD3 and CD4 or CD8. These T-cell leukemias are not uncommon in individuals with ataxia telangiectasia. The molecular biology of T-cell lymphocytic

B-cell receptor (BCR)-signaling pathways are triggered with or without antigen ligation in CLL. After antigenic BCR triggering downstream signaling of the BCR is dominated by the kinases lyn and syk, which transduce survival and antiapoptotic signals [11]. In CLL, the elevated expression of antiapoptotic Mcl-1, which leads to increased survival of malignant

and Pi3K/AKT<sup>3</sup>

pathways and with

with abnormalities of chromosomes 11, 12, and 13 being most commonly seen.

Molecular and cellular mechanisms of CLL can be divided into two parts.

and Fas-inhibitory molecules such as TOSO is the principle mecha-

investigations, 1924–1973; and (3) the modern era, 1973–2002.

Overexpression of Bcl-2<sup>1</sup>

90 Cytotoxicity

CD5, a T-cell-associated antigen.

**1.1. Pathophysiology**

leukemia is distinct from that of B-cell CLL [10].

cells, occurred by prolonged activation of the MEK/ERK2

Mitogen-activated protein kinase/extracellular signal-regulated kinase.

*1.1.1. B-cell receptor-signaling pathways*

AKT after BCR signaling (**Figure 1**) [12].

Phosphatidylinositol-3-kinase and protein kinase B.

1

2

3

B-cell CLL Lymphoma 2.

For establishment of BCR-signaling pathways independent of antigen ligation, CD19 is an important surface marker. It has an important role in regulation and amplification of signal transduction via lyn [13]. ZaP-70 is another tyrosine kinase that has important role in BCR signaling. When syk is not expressed, it can partially restore BCR signaling [14].

### *1.1.2. Aberrant apoptotic signaling pathway*

Apoptosis is a kind of cell death. Extrinsic pathway of apoptosis triggers by death receptors. In CLL, they are CD95/Fas and trail (tumor-necrosis factor-related apoptosis-inducing ligand). After ligation by ligands (like CD40L), these receptors directly feed into a caspase cascade and lead to cell death [16]. The intrinsic pathway, or mitochondrial pathway, is regulated by the balance between antiapoptotic and proapoptotic members of the Bcl-2 family [15]. "BH3-only" proteins (e.g., Bim, Bid, Bmf, Puma, Bad, and noxa) are another class of Bcl-2 family proteins which can modify this balance (**Figure 2**).

Non-death-transmitted signals drive from developmental cues or sensor platforms. Developmental cues like Bim-dependent B-cell killing upon BCR cross-linking [17] and sensor platforms like the DNA damage sensor network involving the ATM (ataxia telangiectasia mutated) and p53 tumor suppressors, which prominently determine survival and treatment outcomes in CLL [15].

Currently, one of the therapeutic strategies that kill CLL cells is the DNA damage response via p53 that leads to a dominant cell-death signal via Puma [18, 19]. A major problem encountered with this strategy is that a number of patients with CLL harbor defects in the DNA damage machinery that leads to deactivation of the pathway. The challenge thus seems to be to bypass such resistance and produce p53-independent cell death [15].

Another therapeutic strategy is the exploitation of CD95 signaling. But it seems to be restricted, as systemic CD95 triggering leads to fulminant liver toxicity [20]. The role of trail receptor

**Figure 2.** The interaction between Bcl-2 family member proteins [15].

targeting is currently under development. CD40 signaling may also have a positive effect on conventional therapy. It has been shown that CD154 (CD40L) application was able to induce the p53-related transcription factor p73, leading to a sensitization of p53-deficient CLL cells to conventional therapeutics such as fludarabine [21].

*2.1.1. Extrinsic pathway*

*2.1.2. Intrinsic pathway*

the cytosol [27].

"Death receptors" transmit apoptotic signals after ligation with specific ligands in extrinsic pathway. Death receptors belong to a superfamily, including TNFR-1, Fas/CD95, and the TRAIL receptors DR-4 and DR-5 [24]. Caspase-8 is the hallmark of this pathway. It is activated by a complex named death-inducing-signaling complex (DISC). Activated death receptor recruited adapter molecules like FADD (Fas-associated protein with death domain) or TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). These adapter molecules form the DISC (**Figure 4**). Caspase-8 then cleave and activate other caspases resulting in cell death. These types of cells, which have the capacity to induce such direct and

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In this pathway, the signal does not come from death receptors. In this case, the signal amplified via mitochondria-dependent apoptotic pathways. Bcl-2 family member, Bid, is cleaved by caspase-8 (tBid) and translocates to the mitochondria. tBid in concert with the proapoptotic Bcl-2 family members Bax (Bcl-2-associated x) and Bak (Bcl-2 homologous antagonist/ killer) induces the release of cytochrome C and other mitochondrial proapoptotic factors into

Cytosolic cytochrome C binds to monomeric Apaf-1 (apoptotic protease-activating factor 1) which then oligomerizes to assemble the apoptosome that triggers the activation of the

mainly caspase-dependent apoptosis pathways, were classified to type I cells [25].

**Figure 3.** Hallmarks of the apoptotic and necrotic cell-death process. Modified from [23].

A number of approaches have been taken to directly modulate the core components of the Bcl-2 cell-death machinery. The Bcl-2 antisense molecule oblimersen is the most advanced agent in clinical testing. "BH3-mimetics" and "pan-Bcl-2 family antagonists" can mimic the BH3 domain of BH3-only death-inducing proteins and are thought to liberate BH3-only proteins from the inhibition by antiapoptotic Bcl-2 proteins, thus making them effective killers.
