Using bionformatic tools Cimmino et al. (Cimmino et al., 2005) found that miR-15a and miR-16- sequences share complementary homology with BCL2 (B-cell CLL/lymphoma 2) mRNA sequence. Bcl2 is an anti-apoptotic protein that is highly expressed in CLL as in other types of human cancer, including leukemias, lymphomas, and carcinomas (Sanchez-Beato et al., 2003)

As the BLC2 gene is overexpressed and that deletions or down-regulation of the miR-15a and miR-16-1 cluster have been reported to occur in the same proportion in CLL samples, it was proposed that miR-15a and miR-16-1 produce their anti-tumorigenic effect by targeting the BCL2 gene (Aqeilan et al., 2010; Cimmino et al., 2005). Its function has also been assayed *in vitro* and *in vivo*. In immunocompromised nude mice, ectopic expression of miR-15a/16-1 was found to cause dramatic suppression of tumorigenicity of MEG-01 leukemic cells exhibiting a loss of endogenous expression of miR-15a/ 16-1 (Calin et al., 2008) On the other hand, and besides the results obtained by Cimmino et al, other authors found no correlation between BCL2 and mir15a and miR-16-1 in cancer (Fulci et al., 2007; Hanlon et al., 2009; Klein et al., 2010; Linsley et al., 2007). Klein et al. (Klein & Dalla-Favera, 2010) confirmed the observation that the miR-15a/miR-16-1 locus controls B cell expansion by modulating proliferation, rather than influencing survival via regulation of BCL2 i*n vivo*.

Nowadays, different methodological approaches revealed that CCND1 (encoding cyclin D1) and WNT3A mRNAs which promote several prostate tumorigenic features, could also be directly affected by miR-15a/16-1 (Bonci et al., 2008), like other cancer genes such as MCL1, ETS1 and PDCD6IP that directly or indirectly affect cell cycle and apoptosis.

Fig. 2. Localization of miR15/16 cluster on human chromosome 13.

The 13q14 tumor suppressor locus deleted in CLL contain DLEU 2 gene where MIR15a/MIR16/b is located inside, between exon 4 and 5.

## **2.2 The miR-34 family**

124 Chronic Lymphocytic Leukemia

cancers ((Ward et al., 2011)). This miR-17-92 cluster consists of seven miRNAs: miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b-1 and miR-92-1 transcribed from the MIR17 Host Gene (MIR17HG) at locus 13q31.3, and members of this cluster are thought to coexpress with the proto-oncogenic transcription factor MYC (He et al., 2005). Several profiling studies show that expression of members of the miR-17-92 cluster is altered to some degree in CLL (Calin et al., 2004a; Fulci et al., 2007). Thus, advances in miRNA biology will likely have an increasing influence in the diagnosis, prognosis and treatment of human cancers, including CLL. In addition, it is discussed below the discovery of the oncogenic and tumor-suppressive properties of various miRNAs that come up with the possibility of

The association of miR-15a/16-1 in the pathogenesis of CLL was not only the first implication of miRNAs in this disease, but also in cancer. The authors of this finding were Calin et al. who determined that miR-15a/16-1 were located in 13q14.3, and were either

As this chromosomal region is also deleted in other types of cancer (mantle cell lymphoma, multiple myeloma, DLBCL, mature T-cell lymphoma, and solid tumors) conducted several authors to search for the presence of tumor suppressor genes in this region. A region of more than 1.0 Mb was sequenced, but none of the protein genes identified were found implicated in the initiation or progression of CLL (Calin et al., 2005).  On this way, a minimal deleted region (MDR) that contains two tightly linked miRNAs, miR-15a and mirR-16-1 was identified (Calin et al., 2002; Lagos-Quintana et al., 2001; Migliazza et al., 2000) (Figure 2). Recent studies demonstrated that other genes located in the same region (deleted in leukemia gene, DLEU 2 and 7), may also function as tumor

In addition to chromosomal abnormalities, other mechanisms as mutation, loss of heterozygosis, epigenetic deregulation and defects in the miRNA biogenesis machinery, could also contribute to deregulation of miRNA expression (Deng et al., 2008). In this respect, Calin et al. demonstrated that mutations in miR-16-1 could be responsible for the altered expression observed in CLL patients compared with subjects without cancer

Using bionformatic tools Cimmino et al. (Cimmino et al., 2005) found that miR-15a and miR-16- sequences share complementary homology with BCL2 (B-cell CLL/lymphoma 2) mRNA sequence. Bcl2 is an anti-apoptotic protein that is highly expressed in CLL as in other types of human cancer, including leukemias, lymphomas, and carcinomas (Sanchez-Beato et

As the BLC2 gene is overexpressed and that deletions or down-regulation of the miR-15a and miR-16-1 cluster have been reported to occur in the same proportion in CLL samples, it was proposed that miR-15a and miR-16-1 produce their anti-tumorigenic effect by targeting the BCL2 gene (Aqeilan et al., 2010; Cimmino et al., 2005). Its function has also been assayed

deleted or down-regulated in 68% of patients with CLL (Calin et al., 2002).

miRNA therapy for cancer in the near future.

suppressors (Palamarchuk et al., 2010).

(Aqeilan et al., 2010).

al., 2003)

**2.1 The miR-15a/16-1**

The miR-34 family has been implicated in several solid and hematological malignancies. The three members of the miR-34 family are encoded by two different genes: miR-34a is encoded by its own transcript, whereas miR-34b and miR-34c share a common primary transcript (Auer et al., 2007; Cole et al., 2008).

Deregulation of the miR-34a and miR-34b/c expression by chromosomal deletion and/or epigenectic inactivation, presumably occurs during tumorigenesis (Hermeking, 2010).

In the case of miR-34a, chromosomal deletion of the region in which it resides (locus 1p36) and epigenetic inactivation were identified in tumors. Moreover the epigenetic inactivation

Contribution of microRNAs to CLL Biology and Their Potential as New Biomarkers 127

Downregulation of miR-29 members have been reported in various human cancers including aggressive chronic lymphocytic leukemia (Garzon et al., 2009). They were demonstrated to have a tumor suppressor activity by targeting several oncogenes as the Tcell leukemia/ lymphoma 1 (TCL1), the BCL2 family member MCL1, the cyclin-dependent kinase CDK6, and the transcriptional repressor Yy1 (Mott et al., 2007; Pekarsky et al., 2006;

Fabbri et al, showed that members of the miR-29 family target also the *de novo* DNA methyltransferases (DNMTs) and can reactivate tumor suppressor genes (Fabbri et al., 2007). Thus, loss of miR-29 family members could cause epigenetic changes associated with CLL

Besides of the antitumoral activity of miR-29 overexpression by inhibition of cell proliferation, it was also observed that miR-29 up-regulation could also initiate acute and chronic leukemias in animal models. Santanam and co-workers (Santanam et al., 2010) developed a transgenic mice overexpressing mir-29 in mouse B cells. They reported that miR-29 is overexpressed in indolent CLL compared with normal B cells. In contrast, miR29

In addition, it was demonstrated that miR-29 inversely correlated with levels of TCL-1 in CLL patients. TCL-1 is a coactivator of AKT, an oncogene that inhibits apoptotic patways and has a critical role in the regulation of many relevant cell processes including cell proliferation and cell death (Santanam et al., 2010; Vasilatou et al., 2010). Taken together, these data led authors to hypothesize that TCL1 is mostly not expressed in indolent CLL and probably does not play an important role in this disease stage. Indeed, miR-29 overexpression is not sufficient to initiate aggressive CLL. In contrast, up-regulation of TCL1 is a critical event in the pathogenesis of the aggressive form of CLL and because miR-29 is down-regulated in aggressive CLL (compared with the indolent form), it contributes to the development of an aggressive phenotype (Santanam et al., 2010). As mir-29b, mir-181b acts as tumor-suppresor in aggressive CLL by targeting also the TCL-1 oncogene, there is an inverse correlation between TCL1 and miR-181 expression at different stages of B-cell

The high expression of these miRNAs is associated with expression of unmutated IgVH and high expression of ZAP-70, indicating an aggressive CLL phenotype (Vasilatou et al.,

As was previously described in this chapter, several miRNAs have been reported to be

The miR-155, for example, was reported overexpressed in solid tumors, including lung, colon, and breast cancer as well as in both acute myeloid leukemia and CLL (Garzon et al., 2009). Frenquelli et al. (Frenquelli et al., 2010) recently showed an inverse relationship between miR-221/222 and p27 expression and validated p27 as a functional target for miR-221/222 in CLL. Fulci and co-workers (Fulci et al., 2007) found an overexpression of miR-150, miR-223, miR-29b, and miR-29c in CLL patients with a mutated IgVH phenotype

**2.4 Other miRNAs potentially implicated in CLL pathogenesis**

compared to the patients with unmutated *IgVH* phenotypes.

differently expressed in B cells from CLL compared to normal B-cells.

Zhao et al. 2010).).

and other cancer types.

was down-regulated in aggressive CLL.

development (Pekarsky et al., 2006).

2010).

of miR-34a was identified in cell lines derived from some of the most common tumors and in primary melanoma. In addition, CpG methylation of miR-34b/c was also found in colorectal cancer, in oral squamous cell carcinoma and in malignant melanoma.

In the case of CLL, the variability of the mir-34a expression observed in patients is not precisely associated to the just mentioned process. It was demonstrated that members of the miR-34 family are direct p53 targets. MiR-34 genes are up-regulated by the tumor-suppressor protein p53, and their overexpression in turn causes senescence, apoptosis, or cell cycle arrest by regulating proteins such as BCL2, Cyclin D1, Cyclin E2, CDK4, and c-MYC Sirt-1, depending on the cell type (Corney et al., 2007; Ward et al., 2011). Further analysis effectively determined that miR-34a expression partially correlated with p53 status and patients with p53 mutations or deletions of 17p13.1, in general had lower miR-34a expression (Figure 3). However, in some patients decreased miR-34a was seen without p53 aberrations. These patients are homozygous for the single nucleotide polymorphism 309 (SNP309) in the intronic promoter of MDM2, a negative regulator of p53 (Asslaber et al., 2010). The down-regulation of miR-34 b/c is also related to a p53 inactivation and to one of the most characteristic chromosomal deletion in CLL: 11q23.1, where the miR-34 b/c cluster is located.

Fig. 3. MicroRNA-34 family regulation and targets involved in CLL. microRNA 34b (*miR-34b*)/microRNA 34c (*miR-34c*) cluster and miR-34a are regulated by tumor suppressor protein p53. After DNA damage or cellular stress, p53 is activated through ATM, ARF or ATR pathways and transactivates target genes including the miR-34 family members. The mature transcripts of the activated miR-34a/b/c induce either translation inhibition or cleavage of the indicated RNA targets.

## **2.3 The miR-29 and miR-181 in CLL**

The members of miR-29 family are arranged in two different loci; the miR-29b-1/miR-29a located at 7q32 and the miR-29b-2/miR-29c at 1q32.

of miR-34a was identified in cell lines derived from some of the most common tumors and in primary melanoma. In addition, CpG methylation of miR-34b/c was also found in

In the case of CLL, the variability of the mir-34a expression observed in patients is not precisely associated to the just mentioned process. It was demonstrated that members of the miR-34 family are direct p53 targets. MiR-34 genes are up-regulated by the tumor-suppressor protein p53, and their overexpression in turn causes senescence, apoptosis, or cell cycle arrest by regulating proteins such as BCL2, Cyclin D1, Cyclin E2, CDK4, and c-MYC Sirt-1, depending on the cell type (Corney et al., 2007; Ward et al., 2011). Further analysis effectively determined that miR-34a expression partially correlated with p53 status and patients with p53 mutations or deletions of 17p13.1, in general had lower miR-34a expression (Figure 3). However, in some patients decreased miR-34a was seen without p53 aberrations. These patients are homozygous for the single nucleotide polymorphism 309 (SNP309) in the intronic promoter of MDM2, a negative regulator of p53 (Asslaber et al., 2010). The down-regulation of miR-34 b/c is also related to a p53 inactivation and to one of the most characteristic chromosomal deletion in CLL:

Fig. 3. MicroRNA-34 family regulation and targets involved in CLL. microRNA 34b (*miR-34b*)/microRNA 34c (*miR-34c*) cluster and miR-34a are regulated by tumor suppressor protein p53. After DNA damage or cellular stress, p53 is activated through ATM, ARF or ATR pathways and transactivates target genes including the miR-34 family members. The mature transcripts of the activated miR-34a/b/c induce either translation inhibition or

The members of miR-29 family are arranged in two different loci; the miR-29b-1/miR-29a

colorectal cancer, in oral squamous cell carcinoma and in malignant melanoma.

11q23.1, where the miR-34 b/c cluster is located.

cleavage of the indicated RNA targets.

**2.3 The miR-29 and miR-181 in CLL**

located at 7q32 and the miR-29b-2/miR-29c at 1q32.

Downregulation of miR-29 members have been reported in various human cancers including aggressive chronic lymphocytic leukemia (Garzon et al., 2009). They were demonstrated to have a tumor suppressor activity by targeting several oncogenes as the Tcell leukemia/ lymphoma 1 (TCL1), the BCL2 family member MCL1, the cyclin-dependent kinase CDK6, and the transcriptional repressor Yy1 (Mott et al., 2007; Pekarsky et al., 2006; Zhao et al. 2010).).

Fabbri et al, showed that members of the miR-29 family target also the *de novo* DNA methyltransferases (DNMTs) and can reactivate tumor suppressor genes (Fabbri et al., 2007). Thus, loss of miR-29 family members could cause epigenetic changes associated with CLL and other cancer types.

Besides of the antitumoral activity of miR-29 overexpression by inhibition of cell proliferation, it was also observed that miR-29 up-regulation could also initiate acute and chronic leukemias in animal models. Santanam and co-workers (Santanam et al., 2010) developed a transgenic mice overexpressing mir-29 in mouse B cells. They reported that miR-29 is overexpressed in indolent CLL compared with normal B cells. In contrast, miR29 was down-regulated in aggressive CLL.

In addition, it was demonstrated that miR-29 inversely correlated with levels of TCL-1 in CLL patients. TCL-1 is a coactivator of AKT, an oncogene that inhibits apoptotic patways and has a critical role in the regulation of many relevant cell processes including cell proliferation and cell death (Santanam et al., 2010; Vasilatou et al., 2010). Taken together, these data led authors to hypothesize that TCL1 is mostly not expressed in indolent CLL and probably does not play an important role in this disease stage. Indeed, miR-29 overexpression is not sufficient to initiate aggressive CLL. In contrast, up-regulation of TCL1 is a critical event in the pathogenesis of the aggressive form of CLL and because miR-29 is down-regulated in aggressive CLL (compared with the indolent form), it contributes to the development of an aggressive phenotype (Santanam et al., 2010). As mir-29b, mir-181b acts as tumor-suppresor in aggressive CLL by targeting also the TCL-1 oncogene, there is an inverse correlation between TCL1 and miR-181 expression at different stages of B-cell development (Pekarsky et al., 2006).

The high expression of these miRNAs is associated with expression of unmutated IgVH and high expression of ZAP-70, indicating an aggressive CLL phenotype (Vasilatou et al., 2010).

## **2.4 Other miRNAs potentially implicated in CLL pathogenesis**

As was previously described in this chapter, several miRNAs have been reported to be differently expressed in B cells from CLL compared to normal B-cells.

The miR-155, for example, was reported overexpressed in solid tumors, including lung, colon, and breast cancer as well as in both acute myeloid leukemia and CLL (Garzon et al., 2009). Frenquelli et al. (Frenquelli et al., 2010) recently showed an inverse relationship between miR-221/222 and p27 expression and validated p27 as a functional target for miR-221/222 in CLL. Fulci and co-workers (Fulci et al., 2007) found an overexpression of miR-150, miR-223, miR-29b, and miR-29c in CLL patients with a mutated IgVH phenotype compared to the patients with unmutated *IgVH* phenotypes.

Contribution of microRNAs to CLL Biology and Their Potential as New Biomarkers 129

conditions that would normally degrade most RNAs. This stability can be partially explained by the discovery of lipoprotein complexes, including small membrane vesicles of endocytic origin called exosomes or microvesicles (30-1000 nm in diameter), containing miRNAs, mRNAs and proteins. Exosomes can be formed through inward budding of endosomal membranes, giving rise to intracellular multi vesicular bodies (MVBs) that later fuse with the plasma membrane, releasing the exosomes to the exterior (Figure 1). In contrast, microvesicles are originated by outward budding from the plasma membrane. The utility of miRNAs as diagnostic markers will be increased because samples of human

Exosomes containing miRNAs were found in blood (Hunter et al., 2008), but also in other types of body fluids such as saliva and urine (Michael et al.). Importantly, exosomes represent a newly discovered mechanism by which donor cells can communicate and influence the gene expression of recipient cells. These findings were first demonstrated by the same study that discovered miRNAs in exosomes, in which mouse mast cell exosomes were added to human mast cells, leading to a subsequent detection of mouse proteins in the

Tumor-specific miRNAs were first discovered in the serum of patients with diffuse large B cell lymphoma where high levels of miR-21 correlated with improved relapse-free survival (Lawrie et al., 2009). In an elegant experiment in a xenograft mouse prostate cancer model, the presence of circulating tumor-derived miR-629 and miR-660 was confirmed in blood with 100% sensitivity and specificity (Mitchell et al., 2008). In addition to showing that both serum and plasma samples are adequate for measuring specific miRNA levels, the investigators reported that by measuring serum levels of miR-141, they were able to distinguish patients with prostate cancer from healthy subjects. Since then, over 100 studies have assessed the potential use of serum or plasma miRNAs as biomarkers in different types of cancer (Cortez et al., 2011). In a comprehensive study, miRNA-expression profiles were identified in the sera of patients with lung or colorectal cancer, or diabetes by extracting miRNA from the serum (Chen et al., 2008). Although a unique expression profile of serum miRNAs was identified for each cancer type, an overlap was found in the profiles of specimens from all diseases analyzed in the study, including diabetes. In addition, this study also showed that miRNA-expression profiles differed between the serum and blood cells of lung cancer patients, while similar miRNA-expression profiles were seen in the serum and blood cells of healthy controls. These findings suggest that tumor-specific miRNAs in serum are derived not only from circulating blood cells but also cancer cells. An actualized compendium of relevant circulating miRNAs with potential as biomarkers for

Because most current approaches to cancer screening are invasive and unable to detect early-stage disease, it is important to determine when tumor-related circulating miRNAs can be detected in the bloodstream during disease development between other important factors. In this respect, it was recently reported that miRNAs can be also sensitive biomarkers for CLL, because certain extracellular miRNAs are present in CLL patient plasma at levels significantly different from healthy controls and from patients affected by other hematologic malignancies (Klein et al., 2010). Moreover, in this study the authors also determined that level of circulating miR-20a correlates reliably with diagnosis-to treatment time and miR-483–5p elevated almost six fold in plasma of CLL patients is predicted to target the mediators of IL-15 that induces CLL proliferation and drives CLL cell migration

plasma and serum can be obtained in a less invasive manner than can tissues.

cancer was recently reviewed by Cortez et al. (Cortez et al., 2011).

human cells (Valadi et al., 2007).

## **3. MicroRNA signatures and prognostic miRNAs in CLL**

In addition to the well known molecular factors (mutational status of *IgVH*, expression levels of ZAP-70 or β2-microglobulin and expression of CD38+) and chromosomal abnormalities (11q, 13q, 17p deletions) associated to the clinical course of CLL, the molecular basis for these correlations was largely unknown. However, several studies identified miRNAs that could act as prognostic indicators in CLL, typically by correlating expression levels of these miRNAs with previously mentioned established prognostic indicators (Ward et al., 2011).

One of the first works that described this relationship was published by Calin et al. in 2005. They described a signature of 13 miRNAs (miR- 15a, miR-195, miR-221, miR-23b, miR-155, miR-223, miR29a-2, miR- 24-1, miR-29b-2, miR-146, miR-16-1, miR-16-2, and miR-29c) differentially expressed between unmutated IgVH/ ZAP70+ and mutated IgVH/ZAP70 CLL patients. Further studies, using different molecular techniques, also confirmed the mentioned correlation (Fulci et al., 2007; Marton et al., 2008; Rossi et al., 2010; Stamatopoulos et al., 2009). They reveled also the implication of miR-150 and miR-181 as prognostic factors.

It was recently reported that the deregulation of miR-181b expression can be monitored throughout the course of the disease, which correlate with the overexpression of 4 genes with great significance in CLL biology and other cancers (i.e. MCL1, TCL1, BCL2 and AID (Visone et al., 2009).

An interesting link between the classical prognostic molecular and chromosomal markers of CLL and two groups of defined miRNAs have been recently described by Fabri et al. (Fabbri et al., 2011). They found that miR-15a/miR-16-1 cluster is associated with reduced expression levels of TP53, miR-34a, miR-34b, and miR-34c and increased protein levels of ZAP70. Low expression levels of ZAP70 have been found to be positively correlated with survival in patients with the indolent course of CLL carrying 13q deletions, and it was associated with increased TP53 levels and transactivation of miR-34b/ miR-34c. In this way, the authors found a microRNA/TP53 feedback circuitry associated with the pathogenesis and prognosis of CLL and revealed a new pathogenic model for human CLL.

As mentioned above, miR-29 expression correlated with the clinical course of CLL. Low expression of miR-29c was associated to patients who had a poor prognosis and shorter treatment-free survival as well as reduced overall survival (Calin & Croce, 2009).

Thus, advances in the identification of miRNAs as CLL biomarkers, as well as the mode of regulation of gene expression and the pathway in which they are involved, should provide a useful prognostic tool for patient stratification and more appropriated treatments.

## **3.1 Circulating miRNAs as novel biomarkers in CLL**

Nowadays, after the identification of extracellular circulating microRNAs in plasma microvesicles or ribonucleoprotein complexes (Cortez et al., 2011; Valadi et al., 2007), they have become an attractive source of new nucleic acid-based biomarkers. Today miRNAs are considered powerful markers for early detection, prognosis, response, and recurrence surveillance of different cancers because they are widely involved in oncogenesis (Taylor & Gercel-Taylor, 2008). The diagnostic and prognostic potential of miRNAs as cancer biomarkers relies mainly on their high stability and resistance to storage handling. It has been consistently shown that serum miRNAs remain stable after being subjected to severe

In addition to the well known molecular factors (mutational status of *IgVH*, expression levels of ZAP-70 or β2-microglobulin and expression of CD38+) and chromosomal abnormalities (11q, 13q, 17p deletions) associated to the clinical course of CLL, the molecular basis for these correlations was largely unknown. However, several studies identified miRNAs that could act as prognostic indicators in CLL, typically by correlating expression levels of these miRNAs with previously mentioned established prognostic indicators (Ward et al., 2011). One of the first works that described this relationship was published by Calin et al. in 2005. They described a signature of 13 miRNAs (miR- 15a, miR-195, miR-221, miR-23b, miR-155, miR-223, miR29a-2, miR- 24-1, miR-29b-2, miR-146, miR-16-1, miR-16-2, and miR-29c) differentially expressed between unmutated IgVH/ ZAP70+ and mutated IgVH/ZAP70 CLL patients. Further studies, using different molecular techniques, also confirmed the mentioned correlation (Fulci et al., 2007; Marton et al., 2008; Rossi et al., 2010; Stamatopoulos et al., 2009). They reveled also the implication of miR-150 and miR-181 as prognostic factors. It was recently reported that the deregulation of miR-181b expression can be monitored throughout the course of the disease, which correlate with the overexpression of 4 genes with great significance in CLL biology and other cancers (i.e. MCL1, TCL1, BCL2 and AID

An interesting link between the classical prognostic molecular and chromosomal markers of CLL and two groups of defined miRNAs have been recently described by Fabri et al. (Fabbri et al., 2011). They found that miR-15a/miR-16-1 cluster is associated with reduced expression levels of TP53, miR-34a, miR-34b, and miR-34c and increased protein levels of ZAP70. Low expression levels of ZAP70 have been found to be positively correlated with survival in patients with the indolent course of CLL carrying 13q deletions, and it was associated with increased TP53 levels and transactivation of miR-34b/ miR-34c. In this way, the authors found a microRNA/TP53 feedback circuitry associated with the pathogenesis

As mentioned above, miR-29 expression correlated with the clinical course of CLL. Low expression of miR-29c was associated to patients who had a poor prognosis and shorter

Thus, advances in the identification of miRNAs as CLL biomarkers, as well as the mode of regulation of gene expression and the pathway in which they are involved, should provide a

Nowadays, after the identification of extracellular circulating microRNAs in plasma microvesicles or ribonucleoprotein complexes (Cortez et al., 2011; Valadi et al., 2007), they have become an attractive source of new nucleic acid-based biomarkers. Today miRNAs are considered powerful markers for early detection, prognosis, response, and recurrence surveillance of different cancers because they are widely involved in oncogenesis (Taylor & Gercel-Taylor, 2008). The diagnostic and prognostic potential of miRNAs as cancer biomarkers relies mainly on their high stability and resistance to storage handling. It has been consistently shown that serum miRNAs remain stable after being subjected to severe

and prognosis of CLL and revealed a new pathogenic model for human CLL.

treatment-free survival as well as reduced overall survival (Calin & Croce, 2009).

useful prognostic tool for patient stratification and more appropriated treatments.

**3.1 Circulating miRNAs as novel biomarkers in CLL**

**3. MicroRNA signatures and prognostic miRNAs in CLL**

(Visone et al., 2009).

conditions that would normally degrade most RNAs. This stability can be partially explained by the discovery of lipoprotein complexes, including small membrane vesicles of endocytic origin called exosomes or microvesicles (30-1000 nm in diameter), containing miRNAs, mRNAs and proteins. Exosomes can be formed through inward budding of endosomal membranes, giving rise to intracellular multi vesicular bodies (MVBs) that later fuse with the plasma membrane, releasing the exosomes to the exterior (Figure 1). In contrast, microvesicles are originated by outward budding from the plasma membrane. The utility of miRNAs as diagnostic markers will be increased because samples of human plasma and serum can be obtained in a less invasive manner than can tissues.

Exosomes containing miRNAs were found in blood (Hunter et al., 2008), but also in other types of body fluids such as saliva and urine (Michael et al.). Importantly, exosomes represent a newly discovered mechanism by which donor cells can communicate and influence the gene expression of recipient cells. These findings were first demonstrated by the same study that discovered miRNAs in exosomes, in which mouse mast cell exosomes were added to human mast cells, leading to a subsequent detection of mouse proteins in the human cells (Valadi et al., 2007).

Tumor-specific miRNAs were first discovered in the serum of patients with diffuse large B cell lymphoma where high levels of miR-21 correlated with improved relapse-free survival (Lawrie et al., 2009). In an elegant experiment in a xenograft mouse prostate cancer model, the presence of circulating tumor-derived miR-629 and miR-660 was confirmed in blood with 100% sensitivity and specificity (Mitchell et al., 2008). In addition to showing that both serum and plasma samples are adequate for measuring specific miRNA levels, the investigators reported that by measuring serum levels of miR-141, they were able to distinguish patients with prostate cancer from healthy subjects. Since then, over 100 studies have assessed the potential use of serum or plasma miRNAs as biomarkers in different types of cancer (Cortez et al., 2011). In a comprehensive study, miRNA-expression profiles were identified in the sera of patients with lung or colorectal cancer, or diabetes by extracting miRNA from the serum (Chen et al., 2008). Although a unique expression profile of serum miRNAs was identified for each cancer type, an overlap was found in the profiles of specimens from all diseases analyzed in the study, including diabetes. In addition, this study also showed that miRNA-expression profiles differed between the serum and blood cells of lung cancer patients, while similar miRNA-expression profiles were seen in the serum and blood cells of healthy controls. These findings suggest that tumor-specific miRNAs in serum are derived not only from circulating blood cells but also cancer cells. An actualized compendium of relevant circulating miRNAs with potential as biomarkers for cancer was recently reviewed by Cortez et al. (Cortez et al., 2011).

Because most current approaches to cancer screening are invasive and unable to detect early-stage disease, it is important to determine when tumor-related circulating miRNAs can be detected in the bloodstream during disease development between other important factors. In this respect, it was recently reported that miRNAs can be also sensitive biomarkers for CLL, because certain extracellular miRNAs are present in CLL patient plasma at levels significantly different from healthy controls and from patients affected by other hematologic malignancies (Klein et al., 2010). Moreover, in this study the authors also determined that level of circulating miR-20a correlates reliably with diagnosis-to treatment time and miR-483–5p elevated almost six fold in plasma of CLL patients is predicted to target the mediators of IL-15 that induces CLL proliferation and drives CLL cell migration

Contribution of microRNAs to CLL Biology and Their Potential as New Biomarkers 131

increasingly recognized in almost all fields of biological and biomedical sciences. Over the last years, microRNAs have emerged as new actors in cancer biology as well as new diagnostic biomarkers and therapeutic targets in human cancer. This review attempts to briefly outline our current knowledge on the abnormalities of miRNAs found to be associated with CLL pathogenesis and possible mechanisms underlying the roles of miRNAs in CLL initiation and progression and to provide a perspective insight in using miRNAs as new CLL biomarkers. Nowadays, microRNAs are proposed as new sensitive, non-invasive and inexpensive biomarkers in CLL for early stage detection, predict outcome, monitor treatment and screen for disease recurrence. Understanding the significance of microRNAs in the pathogenesis of CLL represents an important dimension in miRNA research as it may lead to the development of miRNA-based novel therapeutic strategies or diagnostic/prognostic biomarkers. Additionally, microRNAs should also afford new

Aqeilan, R.I., Calin, G.A., and Croce, C.M. (2010). miR-15a and miR-16-1 in cancer:

Asslaber, D., Pinon, J.D., Seyfried, I., Desch, P., Stocher, M., Tinhofer, I., Egle, A., Merkel, O.,

Auer, R.L., Riaz, S., and Cotter, F.E. (2007). The 13q and 11q B-cell chronic lymphocytic

Bonci, D., Coppola, V., Musumeci, M., Addario, A., Giuffrida, R., Memeo, L., D'Urso, L.,

Calin, G.A., Cimmino, A., Fabbri, M., Ferracin, M., Wojcik, S.E., Shimizu, M., Taccioli, C.,

Calin, G.A., and Croce, C.M. (2009). Chronic lymphocytic leukemia: interplay between

Calin, G.A., Dumitru, C.D., Shimizu, M., Bichi, R., Zupo, S., Noch, E., Aldler, H., Rattan, S.,

Calin, G.A., Liu, C.G., Sevignani, C., Ferracin, M., Felli, N., Dumitru, C.D., Shimizu, M.,

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discovery, function and future perspectives. Cell death and differentiation *17*, 215-

and Greil, R. (2010). microRNA-34a expression correlates with MDM2 SNP309 polymorphism and treatment-free survival in chronic lymphocytic leukemia. Blood

leukaemia-associated regions derive from a common ancestral region in the

Pagliuca, A., Biffoni, M., Labbaye, C.*, et al.* (2008). The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nature medicine

Zanesi, N., Garzon, R., Aqeilan, R.I.*, et al.* (2008). MiR-15a and miR-16-1 cluster functions in human leukemia. Proceedings of the National Academy of Sciences of

Keating, M., Rai, K.*, et al.* (2002). Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America *99*, 15524-15529. Calin, G.A., Ferracin, M., Cimmino, A., Di Leva, G., Shimizu, M., Wojcik, S.E., Iorio, M.V.,

Visone, R., Sever, N.I., Fabbri, M.*, et al.* (2005). A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. The New

Cimmino, A., Zupo, S., Dono, M.*, et al.* (2004a). MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proceedings of the National

avenues for exploring innovative pathways in CLL pathogenesis.

zebrafish. British journal of haematology *137*, 443-453.

the United States of America *105*, 5166-5171.

England journal of medicine *353*, 1793-1801.

**6. References**

220.

*115*, 4191-4197.

*14*, 1271-1277.

and infiltration. Although most of the miRNA–mRNA interactions are yet to be fully validated, the roles of these plasma miRNAs in CLL present intriguing biological questions with medically significant implications (Moussay et al., 2011; Ward et al., 2011).

## **4. MiRNAs as predictors of CLL responses to therapy**

## **4.1 Chemotherapy resistance**

In spite of the existence of effective treatments for patients suffering CLL, present therapeutic regimens are not totally effective and additional therapies are required.

Recently fludarabine-refractory CLL was linked to patients with p53 deletion and low miR-34a expression. It was previously mentioned that miR-34a is positively regulated by p53. Low miR-34a expression levels were statistically significantly associated with impaired DNA damage response, p53 mutations, and fludarabine-refractory CLL either with or without p53 deletion (Calin & Croce, 2009).

In addition, patients with resistant disease exhibited increased miR-181a and decreased miR-29a expression as consequence of their regulation by the myelocytomatosis viral oncogene homolog (MYC), which is increased in patients with fludarabine-resistant disease (Moussay et al., 2010).

These are the first identified miRNAs associated in the complex network of molecules associated to drug resistance and sensitivity in patients with CLL.

## **4.2 Therapy**

According to the novel miRNAs described as biomarkers of prognosis and treatment in CLL, one of the most expecting use of them is the possible utility in the therapy of CLL.

The advantage to use these molecules for therapy, compared to other RNA inhibition strategies, relies on the fact that they are not only tiny and simple, but also they can target more than one mRNA relevant for the pathway of the disease (Calin & Croce, 2009).

Different chemical modification as are 2′-O-methylphosphorothioate oligonucleotides, locked nucleic acid and miRNA sponge are currently being used to improve the blocking capacity and half-life of miRNA. In addition Liposome–oligonucleotide Complexes, among other strategies are been improved for the delivery, specificity and reduction of toxicity of miRNAs.

As for miR-122, which seems to be effective for the treatment hepatitis C in ongoing clinical trials (Lanford et al.), the potential use of miRNAs in CLL treatment has only recently been envisioned and were only used *in vitro*.

## **5. Concluding remarks**

In this chapter, we have analyzed the potential contribution of microRNAs as novel players and biomarkers in CLL pathogenesis.

The role of miRNAs as key regulatory molecules that control a wide variety of fundamental cellular processes, such as proliferation, death, differentiation, motility, invasiveness, etc., is increasingly recognized in almost all fields of biological and biomedical sciences. Over the last years, microRNAs have emerged as new actors in cancer biology as well as new diagnostic biomarkers and therapeutic targets in human cancer. This review attempts to briefly outline our current knowledge on the abnormalities of miRNAs found to be associated with CLL pathogenesis and possible mechanisms underlying the roles of miRNAs in CLL initiation and progression and to provide a perspective insight in using miRNAs as new CLL biomarkers. Nowadays, microRNAs are proposed as new sensitive, non-invasive and inexpensive biomarkers in CLL for early stage detection, predict outcome, monitor treatment and screen for disease recurrence. Understanding the significance of microRNAs in the pathogenesis of CLL represents an important dimension in miRNA research as it may lead to the development of miRNA-based novel therapeutic strategies or diagnostic/prognostic biomarkers. Additionally, microRNAs should also afford new avenues for exploring innovative pathways in CLL pathogenesis.

## **6. References**

130 Chronic Lymphocytic Leukemia

and infiltration. Although most of the miRNA–mRNA interactions are yet to be fully validated, the roles of these plasma miRNAs in CLL present intriguing biological questions

In spite of the existence of effective treatments for patients suffering CLL, present

Recently fludarabine-refractory CLL was linked to patients with p53 deletion and low miR-34a expression. It was previously mentioned that miR-34a is positively regulated by p53. Low miR-34a expression levels were statistically significantly associated with impaired DNA damage response, p53 mutations, and fludarabine-refractory CLL either with or

In addition, patients with resistant disease exhibited increased miR-181a and decreased miR-29a expression as consequence of their regulation by the myelocytomatosis viral oncogene homolog (MYC), which is increased in patients with fludarabine-resistant disease

These are the first identified miRNAs associated in the complex network of molecules

According to the novel miRNAs described as biomarkers of prognosis and treatment in CLL, one of the most expecting use of them is the possible utility in the therapy of CLL.

The advantage to use these molecules for therapy, compared to other RNA inhibition strategies, relies on the fact that they are not only tiny and simple, but also they can target more than one mRNA relevant for the pathway of the disease (Calin & Croce,

Different chemical modification as are 2′-O-methylphosphorothioate oligonucleotides, locked nucleic acid and miRNA sponge are currently being used to improve the blocking capacity and half-life of miRNA. In addition Liposome–oligonucleotide Complexes, among other strategies are been improved for the delivery, specificity and reduction of toxicity of

As for miR-122, which seems to be effective for the treatment hepatitis C in ongoing clinical trials (Lanford et al.), the potential use of miRNAs in CLL treatment has only recently been

In this chapter, we have analyzed the potential contribution of microRNAs as novel players

The role of miRNAs as key regulatory molecules that control a wide variety of fundamental cellular processes, such as proliferation, death, differentiation, motility, invasiveness, etc., is

therapeutic regimens are not totally effective and additional therapies are required.

with medically significant implications (Moussay et al., 2011; Ward et al., 2011).

**4. MiRNAs as predictors of CLL responses to therapy**

associated to drug resistance and sensitivity in patients with CLL.

**4.1 Chemotherapy resistance**

(Moussay et al., 2010).

**4.2 Therapy**

2009).

miRNAs.

without p53 deletion (Calin & Croce, 2009).

envisioned and were only used *in vitro*.

and biomarkers in CLL pathogenesis.

**5. Concluding remarks**

Contribution of microRNAs to CLL Biology and Their Potential as New Biomarkers 133

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**7**

*Ghent University*

*Belgium*

**The Biological Relevance of ZAP-70 in CLL**

Initially, CLL was considered as a homogeneous disease caused by the accumulation of functionally incompetent B lymphocytes carrying no mutations in the immunoglobulin heavy chain variable (IgVH) genes. However, further studies by Schroeder and Dighiero suggested that IgVH genes may be mutated in CLL (Schroeder & Dighiero 1994). This report changed the general view of CLL and gained even more significance when the mutational status of the IgVH genes was linked to the prognosis of the patients in two independent studies (Damle et al. 1999, Hamblin et al. 1999). These studies showed for the first time that patients with CLL cells expressing unmutated IgVH genes presented with a more aggressive disease and shorter survival than those with cells carrying mutated IgVH genes. Determination of the mutational status of the IgVH genes in CLL patients became of great interest, but even today remains difficult to carry out in most medical centers since the technique is laborious, expensive and time-consuming. Therefore many efforts have been made to identify possible surrogate markers with the same prognostic value as the mutation status. In 2001, two independent groups published their studies in which they compared the gene expression profiles for IgVH mutated versus unmutated CLL cells. Because, the description of the two groups of CLL with markedly different prognosis prompted the idea that CLL consisted of two different entities originating from either naive or memory cells, it was unexpected that only a few differentially expressed genes were found (Rosenwald et al. 2001, Klein et al. 2001). Among them, ZAP-70 appeared to be one of the most significant (Rosenwald et al. 2001). Subsequently, the correlation of ZAP-70 expression with the mutational status of the IgVH genes was assessed in larger series of CLL patients, where ZAP-70 was mostly found expressed in unmutated CLL (reviewed in (Van Bockstaele et al. 2009)). Further clinical studies revealed that ZAP-70 was also an independent prognostic marker (Bosch et al. 2006). There were several attempts to standardize the determination of ZAP-70 expression by flow cytometry (Crespo et al. 2003, Wang et al. 2011, Van Bockstaele et al. 2006). Although, a successful standardized procedure was put forward by the European Research Initiative on CLL (ERIC), the determination and interpretation of ZAP-

70 remains difficult (Letestu et al. 2006) and optimalization is still ongoing.

The zeta-chain associated protein kinase with a molecular weight of 70 kDa, ZAP-70, is a member of the Syk family kinases predominantly involved in T cell receptor (TCR) signaling initiation and subsequent T cell activation (Chan et al. 1992). The ZAP-70 gene is located on

**1. Introduction**

**2. Structure of ZAP-70**

Valerie Pede, Ans Rombout, Bruno Verhasselt and Jan Philippé

regulators in B-cell chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America *108*, 6573-6578.

## **The Biological Relevance of ZAP-70 in CLL**

Valerie Pede, Ans Rombout, Bruno Verhasselt and Jan Philippé *Ghent University Belgium*

## **1. Introduction**

134 Chronic Lymphocytic Leukemia

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leukemia and improves disease risk stratification. Blood *113*, 5237-5245. Taylor, D.D., and Gercel-Taylor, C. (2008). MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecologic oncology *110*, 13-21. Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J.J., and Lotvall, J.O. (2007).

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transitioning from laboratory-based investigation to clinical application. Cancer

Croce, C.M., and Pekarsky, Y. (2010). 13q14 deletions in CLL involve cooperating

Alder, H., Liu, C.G., Rassenti, L.*, et al.* (2006). Tcl1 expression in chronic lymphocytic

Lerner, S., Barron, L.L., Rassenti, L.Z.*, et al.* (2010). microRNA fingerprinting of CLL patients with chromosome 17p deletion identify a miR-21 score that stratifies early

Alder, H., Rassenti, L., Kipps, T.*, et al.* (2010). Chronic lymphocytic leukemia modeled in mouse by targeted miR-29 expression. Proceedings of the National

L., Heimann, P., Martiat, P., Bron, D., and Lagneaux, L. (2009). microRNA-29c and microRNA-223 down-regulation has in vivo significance in chronic lymphocytic

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of

role of microRNAs in normal and malignant hematopoiesis. European journal of

Ferracin, M., Palatini, J., Balatti, V.*, et al.* (2009). Karyotype-specific microRNA

Stredney, D., Spitzner, J.*, et al.* (2001). A draft annotation and overview of the

Rezania, D., Dalton, W.S.*, et al*. (2010) microRNA expression profile and identification of miR-29 as a prognostic marker and pathogenetic factor by Initially, CLL was considered as a homogeneous disease caused by the accumulation of functionally incompetent B lymphocytes carrying no mutations in the immunoglobulin heavy chain variable (IgVH) genes. However, further studies by Schroeder and Dighiero suggested that IgVH genes may be mutated in CLL (Schroeder & Dighiero 1994). This report changed the general view of CLL and gained even more significance when the mutational status of the IgVH genes was linked to the prognosis of the patients in two independent studies (Damle et al. 1999, Hamblin et al. 1999). These studies showed for the first time that patients with CLL cells expressing unmutated IgVH genes presented with a more aggressive disease and shorter survival than those with cells carrying mutated IgVH genes. Determination of the mutational status of the IgVH genes in CLL patients became of great interest, but even today remains difficult to carry out in most medical centers since the technique is laborious, expensive and time-consuming. Therefore many efforts have been made to identify possible surrogate markers with the same prognostic value as the mutation status. In 2001, two independent groups published their studies in which they compared the gene expression profiles for IgVH mutated versus unmutated CLL cells. Because, the description of the two groups of CLL with markedly different prognosis prompted the idea that CLL consisted of two different entities originating from either naive or memory cells, it was unexpected that only a few differentially expressed genes were found (Rosenwald et al. 2001, Klein et al. 2001). Among them, ZAP-70 appeared to be one of the most significant (Rosenwald et al. 2001). Subsequently, the correlation of ZAP-70 expression with the mutational status of the IgVH genes was assessed in larger series of CLL patients, where ZAP-70 was mostly found expressed in unmutated CLL (reviewed in (Van Bockstaele et al. 2009)). Further clinical studies revealed that ZAP-70 was also an independent prognostic marker (Bosch et al. 2006). There were several attempts to standardize the determination of ZAP-70 expression by flow cytometry (Crespo et al. 2003, Wang et al. 2011, Van Bockstaele et al. 2006). Although, a successful standardized procedure was put forward by the European Research Initiative on CLL (ERIC), the determination and interpretation of ZAP-70 remains difficult (Letestu et al. 2006) and optimalization is still ongoing.

## **2. Structure of ZAP-70**

The zeta-chain associated protein kinase with a molecular weight of 70 kDa, ZAP-70, is a member of the Syk family kinases predominantly involved in T cell receptor (TCR) signaling initiation and subsequent T cell activation (Chan et al. 1992). The ZAP-70 gene is located on

The Biological Relevance of ZAP-70 in CLL 137

For a long time ZAP-70 expression was considered to be restricted to T and NK cells, until it was found to be expressed as well in leukemic cells of CLL patients. Later studies have revealed ZAP-70 expression in a subset of normal tonsillar and splenic B cells (Cutrona et al. 2006, Nolz et al. 2005) and in bone marrow pro-B cells (Crespo et al. 2006, Guillaume et al. 2005). Besides the occurrence of ZAP-70 in a subset of CLL patients it was also found in a number of other human B cell tumors like some cases of mantle cell lymphoma, splenic marginal zone lymphoma, B-ALL and Burkitt lymphoma (Crespo et al. 2006, Guillaume et al. 2005, Admirand et al. 2004, Chiaretti et al. 2006, Scielzo et al. 2006, Sup et al. 2004, Orchard et al. 2005). A correlation was described between maturation of tumor cells and expression of ZAP-70, ZAP-70 being more expressed in the more mature cases with IgM, higher CD20 expression and pre-B rather than pro-B phenotype (Chiaretti et al. 2006) .

In 2005, Corcoran et al. showed that there was an association between the methylation status of the ZAP-70 gene and expression status of ZAP-70 protein (Corcoran et al. 2005). It is well established that abnormal methylation is frequent in malignancy. Hypermethylation of CpG islands within gene promoters results in gene silencing, while hypomethylaion may cause genomic instability or the upregulation of gene expression (Herman & Baylin 2003). Specifically, they found that the majority of cases expressing ZAP-70 protein lacked methylation in the intron 1-exon 2 boundery region of the ZAP-70 gene. This region was found unmethylated in circulating ZAP-70 expressing T cells, but methylated in ZAP-70 negative normal B cells.  But the absence of an association between ZAP-70 expression

ZAP-70 expression in B cells may also be regulated by the heat shock protein (Hsp)90. Hsp90 is a molecular chaperone that catalyses the conformational maturation of a large number of signaling proteins in cancer that are collectively described as 'clients'. In advanced tumours, it exists in an activated form complexed with other molecular chaperones, whereas in normal tissue it is present in a latent, uncomplexed state (Kamal et al. 2003). Using inhibitors, Castro et al. have demonstrated that ZAP-70 is an Hsp90 client

Ligation of the TCR triggers a cascade of intracellular signals that culminate in cytokine gene expression, proliferation, and the execution of T cell effector functions. After engagement of the TCR with a peptide bound to a major histocompatibility complex molecule, a signaling cascade is activated by the sequential activation of two families of PTKs. First of all, members of the Src family, Lck and FynT, initiate this process by phosphorylating tyrosine residues at the ITAMs of the CD3 ζ subunits. Once the ITAMs in the receptor cytoplasmic tails have been phosphorylated, they can recruit the next player in

**3. Expression of ZAP-70**

**3.1 Expression of ZAP-70 in T and B cells**

**3.2 Regulation of ZAP-70 expression in CLL cells**

and methylation status indicates that other factors must play a role.

protein in tumour cells, but not in T cells (Castro et al. 2005).

**4. ZAP-70 in lymphocyte receptor signaling**

**4.1 ZAP-70 in T cell signaling**

chromosome 2q11.2 and is composed of 14 exons encoding a protein tyrosine kinase (PTK) made out of 619 amino acids building three functional domains; two Src homology 2 (SH2) domains arranged in tandem at the amino-terminus and a tyrosine kinase domain at the carboxy-terminus (Figure 1) (Au-Yeung et al. 2009).

*ID-A: interdomain A; ID-B: interdomain B; Y: Tyrosine residue*

Fig. 1. Zeta-chain-associated protein kinase 70 (ZAP-70) protein structure.

The two SH2 domains are separated by a linker region known as interdomain A (ID-A) and a linker region known as interdomain B (ID-B) connecting the SH2 domains to the kinase domain. SH2 domains are involved in the recruitment of ZAP-70 to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) on the CD3 ζ chain homodimers and both SH2 domains are responsible for ZAP-70 dependent signal transduction. The kinase domain of ZAP-70 contains two tyrosine residues, Tyr492 and Tyr493, which are phosphorylated by the Src tyrosine kinase, Lck, or autophosphorylated by ZAP-70 itself, after TCR engagement. Mutation of Tyr493 impairs ZAP-70 kinase activity, revealing a positive regulatory role of this residue, while mutation of Tyr492 increases kinase activity, indicating its inhibitory role (Wange et al. 1995). The tyrosine 474 of ZAP-70 is required for association with the Src Homology 2 domain Containing (Shc) adaptor protein and coupling of the activated TCR to the Ras/Raf/Erk pathways. Although the catalytic activity of ZAP-70 represents its major function, it is likely that interactions of ZAP-70 with other proteins contribute to its role in signal transduction. Interdomain B contains three tyrosines, Tyr292, Tyr315 and Tyr319, also representing important phosphorylation targets (Au-Yeung et al. 2009). These three tyrosines are phosphorylated by one of the Src family kinases, i.e. Lck or Fyn after TCR stimulation in T cells. Each of these binding sites, once phosphorylated, may bind to a different signaling molecule, conferring a role of ZAP-70 in the recruitment of additional signaling molecules to the antigen receptor complex. Tyr292 is able to bind the E3 ligase c-Cbl, which might be important for the turnover of the signaling complex (Rao et al. 2002). This implies a negative regulatory role for this tyrosine that however was recently shown to function also as a binding site for the p85 regulatory subunit of PI3K, indicating that it may also play a positive role in signal transduction. The two other tyrosines, Tyr315 and Tyr319 are positive regulators of ZAP-70 activity. Their phosphorylation is important for ZAP-70 activity, because it appears to prevent ZAP-70 from returning to an autoinhibited conformation (Au-Yeung et al. 2009). Phosphorylated Tyr315 is a binding site for the SH2 domain of Vav-1. This plays a key role in activation of the Rho family of GTPases involved in cytoskeleton remodeling after receptor stimulation (Wu et al. 1997, Sanchez-Aguilera et al. 2010). Tyr319 is a binding site for the C terminal SH2 domain of phospholipase C γ (PLCγ), and phosphorylation of Tyr319 is required for PLCγ phosphorylation and subsequent activation of downstream signals, such as calcium mobilization or Il-2 production (Williams et al. 1999).

## **3. Expression of ZAP-70**

136 Chronic Lymphocytic Leukemia

chromosome 2q11.2 and is composed of 14 exons encoding a protein tyrosine kinase (PTK) made out of 619 amino acids building three functional domains; two Src homology 2 (SH2) domains arranged in tandem at the amino-terminus and a tyrosine kinase domain at the

The two SH2 domains are separated by a linker region known as interdomain A (ID-A) and a linker region known as interdomain B (ID-B) connecting the SH2 domains to the kinase domain. SH2 domains are involved in the recruitment of ZAP-70 to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) on the CD3 ζ chain homodimers and both SH2 domains are responsible for ZAP-70 dependent signal transduction. The kinase domain of ZAP-70 contains two tyrosine residues, Tyr492 and Tyr493, which are phosphorylated by the Src tyrosine kinase, Lck, or autophosphorylated by ZAP-70 itself, after TCR engagement. Mutation of Tyr493 impairs ZAP-70 kinase activity, revealing a positive regulatory role of this residue, while mutation of Tyr492 increases kinase activity, indicating its inhibitory role (Wange et al. 1995). The tyrosine 474 of ZAP-70 is required for association with the Src Homology 2 domain Containing (Shc) adaptor protein and coupling of the activated TCR to the Ras/Raf/Erk pathways. Although the catalytic activity of ZAP-70 represents its major function, it is likely that interactions of ZAP-70 with other proteins contribute to its role in signal transduction. Interdomain B contains three tyrosines, Tyr292, Tyr315 and Tyr319, also representing important phosphorylation targets (Au-Yeung et al. 2009). These three tyrosines are phosphorylated by one of the Src family kinases, i.e. Lck or Fyn after TCR stimulation in T cells. Each of these binding sites, once phosphorylated, may bind to a different signaling molecule, conferring a role of ZAP-70 in the recruitment of additional signaling molecules to the antigen receptor complex. Tyr292 is able to bind the E3 ligase c-Cbl, which might be important for the turnover of the signaling complex (Rao et al. 2002). This implies a negative regulatory role for this tyrosine that however was recently shown to function also as a binding site for the p85 regulatory subunit of PI3K, indicating that it may also play a positive role in signal transduction. The two other tyrosines, Tyr315 and Tyr319 are positive regulators of ZAP-70 activity. Their phosphorylation is important for ZAP-70 activity, because it appears to prevent ZAP-70 from returning to an autoinhibited conformation (Au-Yeung et al. 2009). Phosphorylated Tyr315 is a binding site for the SH2 domain of Vav-1. This plays a key role in activation of the Rho family of GTPases involved in cytoskeleton remodeling after receptor stimulation (Wu et al. 1997, Sanchez-Aguilera et al. 2010). Tyr319 is a binding site for the C terminal SH2 domain of phospholipase C γ (PLCγ), and phosphorylation of Tyr319 is required for PLCγ phosphorylation and subsequent activation of downstream signals, such as calcium

carboxy-terminus (Figure 1) (Au-Yeung et al. 2009).

*ID-A: interdomain A; ID-B: interdomain B; Y: Tyrosine residue*

mobilization or Il-2 production (Williams et al. 1999).

Fig. 1. Zeta-chain-associated protein kinase 70 (ZAP-70) protein structure.

## **3.1 Expression of ZAP-70 in T and B cells**

For a long time ZAP-70 expression was considered to be restricted to T and NK cells, until it was found to be expressed as well in leukemic cells of CLL patients. Later studies have revealed ZAP-70 expression in a subset of normal tonsillar and splenic B cells (Cutrona et al. 2006, Nolz et al. 2005) and in bone marrow pro-B cells (Crespo et al. 2006, Guillaume et al. 2005). Besides the occurrence of ZAP-70 in a subset of CLL patients it was also found in a number of other human B cell tumors like some cases of mantle cell lymphoma, splenic marginal zone lymphoma, B-ALL and Burkitt lymphoma (Crespo et al. 2006, Guillaume et al. 2005, Admirand et al. 2004, Chiaretti et al. 2006, Scielzo et al. 2006, Sup et al. 2004, Orchard et al. 2005). A correlation was described between maturation of tumor cells and expression of ZAP-70, ZAP-70 being more expressed in the more mature cases with IgM, higher CD20 expression and pre-B rather than pro-B phenotype (Chiaretti et al. 2006) .

## **3.2 Regulation of ZAP-70 expression in CLL cells**

In 2005, Corcoran et al. showed that there was an association between the methylation status of the ZAP-70 gene and expression status of ZAP-70 protein (Corcoran et al. 2005). It is well established that abnormal methylation is frequent in malignancy. Hypermethylation of CpG islands within gene promoters results in gene silencing, while hypomethylaion may cause genomic instability or the upregulation of gene expression (Herman & Baylin 2003). Specifically, they found that the majority of cases expressing ZAP-70 protein lacked methylation in the intron 1-exon 2 boundery region of the ZAP-70 gene. This region was found unmethylated in circulating ZAP-70 expressing T cells, but methylated in ZAP-70 negative normal B cells.  But the absence of an association between ZAP-70 expression and methylation status indicates that other factors must play a role.

ZAP-70 expression in B cells may also be regulated by the heat shock protein (Hsp)90. Hsp90 is a molecular chaperone that catalyses the conformational maturation of a large number of signaling proteins in cancer that are collectively described as 'clients'. In advanced tumours, it exists in an activated form complexed with other molecular chaperones, whereas in normal tissue it is present in a latent, uncomplexed state (Kamal et al. 2003). Using inhibitors, Castro et al. have demonstrated that ZAP-70 is an Hsp90 client protein in tumour cells, but not in T cells (Castro et al. 2005).

## **4. ZAP-70 in lymphocyte receptor signaling**

## **4.1 ZAP-70 in T cell signaling**

Ligation of the TCR triggers a cascade of intracellular signals that culminate in cytokine gene expression, proliferation, and the execution of T cell effector functions. After engagement of the TCR with a peptide bound to a major histocompatibility complex molecule, a signaling cascade is activated by the sequential activation of two families of PTKs. First of all, members of the Src family, Lck and FynT, initiate this process by phosphorylating tyrosine residues at the ITAMs of the CD3 ζ subunits. Once the ITAMs in the receptor cytoplasmic tails have been phosphorylated, they can recruit the next player in

The Biological Relevance of ZAP-70 in CLL 139

In addition to its importance in T cell signaling through the TCR, ZAP-70 has also been shown to be required for effective signaling by the chemokine CXCL12 (Ticchioni et al. 2002). CXCL12 is an important chemoattractant for T cells, driving them from the blood to tissue sites where they are likely to encounter antigens. Binding of CXCL12 to its ligand CXCR4 results in phosphorylation of ZAP-70, which is required for activation of

Signaling in normal B cells occurs after crosslinking of the surface immunoglobulin molecules (Igα and Igβ) with an antigen. On clustering of the receptors, the receptorassociated Src-family protein tyrosine kinases Blk, FynB and Lyn are activated. These activated kinases phosphorylate the ITAMs in the receptor complex, which bind and activate the cytosolic protein kinase Syk. Subsequently, Syk phosphorylates other targets, inlucing the adaptor protein BLNK, which help to recruit Tec kinases that in turn phosphorylate and activate the enzyme PLCγ. Similar to T cell signaling, PLCγ together with Ras, leads to activation of the three main signaling pathways to the nucleus: activation of NFκB, NFAT and AP-1, initiating new gene transcription that results in the

*Schematic presentation of the intracellular signaling pathways initiated by cross-linking of B cell receptors (BCR)*

*by an antigen, leading to activation of three important transcription factors.*

Fig. 3. B cell signaling

downstream proteins, such as ERK and Vav-1, leading to T cell migration.

differentiation, proliferation and effector actions of B cells (Figure 3).

**4.2 ZAP-70 in CLL B cell signaling**

**4.2.1 Normal B cell signaling**

the signaling cascade, ZAP-70, belonging to the second family of the PTKs, the Syk family. ZAP-70 binds to the double phosphorylated ITAMs via its SH2 domains with high affinity. Recruitment of ZAP-70 to the ITAMs leads to ZAP-70 itself becoming phosphorylated. The subsequent activation of bound ZAP-70 by phosphorylation leads to three important signaling pathways. ZAP-70 phosphorylates the adaptor proteins linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein (SLP)-76, which in turn leads to the activation of PLCγ and the Rho-GTPase Ras. PLCγ cleaves phosphatidylinositol biphosphate (PIP2) to yield diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 increases intracellular Ca2+ concentration, activating the phosphatase calcineurin, and subsequently calcineurin activates a transcription factor, nuclear factor of activated T cells (NFAT). DAG and the increase in Ca2+ concentration activate protein kinase C (PKC). PKC will in turn activate the transcription factor, nuclear factor κappaB (NFκB). Another pathway involves the Rho-GTPase Ras, which activates a mitogen-activated protein (MAP) kinase cascade. This Ras-induced kinase cascade induces and activates Fos, a component of the activator protein (AP)-1 transcription factor. The three transcription factors NFκB, NFAT and AP-1 act to induce specific gene transcription, leading to cell proliferation and differentiation (Figure 2).

*Schematic presentation of the intracellular signaling pathways initiated by the T cell receptor complex (TCR) leading to activation of three important transcription factors.*

Fig. 2. ZAP-70 in T cell signaling

the signaling cascade, ZAP-70, belonging to the second family of the PTKs, the Syk family. ZAP-70 binds to the double phosphorylated ITAMs via its SH2 domains with high affinity. Recruitment of ZAP-70 to the ITAMs leads to ZAP-70 itself becoming phosphorylated. The subsequent activation of bound ZAP-70 by phosphorylation leads to three important signaling pathways. ZAP-70 phosphorylates the adaptor proteins linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein (SLP)-76, which in turn leads to the activation of PLCγ and the Rho-GTPase Ras. PLCγ cleaves phosphatidylinositol biphosphate (PIP2) to yield diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 increases intracellular Ca2+ concentration, activating the phosphatase calcineurin, and subsequently calcineurin activates a transcription factor, nuclear factor of activated T cells (NFAT). DAG and the increase in Ca2+ concentration activate protein kinase C (PKC). PKC will in turn activate the transcription factor, nuclear factor κappaB (NFκB). Another pathway involves the Rho-GTPase Ras, which activates a mitogen-activated protein (MAP) kinase cascade. This Ras-induced kinase cascade induces and activates Fos, a component of the activator protein (AP)-1 transcription factor. The three transcription factors NFκB, NFAT and AP-1 act to induce specific gene transcription, leading to cell proliferation and

*Schematic presentation of the intracellular signaling pathways initiated by the T cell receptor complex (TCR)*

*leading to activation of three important transcription factors.*

Fig. 2. ZAP-70 in T cell signaling

differentiation (Figure 2).

In addition to its importance in T cell signaling through the TCR, ZAP-70 has also been shown to be required for effective signaling by the chemokine CXCL12 (Ticchioni et al. 2002). CXCL12 is an important chemoattractant for T cells, driving them from the blood to tissue sites where they are likely to encounter antigens. Binding of CXCL12 to its ligand CXCR4 results in phosphorylation of ZAP-70, which is required for activation of downstream proteins, such as ERK and Vav-1, leading to T cell migration.

## **4.2 ZAP-70 in CLL B cell signaling**

## **4.2.1 Normal B cell signaling**

Signaling in normal B cells occurs after crosslinking of the surface immunoglobulin molecules (Igα and Igβ) with an antigen. On clustering of the receptors, the receptorassociated Src-family protein tyrosine kinases Blk, FynB and Lyn are activated. These activated kinases phosphorylate the ITAMs in the receptor complex, which bind and activate the cytosolic protein kinase Syk. Subsequently, Syk phosphorylates other targets, inlucing the adaptor protein BLNK, which help to recruit Tec kinases that in turn phosphorylate and activate the enzyme PLCγ. Similar to T cell signaling, PLCγ together with Ras, leads to activation of the three main signaling pathways to the nucleus: activation of NFκB, NFAT and AP-1, initiating new gene transcription that results in the differentiation, proliferation and effector actions of B cells (Figure 3).

*Schematic presentation of the intracellular signaling pathways initiated by cross-linking of B cell receptors (BCR) by an antigen, leading to activation of three important transcription factors.*

Fig. 3. B cell signaling

The Biological Relevance of ZAP-70 in CLL 141

tyrosine phosphorylation (Figure 1) not involved in the regulation of its catalytic activity, but regulating the recruitment of downstream signaling molecules and adaptor proteins. With the use of specific antibodies against Tyr292 constitutive phosphorylation of Tyr292 in CLL B cells was demonstrated. Although, BCR stimulation did not induce a significant change in the level of Tyr292 phosphorylation, suggesting that the interactions mediated by this tyrosine do not require additional phosphorylation. Instead, after IgM ligation an association between c-Cbl and PI3K and ZAP-70 (Gobessi et al. 2007) was observed. Additionally, an association between ZAP-70 and Shc, which requires phosphorylation at Tyr474, was detected. The same authors, in agreement with Chen et al. (2005), reported a stronger and prolonged BCR-induced phosphorylation of Syk, ERK and Akt in ZAP-70

To our knowledge, how ZAP-70 enhances BCR signaling in CLL cells is not known. It is conceivable that the capacity of ZAP-70 to enhance BCR signaling is not dependent upon its kinase activity. Two findings support this hypothesis. First, although Syk and ZAP-70 play similar roles in receptor signaling, Syk has approximately a 100-fold greater kinase activity in vitro than does ZAP-70 (Latour et al. 1996). Second, the kinase specific tyrosines are not phosphorylated in CLL cells (Gobessi et al. 2007). To address this, Chen et al. (Chen et al. 2007) transduced ZAP-70 negative cells with an adenovirus containing a ZAP-70 lacking kinase activity. In these cells, similar responses were observed after IgM stimulation compared to CLL cells naturally expressing ZAP-70 or cells being transduced with the wildtype ZAP-70. This unequivocally shows that ZAP-70 doesn't enhance BCR signaling by

Subsequently, Chen et al. as well as Gobessi et al. considered the hypothesis that ZAP-70 instead may enhance BCR signaling indirectly by enhancing the stability of activated Syk following ligation of the BCR. It is known that activated ZAP-70 and Syk are targets of the E3 ubiquitin ligase, c-Cbl, which in turn directs polyubiquitination and proteosomal degradation of the activated PTK (Fournel et al. 1996, Lupher et al. 1998, Rao et al. 2002). Possibly, the activated ZAP-70 may compete with activated Syk for binding to c-Cbl and thereby prolong the half-life of activated Syk. However, transduction of CLL cells with an adenovirus encoding a mutant form of ZAP-70 carrying a mutation at position 292, which abrogates the ability of the mutant form to interact with c-Cbl, also enhanced BCR signaling of ZAP-70 negative CLL cells. So, binding of C-cbl to Tyr292 (Gobessi et al. 2007) is unlikely to account for the capacity of ZAP-70 to enhance BCR signaling in CLL

Another explanation, proposed by both groups, is that ZAP-70 functions as an adaptor protein that facilitates the recruitment of other signaling molecules to the activated BCR. The associations with PI3K and Shc after IgM stimulation are noteworthy in this respect because these proteins are involved in the activation of Akt and ERK, respectively (Gobessi et al. 2007). In addition to this, examination of additional constructs encoding mutant forms of ZAP-70 that lacked a functional SH2 domain revealed that both SH2 domains are required for enhanced IgM signaling. This suggests that docking at the ITAM may be necessary for

ZAP-70 to have an effect on CLL Ig receptor signaling (Chen et al. 2007).

**4.2.4 Molecular interactions of ZAP-70 in CLL and downstream effects**

transfected CLL cells.

means of its intrinsic kinase activity.

B cells.

## **4.2.2 Interplay between ZAP-70, Syk and CLL B cells**

The role of ZAP-70 in proximal signaling after TCR activation and its homology with Syk, suggest that ZAP-70 may augment signaling through the BCR, thus, providing a biological explanation for the more aggressive clinical outcome of the ZAP-70 positive subgroup of patients (Chen et al. 2002). Nevertheless, the molecular mechanisms underlying the role of ZAP-70 in BCR signaling remain largely unknown. In Figure 4 the interactions between ZAP-70, Syk and BCR signaling is shown.

Studies, performed in the eighties and nineties had indicated that CLL cells varied in their capacity to respond to IgM ligation (Hivroz et al. 1986, Karray et al. 1987). In general, CLL cells were less able to respond to BCR stimulation than normal B cells (Lankester et al. 1995). Furtermore, several investigators found that, upon IgM crosslinking, CLL cells with unmutated Ig genes showed significantly increased levels of tyrosine-phosphorylated proteins, including Syk, compared to CLL cells that expressed mutated Ig receptors (Lanham et al. 2003, Chen et al. 2002). Remarkably, this was not related to differences in the levels of Syk protein found (Semichon et al. 1997).

In 2002, Chen et al. linked this phenomenon to the expression of ZAP-70. They found a greater increase in Syk tyrosine phosphorylation in cells expressing ZAP-70 compared to CLL cells lacking the protein. Consistent with this notion, they found that, besides Syk, also ZAP-70 itself undergoes tyrosine phosphorylation and complexes with the proteins of the BCR complex. At that time, two possible explanations were put forward. ZAP-70 might function to enhance BCR signaling by phosphorylation of specific motifs in CLL cells and thereby lower the threshold for Syk phosphorylation. Alternatively, expression of ZAP-70 in CLL cells might enhance the stability of phosphorylated Syk, allowing accumulation of the functional form of Syk in CLL cells.

In a subsequent study, the same group (Chen et al. 2005) examined samples that expressed unmutated IgVH genes without ZAP-70 or samples carrying mutated IgVH genes with ZAP-70, allowing them to examine the relative importance of ZAP-70 versus the IgVH mutational status in influencing the relative intensity of IgM signaling. A greater Ca2+ influx was seen in samples expressing ZAP-70 independently of the mutational status. Therefore, it was concluded that the induced increase in phosphorylated forms of Syk, BLNK or PLCγ and Ca2+ influx after IgM ligation appeared more closely associated with the expression of ZAP-70, rather than with the expression of unmutated IgVH. However, it could not be excluded that secondary factors other than ZAP-70 might also have been associated with the mutational status of the leukemic cells. To challenge this hypothesis it was required to transduce the CLL cells. Transduction studies revealed that expression of ZAP-70 in initially ZAP-70-negative CLL cells was sufficient to enhance IgM signaling.

## **4.2.3 ZAP-70 tyrosine phosphorylation**

Gobessi et al. explored in 2007 the phosphorylation of ZAP-70 in CLL cells (Gobessi et al. 2007). ZAP-70 was found to be inefficiently activated in CLL and relatively weakly activated compared to Syk. Phosphorylation of Tyr319 and Tyr493 could not be detected. Phosphorylation of these tyrosines are required for the catalytic activation of ZAP-70 (Figure 1). Phosphorylation of the corresponding tyrosines in Syk was readily detectable with the same phospho-specific antibodies (Gobessi et al. 2007). ZAP-70 contains additional sites of

The role of ZAP-70 in proximal signaling after TCR activation and its homology with Syk, suggest that ZAP-70 may augment signaling through the BCR, thus, providing a biological explanation for the more aggressive clinical outcome of the ZAP-70 positive subgroup of patients (Chen et al. 2002). Nevertheless, the molecular mechanisms underlying the role of ZAP-70 in BCR signaling remain largely unknown. In Figure 4 the interactions between

Studies, performed in the eighties and nineties had indicated that CLL cells varied in their capacity to respond to IgM ligation (Hivroz et al. 1986, Karray et al. 1987). In general, CLL cells were less able to respond to BCR stimulation than normal B cells (Lankester et al. 1995). Furtermore, several investigators found that, upon IgM crosslinking, CLL cells with unmutated Ig genes showed significantly increased levels of tyrosine-phosphorylated proteins, including Syk, compared to CLL cells that expressed mutated Ig receptors (Lanham et al. 2003, Chen et al. 2002). Remarkably, this was not related to differences in the

In 2002, Chen et al. linked this phenomenon to the expression of ZAP-70. They found a greater increase in Syk tyrosine phosphorylation in cells expressing ZAP-70 compared to CLL cells lacking the protein. Consistent with this notion, they found that, besides Syk, also ZAP-70 itself undergoes tyrosine phosphorylation and complexes with the proteins of the BCR complex. At that time, two possible explanations were put forward. ZAP-70 might function to enhance BCR signaling by phosphorylation of specific motifs in CLL cells and thereby lower the threshold for Syk phosphorylation. Alternatively, expression of ZAP-70 in CLL cells might enhance the stability of phosphorylated Syk, allowing accumulation of the

In a subsequent study, the same group (Chen et al. 2005) examined samples that expressed unmutated IgVH genes without ZAP-70 or samples carrying mutated IgVH genes with ZAP-70, allowing them to examine the relative importance of ZAP-70 versus the IgVH mutational status in influencing the relative intensity of IgM signaling. A greater Ca2+ influx was seen in samples expressing ZAP-70 independently of the mutational status. Therefore, it was concluded that the induced increase in phosphorylated forms of Syk, BLNK or PLCγ and Ca2+ influx after IgM ligation appeared more closely associated with the expression of ZAP-70, rather than with the expression of unmutated IgVH. However, it could not be excluded that secondary factors other than ZAP-70 might also have been associated with the mutational status of the leukemic cells. To challenge this hypothesis it was required to transduce the CLL cells. Transduction studies revealed that expression of ZAP-70 in initially

Gobessi et al. explored in 2007 the phosphorylation of ZAP-70 in CLL cells (Gobessi et al. 2007). ZAP-70 was found to be inefficiently activated in CLL and relatively weakly activated compared to Syk. Phosphorylation of Tyr319 and Tyr493 could not be detected. Phosphorylation of these tyrosines are required for the catalytic activation of ZAP-70 (Figure 1). Phosphorylation of the corresponding tyrosines in Syk was readily detectable with the same phospho-specific antibodies (Gobessi et al. 2007). ZAP-70 contains additional sites of

ZAP-70-negative CLL cells was sufficient to enhance IgM signaling.

**4.2.2 Interplay between ZAP-70, Syk and CLL B cells**

ZAP-70, Syk and BCR signaling is shown.

levels of Syk protein found (Semichon et al. 1997).

functional form of Syk in CLL cells.

**4.2.3 ZAP-70 tyrosine phosphorylation**

tyrosine phosphorylation (Figure 1) not involved in the regulation of its catalytic activity, but regulating the recruitment of downstream signaling molecules and adaptor proteins. With the use of specific antibodies against Tyr292 constitutive phosphorylation of Tyr292 in CLL B cells was demonstrated. Although, BCR stimulation did not induce a significant change in the level of Tyr292 phosphorylation, suggesting that the interactions mediated by this tyrosine do not require additional phosphorylation. Instead, after IgM ligation an association between c-Cbl and PI3K and ZAP-70 (Gobessi et al. 2007) was observed. Additionally, an association between ZAP-70 and Shc, which requires phosphorylation at Tyr474, was detected. The same authors, in agreement with Chen et al. (2005), reported a stronger and prolonged BCR-induced phosphorylation of Syk, ERK and Akt in ZAP-70 transfected CLL cells.

## **4.2.4 Molecular interactions of ZAP-70 in CLL and downstream effects**

To our knowledge, how ZAP-70 enhances BCR signaling in CLL cells is not known. It is conceivable that the capacity of ZAP-70 to enhance BCR signaling is not dependent upon its kinase activity. Two findings support this hypothesis. First, although Syk and ZAP-70 play similar roles in receptor signaling, Syk has approximately a 100-fold greater kinase activity in vitro than does ZAP-70 (Latour et al. 1996). Second, the kinase specific tyrosines are not phosphorylated in CLL cells (Gobessi et al. 2007). To address this, Chen et al. (Chen et al. 2007) transduced ZAP-70 negative cells with an adenovirus containing a ZAP-70 lacking kinase activity. In these cells, similar responses were observed after IgM stimulation compared to CLL cells naturally expressing ZAP-70 or cells being transduced with the wildtype ZAP-70. This unequivocally shows that ZAP-70 doesn't enhance BCR signaling by means of its intrinsic kinase activity.

Subsequently, Chen et al. as well as Gobessi et al. considered the hypothesis that ZAP-70 instead may enhance BCR signaling indirectly by enhancing the stability of activated Syk following ligation of the BCR. It is known that activated ZAP-70 and Syk are targets of the E3 ubiquitin ligase, c-Cbl, which in turn directs polyubiquitination and proteosomal degradation of the activated PTK (Fournel et al. 1996, Lupher et al. 1998, Rao et al. 2002). Possibly, the activated ZAP-70 may compete with activated Syk for binding to c-Cbl and thereby prolong the half-life of activated Syk. However, transduction of CLL cells with an adenovirus encoding a mutant form of ZAP-70 carrying a mutation at position 292, which abrogates the ability of the mutant form to interact with c-Cbl, also enhanced BCR signaling of ZAP-70 negative CLL cells. So, binding of C-cbl to Tyr292 (Gobessi et al. 2007) is unlikely to account for the capacity of ZAP-70 to enhance BCR signaling in CLL B cells.

Another explanation, proposed by both groups, is that ZAP-70 functions as an adaptor protein that facilitates the recruitment of other signaling molecules to the activated BCR. The associations with PI3K and Shc after IgM stimulation are noteworthy in this respect because these proteins are involved in the activation of Akt and ERK, respectively (Gobessi et al. 2007). In addition to this, examination of additional constructs encoding mutant forms of ZAP-70 that lacked a functional SH2 domain revealed that both SH2 domains are required for enhanced IgM signaling. This suggests that docking at the ITAM may be necessary for ZAP-70 to have an effect on CLL Ig receptor signaling (Chen et al. 2007).

The Biological Relevance of ZAP-70 in CLL 143

70 are more sensitive to this IKK inhibitor compared to CLL cells without ZAP-70. This supports the hypothesis that there is a functional link between ZAP-70 and NFκB. On the other hand, these results also imply that the therapeutic combination of NFκB inhibitors with other chemotherapeutic drugs, represents a novel strategy especially for the group with high ZAP-70, known to be more resistant to agents currently in use (Lopez-Guerra et

*Schematic presentation of the interaction of ZAP-70 into the intracellular signaling pathways occurring in a CLL cell after stimulation of the B cell receptor. Syk, BLNK, PLCγ, MAPK, PKC and NFκB (enclosed by a red line) are potentially stimulated by ZAP-70. Also the phosphorylation of Syk (red P) and calcium influx (red arrow) may be*

In contrast with their long-living capacities in the human body, CLL cells tend to undergo rapid apoptotic cell death when incubated in vitro. This has raised the hypothesis of prosurvival environmental factors existing *in vivo*, supporting the CLL cells in their survival

The peripheral blood of CLL patients contains an accumulation of mature B cells that have escaped programmed cell death and have undergone cell cycle arrest in the G0/G1 phase lacking metabolic activity. However, when in *in vivo* experiments patients drank deuterated water (2H2O) cell generation rates were in the range between 0,1 to 1,75 % of the entire CLL clone per day (Messmer et al. 2005). Considering a CLL clone to contain 1012-1014 cells, an

al. 2009).

*augmented by ZAP-70.*

and growth (Ghia et al. 2002).

Fig. 4. ZAP-70 in CLL signaling in poor prognosis CLL

**5. Interaction of ZAP-70 with the leukemic microenvironment**

The finding that ZAP-70 may enhance IgM signaling by functioning as an adaptor protein is further confirmed by zum Buschenfelde et al. (2010). The interaction of Ig receptors with their ligands has been shown to occur within an organized contact zone known as the immune synapse. Formation of the immune synapse is accompanied by a remodeling of specialized membrane microdomains enriched in sphingolipids and cholesterol known as lipid rafts (Viola & Gupta 2007). In T cells, ZAP-70 is known to be required for clustering signaling molecules into lipid rafts (Blanchard et al. 2002). This group investigated the distribution of ZAP-70 and signaling molecules like PKC-βII in lipid raft domains before and after BCR stimulation . They found that ZAP-70 was constitutively expressed in the raft domains. Accordingly, these cells constitutively expressed PKC-βII in lipid rafts, whereas the expression of PKC-βII was negligible in raft fractions of ZAP-70 negative patients, although total amounts of PKC-βII was expressed in the same amounts in whole-cell lysates in both groups. Signaling through the BCR recruited accessory ZAP-70 and PKC-βII into lipid raft domains (zum Buschenfelde et al. 2010). These experiments demonstrate that ZAP-70 may function as an adaptor protein in specific lipid raft domains, and therefore recruits specific signaling molecules towards the immune synapse.

Although reduced BCR internalization by ZAP-70 is never established in CLL cells, it could be a fourth explanation for increasing the magnitude and duration of BCR signaling. Transfection of ZAP-70 in BJAB B cells downmodulates BCR internalization (Gobessi et al. 2007) explaining how ZAP-70 could contribute to the stronger signaling observed in CLL B cells.

As described above, an important mediator of BCR signaling is NFκB, encoded by a family of transcription factors which are key regulators of differentiation and survival in B cells. In humans they include five members: c-Rel, Rel B, p50, p52 and p65 or Rel A. These factors form homo- or heterodimers, which in the resting state are retained in the cytoplasm by binding to the inhibitory IκB proteins. Upon different stimuli, including ligation of BCR, the IκB proteins are phosphorylated by IκB kinases (IKK) and degraded by the proteasome. Consequently, the NFκB dimers become free to translocate to the nucleus and activate the transcription of their target genes. These include antiapoptotic genes (Bcl-2, Mcl-1, Survivin), inflammatory genes (COX-2, MMP-9, VEGF) and genes encoding adhesion molecules, chemokines (IL-1β, IL-6, IL-8) and cell cycle regulatory proteins (Cyclin D1, c-Myc) (Hayden & Ghosh 2008). More generally, NFκB has been implicated in tumorigenesis and survival of a growing list of leukemias and lymphomas (Karin & Lin 2002).

CLL cells have been reported to exhibit high constitutive NFκB activation compared with normal B lymphocytes (Cuni et al. 2004, Furman et al. 2000, Tracey et al. 2005). Although the exact factors responsible for the constitutive expression of NFκB are not fully resolved, many factors, including Akt activation, BCR signaling, CD40 ligation, IL-4 and B cell activating factor (BAFF), have been shown to increase NFκB activity and enhance CLL survival, with members of the Bcl-2 family being principal transcriptional targets (Petlickovski et al. 2005). Hewamana (2008) found an association between ZAP-70 expression and the ability of CLL cells to activate NFκB (Hewamana et al. 2008). Furthermore, they found that the magnitude of the change in NFκB after stimulation with anti-IgM is associated with the suppression of in vitro apoptosis. Lopez-Guerra et al. (2009) tested a specific IκB kinase inhibitor, BMS-435541, and found that CLL cells expressing ZAP-

The finding that ZAP-70 may enhance IgM signaling by functioning as an adaptor protein is further confirmed by zum Buschenfelde et al. (2010). The interaction of Ig receptors with their ligands has been shown to occur within an organized contact zone known as the immune synapse. Formation of the immune synapse is accompanied by a remodeling of specialized membrane microdomains enriched in sphingolipids and cholesterol known as lipid rafts (Viola & Gupta 2007). In T cells, ZAP-70 is known to be required for clustering signaling molecules into lipid rafts (Blanchard et al. 2002). This group investigated the distribution of ZAP-70 and signaling molecules like PKC-βII in lipid raft domains before and after BCR stimulation . They found that ZAP-70 was constitutively expressed in the raft domains. Accordingly, these cells constitutively expressed PKC-βII in lipid rafts, whereas the expression of PKC-βII was negligible in raft fractions of ZAP-70 negative patients, although total amounts of PKC-βII was expressed in the same amounts in whole-cell lysates in both groups. Signaling through the BCR recruited accessory ZAP-70 and PKC-βII into lipid raft domains (zum Buschenfelde et al. 2010). These experiments demonstrate that ZAP-70 may function as an adaptor protein in specific lipid raft domains, and therefore recruits

Although reduced BCR internalization by ZAP-70 is never established in CLL cells, it could be a fourth explanation for increasing the magnitude and duration of BCR signaling. Transfection of ZAP-70 in BJAB B cells downmodulates BCR internalization (Gobessi et al. 2007) explaining how ZAP-70 could contribute to the stronger signaling observed in CLL B

As described above, an important mediator of BCR signaling is NFκB, encoded by a family of transcription factors which are key regulators of differentiation and survival in B cells. In humans they include five members: c-Rel, Rel B, p50, p52 and p65 or Rel A. These factors form homo- or heterodimers, which in the resting state are retained in the cytoplasm by binding to the inhibitory IκB proteins. Upon different stimuli, including ligation of BCR, the IκB proteins are phosphorylated by IκB kinases (IKK) and degraded by the proteasome. Consequently, the NFκB dimers become free to translocate to the nucleus and activate the transcription of their target genes. These include antiapoptotic genes (Bcl-2, Mcl-1, Survivin), inflammatory genes (COX-2, MMP-9, VEGF) and genes encoding adhesion molecules, chemokines (IL-1β, IL-6, IL-8) and cell cycle regulatory proteins (Cyclin D1, c-Myc) (Hayden & Ghosh 2008). More generally, NFκB has been implicated in tumorigenesis and survival of a growing list of leukemias and lymphomas

CLL cells have been reported to exhibit high constitutive NFκB activation compared with normal B lymphocytes (Cuni et al. 2004, Furman et al. 2000, Tracey et al. 2005). Although the exact factors responsible for the constitutive expression of NFκB are not fully resolved, many factors, including Akt activation, BCR signaling, CD40 ligation, IL-4 and B cell activating factor (BAFF), have been shown to increase NFκB activity and enhance CLL survival, with members of the Bcl-2 family being principal transcriptional targets (Petlickovski et al. 2005). Hewamana (2008) found an association between ZAP-70 expression and the ability of CLL cells to activate NFκB (Hewamana et al. 2008). Furthermore, they found that the magnitude of the change in NFκB after stimulation with anti-IgM is associated with the suppression of in vitro apoptosis. Lopez-Guerra et al. (2009) tested a specific IκB kinase inhibitor, BMS-435541, and found that CLL cells expressing ZAP-

specific signaling molecules towards the immune synapse.

cells.

(Karin & Lin 2002).

70 are more sensitive to this IKK inhibitor compared to CLL cells without ZAP-70. This supports the hypothesis that there is a functional link between ZAP-70 and NFκB. On the other hand, these results also imply that the therapeutic combination of NFκB inhibitors with other chemotherapeutic drugs, represents a novel strategy especially for the group with high ZAP-70, known to be more resistant to agents currently in use (Lopez-Guerra et al. 2009).

*Schematic presentation of the interaction of ZAP-70 into the intracellular signaling pathways occurring in a CLL cell after stimulation of the B cell receptor. Syk, BLNK, PLCγ, MAPK, PKC and NFκB (enclosed by a red line) are potentially stimulated by ZAP-70. Also the phosphorylation of Syk (red P) and calcium influx (red arrow) may be augmented by ZAP-70.*

Fig. 4. ZAP-70 in CLL signaling in poor prognosis CLL

## **5. Interaction of ZAP-70 with the leukemic microenvironment**

In contrast with their long-living capacities in the human body, CLL cells tend to undergo rapid apoptotic cell death when incubated in vitro. This has raised the hypothesis of prosurvival environmental factors existing *in vivo*, supporting the CLL cells in their survival and growth (Ghia et al. 2002).

The peripheral blood of CLL patients contains an accumulation of mature B cells that have escaped programmed cell death and have undergone cell cycle arrest in the G0/G1 phase lacking metabolic activity. However, when in *in vivo* experiments patients drank deuterated water (2H2O) cell generation rates were in the range between 0,1 to 1,75 % of the entire CLL clone per day (Messmer et al. 2005). Considering a CLL clone to contain 1012-1014 cells, an

The Biological Relevance of ZAP-70 in CLL 145

CXCR4

CCL19/CCL21

CCL19/CCL21

21

19

*Lower: CLL ZAP+ cells: In ZAP-70 positive cells, increased levels of CCR7 and the interplay between ZAP-70,*

*CD38 and CXCR4 facilitate movement and migratory potential towards lymph nodes.*

Fig. 5. Migration of CLL cells towards the lymph nodes

19

HEVs and stromal cells

21

19

21

CLL ZAP

CLL

19

19

HEVs and stromal cells

CD38

CXCR4

21

19

SDF1a (a)

SDF1a (a)

a

LN

a

a

a

PB

Migration towards LN

a

LN

a

a

a

PB

Migration towards LN

CCR7

CLL

CCR7

*LN: lymph nodes; HEV: high endothelial venules*

 *cells;*

*Upper: CLL ZAP-*

CLL ZAP

extensive number of 109 to 1012 cells are produced each day. This implies that, although there is a virtual absence of proliferative cells in peripheral blood, ill-defined areas of proliferation are existing in the bone marrow and affected lymph nodes (Herishanu et al. 2011). Indeed, interactions with stromal cells, or nurse-like cells, or interactions between CD38 and its ligand CD31 rescue CLL cells from apoptosis in vitro and probably do the same in vivo (Chiorazzi et al. 2005), and furthermore enhance proliferation of the cells. Below, several interactions between CLL cells and by-stander cells will be discussed, especially those findings that correlate with the more aggressive nature of the ZAP-70 positive disease.

## **5.1 CD38 and ZAP-70**

CD38 is a transmembrane glycoprotein that mediates cell-cell interactions (Deaglio et al. 1998). CD38 expression is significantly associated with unmutated IgVH genes in CLL, also with ZAP-70 and subsequently with a more aggressive disease (Damle et al. 1999).

In CLL, chemokines are reported to contribute significantly to the delivery of growth signals to the malignant CLL cells expressing functional receptors. Knowing that ZAP-70 is an essential element in the signaling cascade initiated via CXCR4 in T cells and knowing that the combination of CD38 and ZAP-70 defines a subgroup of patients with the highest migratory potential towards the ligand for CXCR4, SDF1α, it is tempting to speculate that the CD38-ZAP-70 axis synergizes with the SDF1α-CXCR4 pathway (Deaglio et al. 2007).

SDF1α has previously been shown to exert both a chemotactic effect and a prosurvival effect on CLL cells, being a crucial mechanism through which stromal cells or nurse like cells support CLL cells in vitro (Burger et al. 1999, Burger et al. 2000). The finding that CLL cells proliferate at sites where stromal cells are present suggest that SDF1α is an important factor in CLL pathogenesis. Moreover, several groups confirmed an increased level of CXCR4 on the surface of CLL cells when compared with normal B cells (Richardson et al. 2006, Mohle et al. 1999, Burger et al. 1999). Treatment of CLL cells with SDF1α resulted in a rapid and sustained ERK activation profile only in the ZAP-70 positive subgroup. Furthermore, treatment with SDF1α in vitro of ZAP-70 positive but not in ZAP-70 negative cells, resulted in a longer survival. Sustained ERK activation can lead to the initiation of transcription of genes involved in both proliferation and survival (Burger et al. 2000, Xia et al. 1995, Murphy et al. 2002). This could be the explanation for both survival and proliferative advantages seen in ZAP-70 positive cells. These results indicate that ZAP-70 positive cells are more responsive to signals derived from their surrounding environment.

## **5.2 CCR7 and ZAP-70**

Several groups found that CCR7 is upregulated on the surface of circulating peripheral blood CLL cells when compared with healthy control peripheral blood B cells (Richardson et al. 2006, Till et al. 2002, Lopez-Giral et al. 2004, Ghobrial et al. 2004). Moreover, Richardson et al. (2006) demonstrates that CCR7 levels are increased in ZAP-70 positive CLL cells when compared with ZAP-70 negative CLL cells. This upregulation in CCR7 confers an increased ability to respond to its ligands, CCL19 and CCL21. Both chemokines are important in both T and B lymphocyte trafficking (Figure 5). Prior studies in B cells (Reif et al. 2002) have shown that antigen engagement upregulates expression of CCR7 and can facilitate the movement of these cells into the lymph nodes and localization to the B/T cells

extensive number of 109 to 1012 cells are produced each day. This implies that, although there is a virtual absence of proliferative cells in peripheral blood, ill-defined areas of proliferation are existing in the bone marrow and affected lymph nodes (Herishanu et al. 2011). Indeed, interactions with stromal cells, or nurse-like cells, or interactions between CD38 and its ligand CD31 rescue CLL cells from apoptosis in vitro and probably do the same in vivo (Chiorazzi et al. 2005), and furthermore enhance proliferation of the cells. Below, several interactions between CLL cells and by-stander cells will be discussed, especially those findings that

CD38 is a transmembrane glycoprotein that mediates cell-cell interactions (Deaglio et al. 1998). CD38 expression is significantly associated with unmutated IgVH genes in CLL, also

In CLL, chemokines are reported to contribute significantly to the delivery of growth signals to the malignant CLL cells expressing functional receptors. Knowing that ZAP-70 is an essential element in the signaling cascade initiated via CXCR4 in T cells and knowing that the combination of CD38 and ZAP-70 defines a subgroup of patients with the highest migratory potential towards the ligand for CXCR4, SDF1α, it is tempting to speculate that the CD38-ZAP-70 axis synergizes with the SDF1α-CXCR4 pathway (Deaglio et al. 2007).

SDF1α has previously been shown to exert both a chemotactic effect and a prosurvival effect on CLL cells, being a crucial mechanism through which stromal cells or nurse like cells support CLL cells in vitro (Burger et al. 1999, Burger et al. 2000). The finding that CLL cells proliferate at sites where stromal cells are present suggest that SDF1α is an important factor in CLL pathogenesis. Moreover, several groups confirmed an increased level of CXCR4 on the surface of CLL cells when compared with normal B cells (Richardson et al. 2006, Mohle et al. 1999, Burger et al. 1999). Treatment of CLL cells with SDF1α resulted in a rapid and sustained ERK activation profile only in the ZAP-70 positive subgroup. Furthermore, treatment with SDF1α in vitro of ZAP-70 positive but not in ZAP-70 negative cells, resulted in a longer survival. Sustained ERK activation can lead to the initiation of transcription of genes involved in both proliferation and survival (Burger et al. 2000, Xia et al. 1995, Murphy et al. 2002). This could be the explanation for both survival and proliferative advantages seen in ZAP-70 positive cells. These results indicate that ZAP-70 positive cells are more

Several groups found that CCR7 is upregulated on the surface of circulating peripheral blood CLL cells when compared with healthy control peripheral blood B cells (Richardson et al. 2006, Till et al. 2002, Lopez-Giral et al. 2004, Ghobrial et al. 2004). Moreover, Richardson et al. (2006) demonstrates that CCR7 levels are increased in ZAP-70 positive CLL cells when compared with ZAP-70 negative CLL cells. This upregulation in CCR7 confers an increased ability to respond to its ligands, CCL19 and CCL21. Both chemokines are important in both T and B lymphocyte trafficking (Figure 5). Prior studies in B cells (Reif et al. 2002) have shown that antigen engagement upregulates expression of CCR7 and can facilitate the movement of these cells into the lymph nodes and localization to the B/T cells

with ZAP-70 and subsequently with a more aggressive disease (Damle et al. 1999).

correlate with the more aggressive nature of the ZAP-70 positive disease.

responsive to signals derived from their surrounding environment.

**5.1 CD38 and ZAP-70**

**5.2 CCR7 and ZAP-70**

*LN: lymph nodes; HEV: high endothelial venules Upper: CLL ZAP cells;*

*Lower: CLL ZAP+ cells: In ZAP-70 positive cells, increased levels of CCR7 and the interplay between ZAP-70, CD38 and CXCR4 facilitate movement and migratory potential towards lymph nodes.*

Fig. 5. Migration of CLL cells towards the lymph nodes

The Biological Relevance of ZAP-70 in CLL 147

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boundary. Since increased CCR7 expression has been documented following antigen contact, this could be reflecting either an increased level of antigen contact, which is likely to be the case for both ZAP-70 positive and ZAP-70 negative CLL B cells, as both cell types have been shown to resemble activated B cells (Damle et al. 2002, Herishanu et al. 2011), or an increased ability to respond to antigen contact. Herishanu et al. (2011) further described the increased proliferative signature of the CLL cells residing in the lymph nodes and bone marrow, due to a more intensive BCR triggering. ZAP-70 helps in a more sustained activation state after BCR triggering. The finding that CCR7 levels are lower in lymph node CLL cells than in peripheral blood CLL cells is suggestive of it being involved in migration to the lymph nodes (Till et al. 2002, Lopez-Giral et al. 2004, Ghobrial et al. 2004).

## **6. Summary**

It remains largely unkown why ZAP-70 is or is not expressed in CLL. Hsp90 may be important in this respect. Although the precise molecular mechanism of the role of ZAP-70 in CLL B cells is not yet fully resolved, many hypotheses have been put forward suggesting multiple functions. Strikingly, it is improbable that ZAP-70 exerts its role by its kinase activity. ZAP-70 in CLL may be stabilizing SYK, creating more sustained phosphorylation and in association increasing the proliferative capacity. Moreover, ZAP-70 functions as an adaptor protein that facilitates the recruitment of other signaling molecules to the activated BCR. Also the internalization of the BCR after stimulation may be reduced due to intervention of ZAP-70. An important mechanism explaining prolonged survival of CLL cells has been attributed to its interaction with the microenvironment. In this interaction, ZAP-70 may also be of relevance.

## **7. References**

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