El rol del receptor de células B y la señalización en el microambiente en la progresión de la leucemia linfoide crónica

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1 El rol del receptor de células B y la señalización en el microambiente en la progresión de la leucemia linfoide crónica The role of B-cell receptor and microenvironment signalling in chronic lymphocytic leukemia progression Dighiero G Institut Pasteur, Paris, FR. Primer Director del Institut Pasteur de Montevideo gdighiero@gmail.com HEMATOLOGÍA Volumen 22 Número Extraordinario 3º IBAM CELL: Agosto 2018 Palabras claves: receptor de células B, microambiente, señalización. Keywords: B-cell receptor, microenvironment, signaling. Abstract Despite significant progress in treatment, CLL remains an incurable disease. Advances have been made to understand the molecular pathogenesis underlying CLL progression and treatment resistance. We here review the available evidences concerning the role of the B-cell receptor (BCR) and the tumour microenvironment interactions in CLL pathogenesis. Antigen likely plays a key role in the selection of the tumoral clone, the mutational status of Ig genes is a strong prognostic predictor and BCR signaling has been postulated to play a role for CLL trafficking and interaction with the stromal microenvironment. There is also important evidence favoring a role for the microenvironment in CLL pathogenesis. Most, if not all, proliferative events occur in lymph nodes and bone marrow, where leukemic cells receive through microenvironment interactions survival signals aiming to avoid apoptosis and acquire favourable tumoral growing conditions. In addition, the tumoral microenvironment appears to be the site where acquisition of additional genetic lesions in the clone occur, which should greatly influence clinical outcome. The advent of new tyrosine kinase inhibitors which seem to be able to modulate microenvironment interactions and circumvent the p53 deletion have generated significant promise by raising the possibility that they could provide significant progress in disease treatment. In this article we will discuss the following questions: 1) What is the role of the B-cell receptor (BCR) in CLL pathogenesis?, 2) What is the role of microenvironment in CLL progression? 15

2 1) What is the role of the B-cell receptor (BCR) in CLL pathogenesis? The BCR is a multimeric complex formed by the assembly of surface immunoglobulin (SIg) homodimer and the noncovalently bound heterodimer Iga/ Igb (CD79a/CD79b). Both these molecules play a key role in receptor expression and signal transduction through their immunoreceptor tyrosine-based activation motifs (ITAM), by linking the antigen binding Ig chains to intracellular tyrosine kinases of the Src-family. These events are transmitted through various signaling pathways into the cell nucleus to induce a cellular response. Low expression of the BCR is the hallmark of the B-CLL lymphocyte (1). It correlates with a reduced induction of protein tyrosine kinase activity that results in defective intracellular calcium mobilization and tyrosine phosphorylation, and leads to impaired responses of B-CLL cells when stimulated through the BCR pathway (2,3). The mechanisms accounting for poor expression of the BCR in CLL remain elusive. With the exception of one report describing a mutation in the Igb molecule (4), previous work has shown no genetic defects at the level of the BCR components (9,1). In addition, there is normal transcription and intra-cellular synthesis of BCR components, which contrasts with their poor expression at the membrane level (5-7). Proteins comprising several subunits, such as the BCR, require folding and assembly. These processes take place in the endoplasmic reticulum (ER), where the proteins are modified (cleavage of signal peptide, N-glycosylation, formation of disulfide bonds) and folded before they pass into the Golgi apparatus. If folding and maturation are defective then the quality control system retains the defectively folded proteins and eliminates them. In the case of BCR, several chaperones, including calnexin, calreticulin, BiP and GRP94, have been shown to associate with the µ, CD79a and CD79b chains. Work from Vuillier et al (5) demonstrated constant retention of the μ and CD79a chains in the ER of CLL B cells, leading to an impaired assembly of the BCR. This retention was frequently associated with defective glycosylation of both molecules, which paralleled the level of IgM expression on the surface of B cells. The impaired surface expression of IgM could not be accounted for by structural defects in BCR components and chaperone proteins (5). However, the exact mechanism involved in defective assembly of the BCR is unclear. It also remains to be determined whether the CLL B lymphocyte is an anergic cell and whether BCR under-expression is related to the malignant transforming event. Evidence that the CLL cells may be anergic is suggested by the observation that similar underexpression of the BCR and defective signal transduction have been reported in the case of anergic murine B cells. This could be the consequence of BCR crosslinking by autoantigens, since the BCR of B-CLL can often be autoreactive (8-10). Elucidation of the mechanisms implicated in BCR underexpression in B-CLL should be a key step in understanding disease pathogenesis. The vast majority of B-CLL cells express CD5 and IgM/IgD and thus have a mantle zone-like phenotype of naive cells, which, in normal conditions express unmutated Ig genes. However, it has been shown that 50%-70% of CLL harbor somatic mutations of VH genes (11-13) as if they had matured in a lymphoid follicle. Interestingly, the presence or absence of somatic mutations is associated to prognosis and with the use of particular VH genes. Particular alleles of the V1-69 gene and the V4-39 gene display an unmutated profile. A majority of members from the most prominent VH3 family are expressed in a mutated form, whereas given the significant overexpression of V1-69 among VH1 family members a majority of VH1 expressed genes display an unmutated profile (11-13). The fact that some genes like VH1-69 and VH3-07 recombine this VH segment to particular JH segments and the restricted use of CDR3 sequences by CLLs expressing the VH4-39 gene, suggest that the observed differences in BCR structure in B-CLL could result from selection by distinct antigenic epitopes (8,13). Subsequently it has been revealed that over 20% of patients with CLL carry stereotypic receptors (14-16). Of particular interest, the VH3-21 gene displays strikingly homologous VH and Vλ gene rearrangements and is associated to poor prognosis whether expressed in a mutated or unmutated form (17,18). These results strongly suggest that a common antigen epitope is recognized by these highly homologous molecules. Concerning the epitope recognized, it has been shown that unmutated CLL cells express highly polyreactive antibodies whereas most mutated ones do not (8-10). It is presently unclear whether this putative antigen driven process occurs prior to leukemic transfor- 16

3 mation and/or that the precursors were transformed into leukemic cells at distinct maturational stages. Recent studies analyzing signal competence revealed that unmutated CLLs (U CLLs) tended to express higher amounts of the BCR and respond better upon stimulation when compared to mutated CLLs (M CLLs) (3). These results led to the proposal that unmutated CLLs keep their ability to respond upon BCR stimulation, whereas mutated ones resemble anergic B cells (7). However, it remains to be determined whether anergy can induce the folding and assembly defects observed for CLL B cells. Although a recent study confined to membrane Igs demonstrated that some of these immature glycoforms succeed to assemble and reach the membrane, the amount of Ig and particularly CD79a and CD79b that normally reach the membrane is very low and the majority of Ig produced by the cell remains retained in RE compartment (5). Moreover, even if we accept that UM cases respond better to stimulation through the BCR than mutated ones (19), the response is poor in the vast majority of cases and at least one third of these cases display very low responses to this stimulation. These results raise concerns related to the role of BCR stimulation in progression of the disease. Although promising, it may be premature to ascribe positive clinical responses of SYK, BTK and PI3k-δ inhibitors to specific inhibition of BCR-mediated signaling. In particular these kinases also contribute to other signaling pathways independent of the BCR. For instance, in CLL SYK phosphorylation is increased in cells stimulated via chemokine receptors and integrins and SYK inhibition reduced migration toward CXCL12 and adhesion to VCAM- 1. The PI3K inhibitor CAL-101 also interferes with survival effects of CD40L, TNF-α and fibronectin in CLL cells and BTK is required for Toll-like receptor signaling (20,21). Thus, the contribution of specific BCR signaling inhibition to the clinical efficacy of these agents remains unclear as yet. Indeed, it is possible that their clinical effects stem from simultaneous inhibition of multiple signaling responses. 2) What is the role of microenvironment in CLL progression? Evidence indicates that the relationship of CLL B cells with their microenvironment is crucial for the creation of altered key proliferative/apoptotic pathways, which are hallmarks features in CLL pathogenesis. Crosstalk with accessory cells in specialized tissue microenvironments favours disease progression by promoting malignant B-cell growth and the emergence of new genetic alterations which will lead to drug resistance (22-24). Therefore, understanding the crosstalk between malignant B-cells and their milieu could give us new keys on the cellular and molecular biology of CLL that can finally lead to novel strategies for disease treatment. Messmer et al demonstrated that CLL is not only a static disease but also a disease in which different proliferative subsets coexist (25). These observations have turned the attention towards the generation of different sub-populations inside the tumoral clone that either reach a homeostatic balance in patients with good clinical course or an imbalance in patients with poor outcome (26,27). Interactions with the microenvironment led to the overexpression of three unexpected molecules among unmutated CLLs. The first one is the zeta-associated protein 70 (ZAP-70) a receptor associated protein tyrosine kinase, usually found in T and NK cells, but not in normal B cells. High levels of this protein are detected in the majority of unmutated CLLs (28). Different groups (29-32) demonstrated that CLL B cells that express ZAP-70 are more likely to respond to IgM crosslinking with increased tyrosine phosphorylation and calcium flux than ZAP-70-negative CLL B cells. This suggests that the expression of ZAP-70 in CLL allows more effective IgM signaling in CLL B cells, a feature that could contribute to the more aggressive course observed in these forms. Corcoran et al (33) demonstrated that abnormal ZAP-70 expression was associated with the methylation status of the intron1-exon2 boundary region of ZAP-70 and methylation status of C-334 determined by COBRA. We previously described that Unmutated (Um) patients could be differentiated from mutated (Mut) ones by the abnormal expression of lipoprotein lipase (LPL) gene, which is not expressed in normal B cells and proposed the prognostic value of LPL expression as a surrogate of the mutational status. Since then, a body of evidence has confirmed that the expression of LPL mrna is associated to bad prognosis and that it is one of the most robust molecular marker in CLL (34). In a recent work, we have investigated the mechanisms accounting for the ab- 17

4 errant expression of the lipoprotein lipase (LPL) enzyme in UM CLL patients. Our results demonstrated that this aberrant expression resulted from the lack of methylation in the LPL CpG island. Interestingly, this epigenetic mechanism appeared to be mainly triggered by T cell-dependent microenvironments signals (CD40L+IL-4), whereas cross-linking of the BCR poorly induced this demethylation process and signaling through TLR9 or TLR1/2 pathways were found unable to induce it (35). The third molecule is the activation induced cytidine deaminase (AID), a B cell-restricted enzyme, required for somatic mutation and isotype switching. AID is upregulated, at least at the mrna level, in unmutated CLL cells (36-38). While there is evidence that AID expression could be confined to a small proportion of the clone (39) it appears to be functional, since U-CLL cases can generate isotype-switched transcripts and protein and mutations in the pre-switch µ region (36). Since AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. Thus, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to interaction with microenvironment. Our results showed that among UM patients: 1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; 2) this small subset expresses high levels of proliferation, anti-apoptotic and progression markers (Ki- 67, c-myc, Bcl-2, CD49d and CCL3/4 chemokines). Overall, this work outlined the importance of a subset in PB of UM CLL patients displaying high AID levels and ongoing CSR, whose presence could be a hallmark of a recent contact with the microenvironment and more importantly, is associated to a clinical outcome (26). Individual cancer samples are heterogeneous and include subclonal populations and tumors likely evolve through competition and interaction between different subclones. In the case of CLL, this so called proliferative pool may be the site where acquisition of additional genetic lesions in the clone occur. In this regard, isolation and analysis of the tumoral subset that is being triggered in the proliferative compartments of progressive CLL cases is an important aim to understand CLL pathogenesis. Different groups have tried to assess this question by studying different CLL proliferative subsets like that expressing CD38 marker (40), or those expressing activation induced cytidine deaminase (AID) (26) or CD5/CXCL4 molecules (41). A recent study compared gene expression profiling of PB to LN and BM tumoral cells, in 24 treatment naïve patients. This study identified LN as a key site for proliferation (8). CLL cells in the LN showed up-regulation of gene signatures indicating BCR and NF-kβ activation. In addition, expression of these genes was stronger in clinically more aggressive CLLs and tumor proliferation as assessed by E2F and c-myc and Ki-67 expression was higher in LN and was correlated to disease progression. The authors concluded that these results could be useful to identify target molecules able to disrupt the tumoral microenvironment interactions and to inhibit the BCR signaling as promising therapeutic strategies in CLL (42). The BCR is an essential signal transduction pathway for the survival and proliferation of mature B lymphocytes and likely plays a major role in the context of positive selection of the precursor tumoral cell. Thus, it constitutes a foundational event in CLL pathogenesis and as suggested by the fact that 20% of CLL cases from unrelated patients can have extremely similar, sometimes even identical antigen receptors, this probably occurs as a consequence of a stringent selective process induced by the antigen (14-19). Thus, antigen likely plays an important role at one point during the ontogeny of CLL in the context of positive selection of the precursor cell. It is presently unclear whether it plays a major role during the evolution of disease, as shown in other tumoral models like mouse lymphoma models or in some mucosa-associated lymphoid tissue lymphomas, where continuous antigen stimulation plays a key role and antigen withdrawal leads to regression of disease. Recently, evidence accumulated suggesting that signaling via the BCR could play an important role in the development of CLL and that it could determine the variable clinical behavior of the disease. This could likely occur through antigen exposure in vivo which could induce signaling events for constitutive activation of kinases and of NFkB in CLL B cells. Although, BCR may play a role in 18

5 CLL progression, the low signaling ability of the CLL BCR, raises concerns related to its role in CLL progression. In contrast with in vivo results, apoptosis occurs after in vitro culture, suggesting a role of the microenvironment in CLL cell survival (23,43). Within the leukemic microenvironment, two cellular components appear to be potential players: stromal cells and T-lymphocytes. In vitro, the spontaneous apoptosis of B-CLL can be rescued by stimulation via surface CD40 and IL-4 (26,27), by the co-culture with stromal cells (44) and/or nurse-like cells (45). In our laboratory, we first reported that in contrast to normal circulating B-lymphocytes, which only express AID transcripts following CD40L or LPS stimulation, in most CLL cases expressing UM VH genes, tumoral cells are able to express high levels of an active AID enzyme (36-38), though this expression is confined to a small proportion of the CLL clone (26,39). Since AID expression results from signals mainly received through the CD40-CD40L pathway, we subsequently investigated whether its expression was related to class switch recombination (CSR) occurring in a small subset of CLL B-cells and whether it could have an increased proliferative potential. Our results showed that high AID expression is almost exclusively restricted to the subpopulation of tumoral cells having an ongoing CSR process and more important, the presence of this subpopulation in CLL is closely related to an aggressive course of the disease. This small clonal subset having achieved CSR and expressing either IgG at the membrane or both IgM and IgG, displays higher levels of AID, and higher levels of proliferation and anti-apoptotic molecules like Ki-67, c-myc and Bcl-2. Additionally, it also expresses high levels of proteins associated to progression such as CD49d and CCL3/CCL4 chemokines, as well as a decreased expression of the cell cycle inhibitor p27-kip1 when compared with its quiescent tumoral counterpart expressing exclusively IgM (26). The subset recently described by our group displays very similar markers to those found by Herishanu et al (42) in LN from CLL patients. This small subpopulation expressing AID in the PB of progressive UM CLL patients, probably results from the passage into the circulation of this proliferative pool occurring in LN. In agreement with this hypothesis this pool can be identified in the PB of some UM CLL patients displaying a highly aggressive disease. Being aware that AID overexpression is associated with loss of target specificity resulting in mutations in non-immunoglobulin genes, (BCL-6, MYC, PAX-5 and RHOH) (46), it is logical to assume that progressive disease could be related to clonal CLL evolution. In this regard, it has been also suggested that mutations in TP53 genes could be related with AID expression in CLL (47). If true, the constitutive expression of AID in the leukemic clone history could be a key event in disease progression. Aiming to better characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis comparing the global mrna and microrna expression of the above mentioned leukemic subpopulation and compared it with their quiescent counterparts. Our results suggest that proliferation of this fraction depend on microrna-22 overexpression that induces phosphatase and tensin homolog downregulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. Transfection experiments demonstrated that mir-22 overexpression in CLL B cells switches on PI3K/AKT, leading to downregulation of p27(-kip1) and overexpression of survivin and Ki-67 proteins. We also demonstrated that this pathway could be triggered by microenvironment signals like CD40 ligand/interleukin-4 and, more importantly, that this regulatory loop is also present in lymph nodes from progressive unmutated patients. Altogether, these results underline the key role of PI3K/AKT pathway in the generation of the CLL proliferative pool and provide additional rationale for the usage of PI3K inhibitors (21). Overall, these new approaches have allowed better understanding of CLL pathophysiology and provided a more effective and rationale criteria for the management of this disease. How do these data help defining treatment in CLL? Current chemotherapy options are able to obtain complete and long remissions, though relapse ineluctably occurs and a second round of chemotherapy usually results in inferior response. This probably results because we are presently unable to eliminate residual malignant clones located at LN and BM, which, through their interaction with stromal and T cells, receive survival signals and are able to produce new genetic lesions like the 17p or 11q deletion. Results pointing to a role of BCR and different mi- 19

6 croenvironment signaling pathways in tumor progression may redirect therapeutic options to treatment aiming to selectively attack the proliferative pool existing in this protective microenvironment. The advent of new tyrosine kinase inhibitors which seem to be able to modulate microenvironment interactions and circumvent the p53 deletion have generated significant promise by raising the possibility that they could provide significant progress in disease treatment. However, it may be premature to ascribe positive clinical responses of SYK, BTK and PI3k-δ inhibitors to specific inhibition of BCR-mediated signaling, since these kinases also contribute to other important signaling pathways independent of the BCR. Declaración de conflictos de interés: El autor declara que no posee conflictos de interés: References 1. 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