ALBERT

Description: Abstract 3593 Unlike other hematologic disorders, chronic lymphocytic leukemia(CLL) exhibits remarkable heterogeneity in the rates of disease progression among cases. CLL cells survive by receiving signals from the microenvironment via various receptors: B-cell antigen receptor (BCR), Toll-like receptors (TLRs) and cytokine and chemokine receptors. We previously reported that CLL clones with somatically mutated IGHVs and high (≥30%) percentage of CD38 expressing cells have the highest percentage of CCR4-expressing cells. To further explore the functional contribution of the CCR4:CCL17 axis in CLL, we studied CCL17-induced chemotactic behavior in 16 CLL cases. In transwell cultures we observed a bimodal migratory response to CCL17 at 2 doses in a dose range of 0.78– 25ng/ml, in ~60% of cases; the remaining cases showed maximal migration at a single dose (1.56 or 3.12ng/ml). A comparison of phenotypes of the migrated and non-migrated cell populations was undertaken in 10 cases, analyzing CXCR3, CXCR4, CCR4 and CCR7 that are involved in homing of cells to sites favoring growth, and CD31, CD38 and CD69, activation related molecules. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 in the CCR4-mediated downstream pathway. CCR4 ligand, CCL17, is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes and fibroblasts, whereas CCL22 is produced by tumor cells and the tumor microenvironment. Serum levels of both these ligands in untreated patients were quantified by ELISA. CCL17 levels ranged between 45-1, 229 pg/ml in U-CLL cases (n=23) and between 43-1, 418 pg/ml in M-CLL cases (n=30). CCL22 levels ranged between 121-5, 497 pg/ml in U-CLL cases (n=23) and 409-5, 502 pg/ml in M-CLL cases (n=30). The percentages of CCR4- expressing B cells directly correlated with percentages of T cells expressing CCR4 in individual cases, whereas they inversely correlated with both, serum levels of CCL17 (p〈 0.01) and CCL22 (p〈 0.05). CCL17 produced by DCs in peripheral organs may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells. We therefore tested if CCL17 supported BCR- and TLR-mediated proliferative responses in a cohort of 31 (16 U-CLL and 15M-CLL) CLL cases. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, but only in 3/15 (20%) M-CLL cases. On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 (87%) M-CLL cases at a dose of 2ng/nl (approximating that detected in serum); it also augmented TLR-9 mediated B cell proliferation in 6/16 U-CLL cases but at a 5-fold or higher dose (10-25 ng/ml). In a subset of this cohort (8 cases) CCL17-induced modulation of molecules involved in the apoptotic process was studied. We found upregulation of anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulation of pro-apoptotic molecules Bim, PUMA, and Bid in 5 of these cases. The pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating TLR-9-mediated signaling and migration in CLL. Therefore, inhibition of CCR4:CCL17 interaction in vivo represents a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival. Disclosures: No relevant conflicts of interest to declare.

Description: Chronic lymphocytic leukemia (CLL) is characterized by the clonal amplification of CD5-expressing B cells that appear to develop and evolve based on signals from the microenvironment. In vitro and in vivo evidence suggests that the B-cell antigen receptor (BCR) and Toll-like receptors (TLRs) may be keys to this stimulation. Because clonal turnover can lead to the release of naked nuclear material into the cellular microenvironment, these remnants of dying/dead cells may contribute to disease progression by repeated low level T-independent activation of CLL cells through the combination of the BCR and TLRs. To test this hypothesis, we assessed TLR9-driven or BCR + TLR9-driven CLL B-cell activation, focusing on its impact on telomerase activation in CLL cells, which is known to be important in the disease and which we have shown to be selectively activated by BCR stimulation in Ig V-unmutated (U-CLL) clones but not in Ig V-mutated (M-CLL) clones. B cells, isolated by negative selection from peripheral blood of IgM+ CLL patients and cryopreserved until use, were cultured for 16 hr without/ with TLR9 agonist, ODN 2006, alone and were assayed for apoptosis using Annexin V and flow cytometry. To study the relative contribution of simultaneous TLR9 activation and BCR activation, B cells were exposed to ODN2006 alone or HB57dex (monoclonal anti IgM Ab conjugated onto dextran) alone or a combination of the two reagents. Extracts from cells cultured for a period of 3 days were assayed for functional telomerase activity using TRAP. Parallel cultures of B cells exposed to the same stimuli were harvested at day 3 and assayed for cell activation and proliferation, which was assessed by 3H thymidine incorporation. CLL cells cultured with ODN2006 exhibited significant apoptosis within 16 hours in 6/12 cases. However at day 3, the same stimulus elicited significant increases in percentages of CD69-expressing cells and densities of HLA-DR in all CLL cases studied. As compared to BCR activation, which upregulates telomerase activity in U-CLL only, TLR9-mediated activation of CLL induced telomerase activation in all CLL cases. Furthermore, ODN2006 elicited significantly higher induction of telomerase activity in M-CLL cases compared to U-CLL cases (p=0.01). In addition, in M-CLL cases, simultaneous activation via TLR9 and BCR significantly upregulated the telomerase activity (p=0.05) that was induced by TLR9 activation alone. IRAK-1/4 inhibitor down modulated both TLR9 mediated and TLR9 +BCR mediated telomerase activity to a greater extent in M-CLL cases than in U-CLL cases. TLR9 activation of CLL cells induced a 3.75 + 0.8 fold (range 1.1 to 19.6; n=32) increase in cell proliferation. When segregated by Ig V mutation, U-CLL cells (n=16) responded significantly better (6.0 + 1.6 fold) compared to M-CLL cells (2.1 + 0.3 fold, n=16; p=0.03). However, co-stimulation of cells via their BCR significantly increased TLR-mediated responses only in M-CLL cases (from 2.3 + 0.4 fold to 5.4 + 1.7 fold; p=0.05). IRAK-1/4 inhibitor did not exert a significant effect on TLR9 mediated cell proliferation in either the U-CLL or M-CLL cases. Co-culture of CLL cells with human stromal cells, HS5, further upregulated the concerted TLR9 + BCR induced proliferative responses in 70% of the cases studied. Together, these results indicate that simultaneous stimulation of CLL cells via both their TLR9 and BCR molecules positively impacts on telomerase activity in all patients studied. Since telomerase is crucial in maintaining longevity of repeatedly stimulated cells, this could represent a mechanism for worse clinical outcome in CLL. These studies stress the need for devising therapeutic agents or combinations thereof to effectively target multiple pathways downstream of these signaling receptors and to ultimately eradicate newly evolving CLL cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3690 Mantle cell lymphoma (MCL), a less common non-Hodgkin's lymphoma (NHL), often has a poor prognosis and a median survival time of 3–5 years. Historically, MCLs were believed to originate from mature but naive B cells; this notion has now changed based on the demonstration of somatically mutated IgHV sequences in the lymphoma cells from a subset of cases. Indirect evidence suggesting that the B-cell receptor (BCR) pathway may be at the base of the observed activation in the disease exists; however, that extent that this activation results from Toll-like receptor (TLR), B-cell antigen receptor (BCR), or a combination of signaling from both has not been adequately addressed. In this study, the responsiveness of purified primary B cells isolated from peripheral blood (PB) and/or bone marrow (BM) of MCL patients in the leukemic phase of the disease to triggering via the BCR or via TLR-9 alone or in context with selected chemokines – CCL17, CCL22, or CXCL12 - was assessed using various early and late cell signaling readouts. Phosphoflow analysis revealed that within 5 minutes of stimulation both PB and BM B cells significantly increased levels of pAkt and pNFkB in response to BCR crosslinking by an anti-IgM monoclonal antibody (mAb). When PB B cells were cultured for 3 days in the presence of various stimuli to evaluate their proliferative response (uptake of 3H-thymidine), anti-BCR triggering stimulated 2 to 5.5 fold increases in DNA synthesis, whereas the TLR-9 agonist ODN2006 elicited 55 to 235 fold increases. In addition, conditions simulating T-cell help (anti-CD40 mAb + IL-4 in the presence of CD32-transfected fibroblasts) stimulated significant (40–65 fold) proliferative responses in MCL B cells. Simultaneously, a significant increase in HLA-DR (anti-BCR: 49%; ODN2006: 61%; T-cell help: 20%) and Bcl-2 expression (anti-BCR: 21%; ODN2006: 36%; T-cell help: 25%) was induced by these stimuli. Furthermore, B cells from the BM of the same cases differed in their proliferative responses based on the agonist. Thus, in response to BCR triggering, B cells from BM proliferated to a greater extent compared with PB B cells, whereas in response to TLR-9 stimulation PB B cells proliferated to a greater extent than those from BM. In independent experiments, B cells were incubated with various stimuli including those simulating T-cell help and chemokines for 3 days. Cells were harvested and extracts prepared from viable cells to determine telomerase activity using the telomere repeat amplification protocol (TRAP). Anti-BCR stimulation and anti-TLR-9 stimulation independently increased telomerase activity 1.7 and 1.9 fold, respectively, whereas in combination with CCL17 and CCL22, anti-TLR-9 stimulation further increased telomerase activity to 2.28 and 2.36 fold, respectively. In summary, these findings suggest an important role for commonly encountered microenvironmental influences interacting with TLR9 and to a lesser extent the BCR in promoting the aggressiveness of MCL. They also suggest that responses to these stimuli differ between MCL cells residing in the BM and those circulating in the blood. Finally, the data suggest that ligands for CCR4 may play an enhancing role for signals transduced by the BCR and TLR-9 in this disease. If documented in a larger number of cases, treatment regimens that target these signaling pathways might be of therapeutic value. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 804 Significant inter-and intra-clonal heterogeneity, manifested by phenotypic and molecular differences among cell subsets, is a key feature of chronic lymphocytic leukemia (CLL). However, the definition of the subset of cells within a clone more likely to diversify and understanding the likelihood that these cells would have a survival advantage once altered have not been addressed. Most CLL cells express CXCR4, and interaction with its ligand, CXCL12, promotes survival as well as directing migration, two key factors for perpetuation of genetically-modified subclones. We have shown that clonal fractions with lower CXCR4 density contain cells with increased expression of activation markers and represent recently divided/born cells (“proliferative compartment”) as evidenced by enrichment of Ki-67+ cells and in vivo2H-labeled DNA. The current study was undertaken to better characterize the proliferative compartment and track the evolution of this more actively cycling fraction to determine if it may be the wellspring of clonal heterogeneity in CLL. To follow the natural history of the cells comprising the proliferative compartment, we analyzed changes in 10 CLL (5 U-CLL and 5 M-CLL) cases for whom cryopreserved PBMCs were available over a 3–11yr period during the course of the disease. PBMCs from 3 or 4 successive time points for every case were subjected to immunofluorescent staining and the CXCR4brCD5dim, CXCR4intCD5int, and CXCR4dimCD5br fractions analyzed at every time point for expression of a series of surface and intracellular molecules of functional relevance: CD11a, CD23, CD27, CD38, Ki67, Mcm6, MDR (Pgp120), Survivin, and hTERT. The CXCR4dimCD5br subset exhibited the highest percentages and levels of CD11a, CD23, CD27, and CD38 on all CLL clones at all time points tested. Although Ki-67, Mcm6, MDR (Pgp120), Survivin, and hTERT also followed the same trend, with disease course there was an overall increase in Survivin, MDR, and Mcm6 levels specifically in the CXCR4dimCD5br subset, which were paralleled by a decrease in the Bim:Mcl-1 ratio. Levels of TERT protein and telomerase enzyme activity in B cells at each time point from 8 of 10 cases were consistently higher in the CXCR4dimCD5br B-cell subclones than the corresponding CXCR4brCD5dim and CXCR4intCD5int fractions. Therefore, CXCR4/CD5 B-cell subsets from 8 B-CLL cases frozen over a period of time were flow-sorted and differences in telomere lengths over time analyzed by flow-FISH. Post-sort, in ∼95% of the measurements the CXCR4dimCD5br fraction had significantly longer telomeres than the total B-cell fraction and the CXCR4intCD5int fractions, implying elevated telomerase activity counteracted telomere erosion. Finally, since activation of CLL cells can be achieved through BCR stimulation, we used phosphoflow to analyze differences in the response of CXCR4brCD5dim, CXCR4intCD5int, and CXCR4dimCD5br fractions to anti-IgM stimulation, measured by changes in phospho- Akt, Erk, p38MAPK, NFkB, Syk, Btk and STAT5 levels after a 5 minute exposure to the agonist. CXCR4brCD5dim cells had the highest constitutive phosphorylation levels for all molecules except NFkB. However after anti-IgM stimulation, the greatest increase in phospho- Akt, Btk, Erk and Syk levels (〉25%) occurred in the CXCR4dimCD5br fraction. Notably, CXCL12 elicited the highest (〉40%) phosphorylation of NFkB and Erk among CXCR4brCD5dim cells. Collectively, these findings indicate that the CXCR4dimCD5br fraction of CLL clones contains cells with the highest levels of molecules involved in cell proliferation and longevity, whereas the CXCR4brCD5dim fraction responds best to the pro-survival and trafficking influences of CXCL12. This is consistent with new DNA abnormalities more likely originating and surviving among the proliferating cells of the CXCR4dimCD5br fraction and when these mature to a CXCR4brCD5dim phenotype having the best chance of trafficking back to solid tissues and entering a long-lived pool. This sequence would lead to a survival advantage to altered clonal members and permit ongoing clonal heterogeneity and evolution. Disclosures: No relevant conflicts of interest to declare.