Ghosh, Asish K.

Abstract Purpose: Although the B-cell receptor (BCR) signal plays a critical role in chronic lymphocytic leukemia (CLL) cell survival and a target of current therapies (ibrutinib targets Bruton’s tyrosine kinase; idelalisib targets PI3Kδ), contribution of the cytokine-driven JAK2 pathway to the “CLL cell-survival signaling network” is largely undefined. Experimental Design: Patients with CLL were enrolled to investigate expression/activation of JAK2 and acylglycerol kinase (AGK), and their functional implication in primary CLL cell survival. A series of biochemical and molecular biology assays were employed to uncover the underlying mechanism. Results: We detected that compared with normal B cells, CLL cells aberrantly express constitutively active JAK2. Mechanistically, HSP90 forms a chaperoning complex with JAK2, resulting in its aberrant accumulation in CLL cells. We also discovered aberrant upregulation of a novel mitochondrial lipid kinase, AGK, which remains complexed with HSP90 in CLL cells activating JAK2. Although AGK is typically mitochondrial, we detected its nuclear localization in association with JAK2 in some CLL cells. Functionally, JAK2 phosphorylates its noncanonical substrate, histone H3(Y41), but not STAT3, activating transcription of diverse sets of genes in a patient-specific manner. Additionally, JAK2 activates the BCR signal in CLL cells via LYN/Bruton’s tyrosine kinase axis. Targeted inhibition of JAK2 as monotherapy, or in combination with the BCR inhibitors or venetoclax (a BCL2 inhibitor), induced apoptosis synergistically in CLL cells. Conclusions: These findings suggest that aberrantly expressed AGK activates JAK2, independent of cytokine, leading to activation of diverse sets of gene transcription in CLL cells. Combined targeting of JAK2 and BCR signals or BCL2 may be effective in some patients with CLL.

Abstract Background: B-Chronic Lymphocytic Leukemia (CLL) is predominantly characterized as a clonal B-cell disorder where a minor fraction of CLL B cells proliferate, but the majority of these cells are non-cycling. Importantly, CLL B cells also exhibit apoptosis resistance that results from a complex set of redundant mechanisms. This latter feature presents a significant problem in relation to disease progression and developing effective therapies in CLL. Previously, we and others have reported that leukemic CLL cells spontaneously secrete pro-angiogenic vascular endothelial growth factor (VEGF) that can significantly enhance CLL leukemic B cell apoptosis resistance via unknown mechanisms. In addition, the molecular mechanism of the constitutive upregulation of VEGF in CLL B cells is unknown. Methods and Results: Here, we report that CLL B cells express a high level of HIF-1α protein under normoxic conditions as compared to that of normal B cells in Western blot analysis using a specific antibody. Accumulation of HIF-1α in CLL B cells resulted in upregulation of its downstream target VEGF. To comprehend this abnormal overexpression of HIF-1α, we have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1α degradation by Western blot analysis and found it to be either absent or notably low level in CLL B cells as compared to that of normal B cells. Furthermore, we have shown by in vitro reporter gene assays that miR-92a, a microRNA known to be overexpressed in CLL B cells, can target the VHL 3′-untranslated region (UTR) and is likely a reason for the depressed levels of pVHL in CLL B cells. To validate our hypothesis, we transiently transfected the human embryonic kidney cell line (293T) with miR-92a and observed a dose-dependent repression of pVHL level. Post-transcriptional regulation of the VHL gene was further confirmed when we observed a subtle but definite increase of the pVHL levels monitored by immunoblot following nucleofection of the antisense oligo targeted to miR-92a into primary CLL B cells. To examine whether HIF-1α is functionally active in CLL B cells, we performed co-immunoprecipitation experiments and found that HIF-1α forms an active complex by physical association with the transcriptional co-activator p300 and phospho-STAT3 in CLL B cells. The latter molecule is known to be constitutively expressed in CLL B cells but its function has been unknown. Subsequently, we have found via chromatin immunoprecipitation analyses in CLL B cells that HIF-1α and STAT3 are bound to the VEGF promoter and also recruit RNA polymerase II, further substantiating that this complex likely functions in the activation of VEGF transcription. Summary: Overall, our data show that CLL B cells express constitutively elevated HIF-1α levels in normoxia due, at least in part, to miR-92a regulated diminished pVHL expression. This is a unique previously undescribed mechanism for enhanced HIF-1α levels in human malignancy. We believe that Increased VEGF expression is associated with the combined effects of HIF-1α, p300 and constitutively active STAT3 as a functional complex and is an explanation for the VEGF-based autocrine pathway found in CLL B cells. These findings now provide additional strategy points for interrupting the VEGF autocrine pathway in CLL B cells.

Introduction: Bone marrow stromal cells (BMSCs), a major component of CLL microenvironment influences CLL B-cell survival. We have utilized an in vitro BMSC model system and found unique alterations in CLL B-cells with short term BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and their drug resistance. Methods: Purified primary CLL B-cells (n=41) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=24) or CLL patients (n=18) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses to assess the presence of intracellular signal proteins and real time PCR to determine the RNA level expression. In separate experiments to assess CLL B-cell response in co-culture with BMSC, purified CLL B-cells were treated with the following agents at sub lethal doses; fludarabine, chlorambucil, ibrutinib and venetoclax as single agents. Results: Consistent and significant increase in expression of Axl receptor tyrosine kinase for both mRNA and protein levels was observed in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSCs. In contrast there was no significant change in β-catenin or Axl protein expression in the co-cultured BMSCs. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. Experiments showed that the increased Axl level in co-cultured CLL B-cells was independent of Axl kinase activity. Furthermore, the CLL B-cells from co-culture also had clear upregulation of downstream P-ERK-1/2 but no change in P-AKT(Ser473). Next we reasoned that if increases in Axl and β-catenin are related to drug resistance then CLL B-cells should have increases in those proteins when cultured with drugs and in presence of BMSCs. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil, ibrutinib, venetoclax) with sublethal doses was found to lead to increase in expression levels of both β-catenin and Axl in co-cultured CLL B-cells. However these increases were significantly over that seen with simple co-culture of CLL B-cells with BMSCs. Since CLL B-cells were less sensitive to these latter chemotherapy drugs in presence of BMSCs, this suggests to us a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. Moreover, high nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. It is also known that ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We found upregulation in P-GSK-3β (Ser9) an inactive molecule which can result in increasing accumulation of β-catenin. Inhibition of P-ERK-1/2 using the ERK inhibitor PD98059 in co-cultured CLL B-cells inhibited β-catenin as well as Axl expression levels. Furthermore, it has been shown that Axl expression can be regulated by c-Jun activity. In that regard we have observed upregulation of P-c-Jun(Ser73) which can enhance Axl level in co-cultured CLL B-cells and inhibition of c-Jun activity using SP600125 (c-jun upstream JNK inhibitor), as well inhibited the Axl expression. Finally, we studied 5 CLL patients before and while being treated with ibrutinib/chemo-immuno therapy for the expression of CLL B-cell Axl, β-catenin, P-ERK-1/2, P-c-Jun(Ser73) levels. We found that 2 patients had increase in Axl expression, 4 patients had increased β-catenin and P-ERK-1/2 levels and 3 patients showed increase in P-c-Jun(S73) level after the therapy. Conclusion: Here we show that marrow stromal cell contact with CLL B-cells consistently mediate increased expression in both β-catenin and Axl in CLL B-cells (Figure1). The mechanism for this may, in part, via activated ERK and c-Jun levels (Figure1). We believe that these changes in both molecules are associated with leukemic B-cell survival and drug resistance (Figure1). These studies suggest that a further understanding of the roles of Axl and β-catenin in the CLL B-cells mediated by contact with BMSC will help to develop potential strategies for the management of CLL disease resistant to various drugs. Disclosures Warner: Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Kay:Agios: Other: DSMB; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; MorphoSys: Other: Data Safety Monitoring Board.

Abstract Introduction CLL remains an incurable disease and represents a significant health problem in the western world. Increasing evidence highlights that the impact of marrow stromal cells is a key component influencing CLL B-cell survival. We have utilized an in vitro bone marrow stromal cell (BMSC) model system and found unique alterations in CLL B-cells with BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and drug resistance. Methods Purified primary CLL B-cells (n= 39) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=26) or CLL patients (n=17) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and where needed real time PCR was done to assess the presence of intracellular proteins. In separate experiments to assess CLL B-cell killing, purified CLL B-cells were treated with TP-0903, fludarabine, chlorambucil and ibrutinib as single agents with or without BMSC co-culture. Results We observed significant increases in expression of Axl for both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSC. But, we did not see any significant change in β-catenin or Axl protein expression in BMSCs co-cultured with CLL B-cells. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. The CLL B-cells from co-culture also had upregulation in phosphorylated (P)-ERK-1/2 but no change in P-AKT(Ser473). High nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β (Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β (Ser9) detected in co-cultured CLL B-cells suggests inactivation of GSK-3β and increasing β-catenin accumulation in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. We also determined the phosphorylation status of Axl in CLL B-cells in co-culture with BMSC but found no change either at Y702 (Axl kinase domain) or total tyrosine phosphorylation levels for Axl in CLL B-cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of the highly specific Axl inhibitor TP-0903 on CLL B-cell status of Axl and b-catenin while in BMSC co-culture. Interestingly, both Axl and β-catenin protein expression levels were found to be further upregulated in CLL B-cells exposed to sub-lethal doses of TP-0903 in co-culture with BMSC. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil or ibrutinib) also led to increase in expression levels of both β-catenin and Axl CLL B-cells co-cultured with BMSC. Of interest CLL B-cells were less sensitive to the chemotherapy drugs in presence of BMSCs, suggesting a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. This was not true for the Axl inhibitor as TP-0903 was able to induce robust cell death by targeting P-Axl and overcome BMSC mediated protection even in the presence of increased Axl and b-catenin. We also found that TP-0903 decreased P-Axl as well as the Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells in BMSC co-culture. Conclusions Here we show that marrow stromal cell mediated increased expression in both β-catenin and Axl in CLL B-cells is associated with leukemic B cell survival and drug resistance. The mechanism for this may in part be via activated ERK levels that also occur when CLL B-cells contact BMSC. The BMSC resistance appears to be more profound for chemotherapeutic agents since Axl inhibitor can still induce CLL B-cell killing with BMSC co-culture. These studies suggest that a further understanding of the roles of Axl and β-catenin in the resistance status of CLL B-cells mediated by contact with BMSC are warranted. Disclosures Warner: Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals, Inc: Employment. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 799 Background: Axl is a constitutively active receptor tyrosine kinase (RTK) in CLL B-cells. We have shown that Axl is physically associated with multiple non-receptor cellular kinases and regulates their activities in CLL B-cells including Lyn kinase (a member of Src-family kinases reported to be overexpressed in CLL), PI3K, Syk and phospholipase γ2 (PLCγ2). These aspects likely relate to drug resistance and survival. Importantly, inhibition of Axl by pharmacological inhibitor (R428) induces massive cell death suggesting its critical role in CLL B-cell survival. We hypothesized that in addition to its role in regulating cellular kinases, Axl might modulate the tumor suppressor p53 to make CLL B-cells more resistant to conventional therapeutic agents. This is based on our unpublished findings that CLL B-cells from patients who have undergone treatment without any reduction in tumor burden expressed increased levels of Axl vs. their pre-therapy levels. In addition, we have detected higher levels of Axl in 17p-deleted CLL patients. Thus to understand the mechanism of Axl RTK-mediated regulation of CLL B-cell growth and survival, we interrogated the functional association between Axl and the HDM2 (human homolog of mouse MDM2)/p53 axis. Methods: We cloned the entire (∼2 kb) 3'-untranslated region (UTR) of the Axl transcript into pMIR-Report vector downstream of the fire-fly luciferase gene. Human embryonic kidney cells (HEK293) were transfected with a full length Axl gene containing the first 500 nucleotides of its 3'-UTR or the pMIR-Report plasmid with or without miR-34a. Cell lysates were examined for the expression of Axl by Western blot or performed luciferase assay. Purified CLL B-cells were also treated with various kinase inhibitors including, Wortmannin (pan-PI3K-inhibitor), PP1 (Src-inhibitor) and SKI-606 (targets both Src and Axl) to detect signaling pathway components downstream of Axl involved in regulating HDM2 function. Results: CLL B-cells from majority of CLL patients (n=15) showed constitutive levels of Axl phosphorylation which we found was associated with the phosphorylation level of HDM2 at Ser-166. HDM2 has an important function in controlling p53 stability in response to genotoxic stress. Recent studies have shown that HDM2 can be phosphorylated on Ser-166 by AKT, MAPKs, and other kinases and this phosphorylation regulates HDM2 nuclearcytoplasmic shuttling under certain conditions. However, we found that HDM2 phosphorylation in CLL is independent of PI3K/AKT, p70S6K, and Erk1/2 activation. Interestingly, we found that inhibition of Axl and/or Src kinases (using SKI-606 and PP1) inhibited HDM2 phosphorylation suggesting that Axl and Src kinases are critically involved in regulating HDM2 affinity towards p53 in CLL B-cells. We also identified a complementary binding sequence of miR-34a microRNA on the first 100 nucleotides of Axl 3'-UTR, suggesting a possible post-translational regulation of Axl. Interestingly, miR-34 family transcription was reported to be regulated by p53, and miR-34a was found differentially expressed in CLL versus normal CD5+ B-cells and is linked to response to therapy in CLL. Indeed, exogenous expression of the Axl gene containing the first 500 nucleotides of the Axl 3'-UTR in HEK293 cells exhibited a dose-dependent down regulation of the Axl protein upon co-transfection with miR-34a using Western blot analysis. Furthermore, activation of p53 in CLL B-cells upon treatment with the DNA-damage agent doxorubicin showed a 4–5 fold decrease of Axl protein by flow cytometry. Activation of p53 by doxorubicin was verified by detecting increased expression of p53 and its acetylation at lysine-382 by Western blot. In addition, luciferase assay data using the Axl-3'-UTR reporter plasmid DNA and miR-34a revealed a dose-dependent inhibition of the luciferase activity. Further studies are underway to fully dissect these novel regulatory signaling pathways. Conclusions: Together, these findings suggest that the two functionally distinct cell growth regulatory pathways can modulate CLL B-cell survival in response to various stimuli. This study has established a link between Axl RTK cell-survival signaling pathway and the tumor suppressor p53-mediated apoptosis-signaling pathway in CLL. These first of a kind observations also underscore that the relative balance of p53 status and Axl levels in tumor cells could relate to CLL therapeutic outcome. Disclosures: Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 2851 Background: In an attempt to better treat B-Chronic Lymphocytic Leukemia (CLL) patients, we have examined a unique organometallic complex called Tris (dibenzylideneacetone) dipalladium (Tris DBA). This novel agent is a small-molecule palladium complex shown to have antiproliferative activity against melanoma cells and possible antitumor activity. Tris DBA is known to inhibit activation of signaling molecules such as PI3K and STAT-3, critical factors in CLL B-cell survival and resistance to therapeutic agents. Because B-CLL remains incurable and treatment strategies challenging, our findings may be useful in pursing new therapies to improve patient care. Therefore, this information prompted us to investigate whether Tris DBA could induce apoptosis in CLL B-cells. Method of Approach: Primary leukemic B-cells from CLL patients were cultured in serum free media and treated at 2 × 106 cells per ml with vehicle or various doses of Tris DBA (1 – 10 μg/ml) for 24 – 72 hours. After treatment, cells were analyzed for apoptosis by a FITC-labeled annexin-V/propidium iodide assay using the FACScan. To assess changes in anti-apoptotic proteins, CLL-B cells were cultured for 24 hours with either vehicle or 2.5μg/mL of Tris DBA. Total protein extracts were used for immunoblot methodology with specific antibodies. In some experiments we developed primary marrow stromal cells (MSC) that when nearly confluent were used for co-culture experiments in the presence of freshly isolated CLL B-cells with various chemotherapeutic drugs and/or Tris DBA. Finally, we compared CLL patients with known immunoglobulin heavy chain variable region (IgVH) status because of the clear differences in clinical outcome for these two prognostic subgroups. Results: CLL B-cells were shown to be sensitive to Tris-DBA with a steep dose response curve that significantly increased apoptosis over control levels as early as 24 hours of culture. Thus the LD50 of Tris DBA was between 2.5 and 5.0 μg/ml for the CLL B-cells who were IgVH mutated while between 5 and 7.5μg/ml for the unmutated status CLL B cells (n=10). Normal B or T lymphocytes were found to be equally sensitive to Tris DBA when compared to CLL B-cells however mesenchymal stromal cells (MSC) from CLL marrow (CLL MSC) were far less sensitive to the Tris DBA drug. Thus the LD50 for normal T cells, B cells or NK cells was between 2.5 and 5.0 μg/ml while the LD50 for the MSC was not reached over the dose range of 1–10 μg/ml. When CLL B-cells were added to CLL derived primary MSC in the presence of increasing concentrations of Tris DBA (2.5–10 μg/ml), the cytotoxic impact of Tris DBA on CLL B-cells could be overcome in the presence of MSC. To delineate the mechanism of apoptosis induction of CLL B-cells, we treated CLL B-cells with sub-lethal dose of Tris DBA and prepared cell lysates for Western blot analyses. We found that the anti-apoptotic proteins XIAP and Bcl-2, but not Mcl-1, were down regulated in Tris- DBA-treated leukemic B-cells. In addition, phosphorylation levels of Akt and STAT-3 were inhibited with exposure to Tris DBA as measured by Western blot using specific antibodies. Finally, we studied if Tris DBA could be synergistic or additive with fludarabine in induction of apoptosis of CLL B- cells. Interestingly, we found that in 2 of 3 assays there was synergy between Tris DBA and fludarabine in the enhancement of CLL B-cell apoptosis. Conclusions: We have established that a unique compound, Tris dipalladium, is effective at inducing apoptosis in CLL B-cells whether these leukemic cells are IGVH mutated (low risk) or unmutated (high risk). In addition this compound can modify the levels of XIAP and Bcl-2, Akt and STAT3 activation in the leukemic B-cells. Importantly, by itself the Tris DBA is effective even in the presence of stromal cells albeit losing some effectiveness at high doses in the co-culture and can generate synergy when added to a purine nucleoside for induction of B-cell apoptosis. In total these findings support future clinical applications of this compound in treating CLL patients. Disclosures: Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees. Arbiser:Natuderm: Patents & Royalties.

Abstract Background: B-cell chronic lymphocytic leukemia (CLL) is characterized by defects in the level of apoptosis rather than accelerated leukemic cell proliferation. We have been interested in identification of naturally occurring substances that induce apoptosis in CLL B cells with potential for therapeutic application with a favorable toxicity profile. Two such agents, epigallocatechin-3-gallate (EGCG) in green tea and curcumin from the spice turmeric have recently been studied by our laboratory. We have shown previously that in vitro treatment of CLL B cells with EGCG induces apoptosis [Blood, 2004, 104:788] and is now being tested in a Phase I/II clinical trial. In this study, we explored the possibility of using curcumin in the induction of CLL B cell apoptosis either alone or in combination with EGCG. We also tested their ability to induce CLL B cell apoptosis in the presence of human stromal cells. Results: In vitro treatment of CLL B cells with curcumin induces apoptosis in a dose (5–20 μM) and time dependent manner. Further analysis suggested that curcumin treatment inhibited the effects of the constitutively active proteins STAT3, AKT and NF-κB and suppressed expression of XIAP in CLL B cells. Interestingly, we observed that curcumin/EGCG combination was more effective in the induction of apoptosis in CLL B cells as compared to the corresponding individual dose of the drug alone. We next evaluated the effects of curcumin when CLL B cells were co-cultured with the human stromal cell line HS-5. Curcumin-induced apoptosis was significantly inhibited when CLL B cells were co-cultured in physical contact with HS-5 cells. We also found protection against curcumin-induced cell death, albeit less, when CLL B cells were co-cultured with HS-5 separated by transwells. However, higher doses of curcumin can overcome HS-5 mediated apoptosis-protection of CLL B cells in transwells but, did not overcome the protective effect of physical contact with HS-5. Further analysis of CLL B cells co-cultured with HS-5 in transwells demonstrated elevated levels of STAT3 with increased phosphorylation at serine-727 and increased expression of Mcl-1 and XIAP. At this stage, we employed combined therapeutic approach to evaluate whether combining curcumin with EGCG could overcome stromal cell-mediated protection. Interestingly, combined treatment of CLL B cells could significantly overcome stromal cell-mediated protection against apoptotic cell death, although the effect was more pronounced when CLL B cells were separated by transwells (see figure below). Conclusion: Together, these results suggest that curcumin induces apoptosis in CLL B cells in vitro and may mediate its effects by suppressing the constitutively active signaling pathways that are known to be important in apoptosis resistance This work also demonstrates that stromal cells and their soluble products can resist drug-induced apoptosis of leukemic CLL B cells. Importantly, the apoptosis-inducing activity of curcumin and EGCG is not impaired completely in the presence of stromal cells, suggesting that the combination of curcumin/EGCG may warrant clinical testing in patients with CLL. Figure. Combined treatment of CLL B cells with curcumin/EGCG reduces stromal cell mediated protection against apoptosis. CLL B cells were cultured alone or co-cultured with HS-5 cells and treated with the indicated doses of curcumin (C) and/or EGCG (E). After 24h, cells were collected for Annexin/PI staining for flow cytometric analysis. Mean percent of annexin/PI positive cells are presented with standard error bars. Figure. Combined treatment of CLL B cells with curcumin/EGCG reduces stromal cell mediated protection against apoptosis. CLL B cells were cultured alone or co-cultured with HS-5 cells and treated with the indicated doses of curcumin (C) and/or EGCG (E). After 24h, cells were collected for Annexin/PI staining for flow cytometric analysis. Mean percent of annexin/PI positive cells are presented with standard error bars.

Abstract It is believed that malignant cells “condition” the microenvironment to facilitate tumor cell survival. We hypothesized that crosstalk between CLL B-cells and marrow stromal cells impacts both cell types bi-directionally and ultimately contributes to leukemic cell apoptotic resistance. To test this hypotheses, bone marrow stromal cells from core bone biopsies from CLL patients were isolated and cultured using methods we have previously described (Leuk Res 2007 31(7):899). Subsequently, we determined the impact of co-culture on CLL B-cell features including apoptosis and CD38 expression. In addition, we evaluated the release of angiogenic cytokines on co-culture and signal events in the stromal cells. Immunophenotyping demonstrated that cultured bone biopsy derived stromal cells were CD73+, CD105+, CD146+, CD14−, CD45−, CD34−, HLA-DR-, suggesting they were mesenchymal stem cells (MSC). Co-culture of these MSC with CLL B-cells protected CLL B-cells from both spontaneous apoptosis (SA) and drug-induced (fludarabine and chlorambucil) apoptosis (DA). For SA, the mean survival of CLL B-cells with or without co-culture of MSC for 5 days were 56.9 ± 10.0 and 7.7 ±3.7 (p<0.05), respectively. When CLL B cells were treated with fludarabine or chlorambucil, the fraction of CLL cells tightly adherent to MSC (TA-CLL) showed higher survival than a less adherent but viable fraction of CLL B-cells. The mean survival of TA-CLL cells treated with 10 μM of fludarabine for 48 hours in the presence of MSC were 67.5 ± 3.6 vs 29.8 ± 11.1 without MSC (P<0.05), respectively. When CLL cells with evidence for CD38 expression were co-cultured with MSC, both the percentage of CD38 positive cells and level of expression of CD38 per cell were up-regulated (mean fold change: CD38 percentage, 2.7, p<0.05; CD38 MFI, 1.9, p<0.05) after 2 weeks. In contrast, the CD38 percentage and expression were not changed in cells with minimal CD38 expression when these CLL B-cells were co-cultured with MSC. In addition, co-culture of MSC with CLL cells induced rapid ERK and AKT phosphorylation (within 30 min) in the MSC on immunoblot analysis. When CLL B cells and MSCs were cultured in transwells, the activation of ERK and AKT in MSC occurred at similar levels, indicating that activation of MSC was mediated by soluble factors. In addition, co-culture led to increased secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as well as a decrease of thrombospondin-1 (TSP-1) in the culture medium. These findings confirm that co-culture of CLL B-cells and MSC culminates in “angiogenic switch.” Taken together, these results strongly suggest interactions between MSC and CLL B cells are a bi-directional process. In leukemic cells, the interaction not only protects against spontaneous and drug induced apoptosis but also leads to an increase in CD38 expression consistent with an activated status. In MSC, the interaction leads to activation of ERK and AKT. Co-culture also facilitates angiogenic switching. These results underscore the dynamic and complex nature of the interactions between bone marrow stromal cells and CLL B-cells. Further studies are needed to dissect how crosstalk between CLL B-cells and MSC relates to disease progression, and determines whether these interactions can be targeted with therapeutic intent.

Abstract Abstract 3882 PURPOSE: Nurturing of leukemic B-cells by the microenvironment appears to be an important mechanism of chemoresistance. Recently, several groups have shown that immune cytokines such as MIP-1a (CCL3) and MIP-1b (CCL4) are upregulated when CLL B-cells interact with stroma or nurse-like cells. It appears that stroma may nurture CLL B-cells by providing critical growth factors and signals enhancing survival. Disruption of these signals could be a strategy to overcome stromal cell mediated chemoresistance. METHODS: We cultured CLL B-cells alone or with human stromal cells (HS-5) and evaluated levels of immune cytokines in the media using multiplex cytokine arrays (Invitrogen). To explore the effect of different drugs on cytokine release, CLL B-cells were treated with sub-lethal doses of fludarabine (F) and chlorambucil (C) in co-culture with the stromal cell line HS-5 cells. Based on our previous observation that pharmacologically achievable doses of EGCG are able to overcome stromal rescue of CLL B-cells exposed to purine nucleoside analogues and alkylating agents (ASH 2010), we examined the effects of EGCG on stromal cell mediated cytokine release in co-cultures. In addition, plasma samples collected prior to and after 1 month of treatment from Rai stage 0-II CLL patients participating in a phase II trial of EGCG were used to explore whether there was a clinical correlation of the in vitro effects of EGCG. RESULTS: Co-culture of CLL B-cells with HS-5 cells resulted in upregulation of multiple cytokines. In addition to the previously reported upregulation in MIP-1a and MIP-1b, increases in IL-1RA (5 fold) and MCP-1 (4 fold) were also observed. Upregulation of these cytokines in co-culture was largely contact dependent. When CLL B-cells and HS5 cells were co-cultured with sub-lethal doses of F, modest reductions in the upregulation of IL-1RA (12%) and MCP-1 (18%) were observed compared to co-culture without drug exposure. Similar experiments using sub-lethal doses of C resulted in more substantial reductions in IL-1RA (68%) and MCP-1 (48%) upregulation. Interestingly, the effect of sub-lethal doses of EGCG was similar to C with respect to reducing upregulation of IL-1RA (69%) and greater than both F and C with respect to upregulation of MCP-1 (76%; Figures 1A and B). To explore the potential clinical correlations of these in vitro observations, we assessed the effects of EGCG on plasma IL-1RA and MCP-1 levels in 42 CLL patients participating in a phase II trial evaluating the efficacy of EGCG (2000 mg BID). Of these 42 patients, 13 (31%) were Rai stage 0 and 29 (69%) Rai stage I-II at enrollment. As previously reported (Shanafelt, ASCO 2010), clinical activity was observed with 13 (31%) patients experiencing a sustained ≥20% reduction in absolute lymphocyte count (ALC) and 20 of 29 (69%) patients with palpable adenopathy experiencing at least a 50% reduction in the sum of nodal products with treatment. Median baseline plasma MCP-1 levels prior to treatment were 687 pg/ml and median IL-1RA levels were 468 pg/ml. At the cohort level, no change in median MCP-1 (p=0.21) or IL1-RA (p=0.95) levels were observed after 1 month of EGCG treatment. We next explored whether changes in the MCP-1 or IL1-RA levels of individual patients correlated with response. Although no correlation between reductions these cytokines and declines in ALC were observed, a significant association between reductions of both MCP-1 (Spearman coefficient: 0.51; p=0.02) and IL1-RA (Spearman co-efficient: 0.60; p=0.006) and reductions in lymphadenopathy was found suggesting EGCG effects on these cytokines may relate to clinical efficacy. CONCLUSIONS: Co-culture of CLL B-cells with marrow stromal cells resulted in contact dependent up-regulation of multiple immune cytokines including IL-1Ra and MCP-1 which we speculate may partially facilitate stromal cell mediated chemoresistance. Several drugs, including EGCG appear to modulate this up-regulation. An association between changes in these cytokine levels and reductions in lymphadenopathy in a phase II trial of EGCG suggests that interrupting signaling by these cytokines may be a potential therapeutic target. Disclosures: Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees. Shanafelt:Polyphenon E International: Research Funding.

Abstract Abstract 54 B-cell chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of CD5+ B-lymphocytes in the peripheral blood, lymphoid organs and bone marrow. Despite aggressive therapy, CLL is still incurable partly because of intrinsic defect in apoptosis induction. Deregulated expression of protein kinases by gene deletion, mutation, or amplification has been found to be important for tumor initiation and progression involving cancer cell proliferation, survival, motility, and invasiveness as well as tumor angiogenesis and chemotherapy resistance. Because of their critical functions in oncogenesis, protein kinases have been at the forefront of targeted cancer therapy development since the 1980s. A novel receptor tyrosine kinase (RTK) Axl has been reported to be overexpressed in several types of human cancers including colon, prostatic, thyroid, breast, gastric, renal and lung. Indeed, our recent investigation (ASH 2009, Abstract #2368) identified Axl to be constitutively phosphorylated in CLL B-cells obtained from a majority of CLL patients. We wished to examine whether the constitutively phosphorylated Axl plays a pivotal role in regulating leukemic CLL B-cell survival and could be a potential target to treat this disease. We used freshly isolated purified CLL B-cells after obtaining informed written consent from the patients. Expression status of various kinases in relation to Axl expression and their functional involvement with this novel RTK to drive downstream cell survival signaling pathway(s) were analyzed in CLL B-cell lysates by immunoprecipitation/Western blot analyses using specific antibodies. Finally, we evaluated the functional importance of Axl in CLL by employing two RTK inhibitory agents (Bosutinib [SKI-606] and R428) of different specificity and affinity for receptor tyrosine kinases in order to target Axl and determined the impact on apoptotic/cytotoxic effects on CLL B-cells by flow cytometric analysis. In some experiments, we used bone marrow stromal cells to evaluate if they could diminish the impact of the RTK agent R428. To further our initial examination of Axl status in CLL, we assessed the expression of phosphorylated-Axl (P-Axl) in freshly isolated CLL B-cells by Western blot analysis. Indeed, we detected differential levels of P-Axl in CLL B-cells (n=10). Further analysis revealed that the expression of P-Axl was associated with co-expression of other constitutively phosphorylated kinases/enzymes including Src family kinases (SFKs), PI3K, SyK/ZAP70 and PLC-g2 in CLL B-cells. Importantly, we found by immunoprecipitation of Axl and subsequent Western blotting that these intracellular signaling molecules were complexed with P-Axl in primary CLL B-cells. Further analysis using a siRNA approach targeting Axl suggests that phosphorylation of Axl is an upstream event of SFK-activation in CLL B-cells indicating a pivotal role of Axl in regulating CLL B-cell survival. Finally, when Axl and Src kinases were targeted by a Src/Abl kinase inhibitor, SKI-606 (dose range 2.5–20mM) or the specific-inhibitor of Axl, R428 (dose range 0.5–10 mM) robust induction of CLL B-cell apoptosis was observed in both a dose- and time-dependent manner (Figure). Although both RTK inhibitory agents showed clear induction of apoptosis in CLL B-cells, R428 was found to be the most potent in inducing apoptosis exhibiting an IC50 ∼4-fold lower than SKI-606. Furthermore, we have observed that R428 could target P-Axl resulting in inhibition of SFK-activation further suggesting that Axl regulates SFK-activity in CLL B-cells. Interestingly, R428-induced apoptosis in CLL B-cells (doses of 1.0 mM and 2.5 mM) was significantly inhibited when these leukemic cells were co-cultured with bone marrow stromal cells, but when we tested a higher dose (5 mM) of R428 this stroma-mediated protection was overcome. Further studies are underway to test R428 in combination other chemotherapeutic agents. Thus, we have identified a novel RTK, Axl, in CLL B-cells which appears to work as a docking site for multiple non-RTKs and drives cell survival signals. These findings indicate a unique therapeutic target for CLL treatment. Importantly, there are currently several Axl inhibitory agents that are available to be tested in CLL patients. Disclosures: Holland: Rigel: Employment.