Secreto, Charla

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 Despite recent advances and improvement in treatment options, chronic lymphocytic leukemia (CLL) is still an incurable disease. Recent advancement in parallel sequencing technology has provided better understanding of genetic landscape and clonal architecture underlying disease course. Furthermore, mutation status of TP53, SF3B1, NOTCH1, and BIRC3 has improved current CLL risk stratification. In order to better understand the genetic basis and clonal evolution underlying disease progression we analyzed a homogeneously treated cohort of CLL cases at multiple time points before and after therapy. We performed WES in 30 sequential samples from 12 CLL cases. All 12 cases received PCR (pentostatin, cyclophosphamide and rituximab) based chemoimmunotherapy as initial treatment. Of these 12 cases, two had samples available both >6 months prior to enrollment and at treatment initiation, seven patients who experienced relapse had samples available at both treatment initiation and at and the time of relapse, and three patients had samples >6 months prior to enrollment, at treatment initiation, and at relapse. These cases were previously characterized by aCGH/FISH. R function Mclust and kernel density estimate (KDE) plots were used to identify subclonal heterogeneity. An average of 102-fold depth coverage was obtained with an average of 80% of the targeted regions being covered by at least 30X. Overall, we detected a total of 219 nonsynonymous Single Nucleotide Variants (SNV) and indels (average 19, range 11-33). Del13q (42%) was identified as most prevalent copy number abnormality followed by trisomy 12 (33%), del11q and del17p (17% each). Recurrent mutations were identified in NOTCH1 (33%), DDX3X (25%), TP53, SF3B1 XPO1 and MED12 (17% each). Other tumor implicated genes such as CARD11, NOTCH2, NOTCH4, DIS3, TRAF2, NFKBID, CHK2 and RB1 were found mutated in individual cases. In all 12 cases we identified at least one relevant cytogenetic abnormalities (del11q32, del13q14, del17p13, trisomy 12) and/or a mutated driver gene. Interestingly we found NOTCH1, DDX3X and XPO1 mutated at a significantly higher prevalence than previous studies (10%, 2% and 4%, respectively). Since all cases in our cohort correspond to individuals with progressive disease, a plausible explanation for the higher prevalence of mutations in our cohort is that these mutations are associated with disease progression. By WES we identified multiple subclones in 58% of cases (7 of 12 cases), as compared to previously identified 33% (4 of 12 cases) by CGH/FISH. In 5 chemotherapy naïve cases, no significant increase in genomic complexity was detected with disease progression. In contrast, 5 out of 10 cases with samples analyzed before CIT and at the time of relapse had changes in clonal dynamics, with initially dominant subclones diminishing in response to therapy and the available empty niche being occupied by expansion of another “fitter” subclone at the time of relapse. No acquisition of tumor associated gene mutations was seen post therapy or associated with relapse in 9 of 10 cases. In the remaining case we identified an interesting example of convergent evolution. The pre-therapy sample was comprised of a unique clone characterized with del11q13, mutations in SF3B1 (G742D) and DDX3X (pE196fs). After therapy that clone was either eradicated or reduced to below detection level. Interestingly another clone emerged after relapse that harbored an independent del11q22 and mutations affecting different aminoacids of SF3B1 (K700E) and DDX3X (pL21fs). The strong selection of impairment in these genes during disease progression suggests a key role of these pathways/genes in the pathogenesis of this patient. In conclusion, these studies provide additional insight into the genomic landscape and clonal architecture in a cohort of homogeneously treated CLL cases before and after therapy. Stable clonal architecture was seen in prior to treatment, whereas clonal evolution leading to increased tumor heterogeneity occurred after the under selection selective pressure of therapy in relapsed cases. Early identification of tumoral heterogeneity and underlying genetic abnormalities might provide better targets for therapy to decrease the likelihood of relapse. Disclosures: Fonseca: Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract Backgrounds B cell receptor (BCR) signals and microenvironment protection have been recognized to have key influences on CLL survival and activation. Akt activation is down-stream of the BCR signaling cascade and is critical in the mediation of the bi-directional interactions between CLL B-cells and bone marrow stroma. MK2206 is an allosteric Akt inhibitor previously tested for safety in solid tumor patients. Our preclinical data has demonstrated the synergy of MK2206 with bendamustine (B) to induce CLL apoptosis in vitro. MK2206 also abolished the signal activation and cytokine production induced by BCR ligation in CLL leukemic cells (Ding, ASH 2012). Therefore, we conducted a phase 1 trial testing the safety and efficacy of MK2206 in combination with bendamustine and rituximab (BR) for patients with relapsed/refractory CLL. Methods A phase 1 dose-escalation study was designed to define the maximum tolerated dose (MTD) as well as the safety and efficacy of 3 dose levels (90 mg or 135 mg or 200 mg weekly) of the oral Akt inhibitor MK2206 in combination with bendamustine (70 mg/m2 daily for 2 days in each cycle) and rituximab (cycle 1: 375 mg/m2, cycle 2-6: 500 mg/m2) therapy in relapse/refractory CLL patients. To test the targeting efficacy of MK2206 on Akt and downstream signals, we designed a one-week “run-in” of single agent MK2206 before the initiation of BR during cycle 1. MK2206 was then given along with BR on day 1 of cycles 2-6. Tumor response was evaluated 2 months after the 6th/last cycle of therapy based on IWCLL 2008 criteria. Correlative laboratory studies included assessment of Akt signaling and the plasma cytokine levels (multiplex beads assay (Invitrogen)) at baseline and after one week of first dose of single agent MK2206. Results Nine patients (median age 68) were enrolled in the phase I portion of this trial. All 9 patients had unmutated immunoglobulin heavy chain variable region genes (IGHV) and 4 had del(11q). Six had received prior chemoimmunotherapy (CIT: FCR or PCR or PCO) and 5 had high risk disease defined as early progression within 26 months of the last CIT. Three had received alemtuzumab containing regimen. We have observed potent lymphocyte mobilization in all evaluated patients (n=7) after one-dose of MK2206 during week 1 (mean increase ALC: 35%) as well as reductions of plasma CCL3, CCL4 and CCL2 in the majority of the patients. A single dose-limiting toxicity (DLT) (febrile neutropenia and hemolytic anemia) was observed at dose level 1 (90 mg weekly) among 6 patients treated at this dose. Two patients with dose-limiting toxicities at dose level 2 (135 mg weekly) were observed where one patient had a rash and one patient had dehydration and febrile neutropenia among three patients treated at this dose. Accordingly, the 90 mg once weekly dose was determined to be the MTD for phase 2 trial testing. The most frequent grade 3 or 4 adverse events observed to date were neutropenia (44%) including febrile neutropenia (22%), rash (22%), diarrhea (22%), nausea and vomiting (11%), dehydration (11%), hemolytic anemia (11%) and thrombocytopenia (11%). The frequency of cytopenias was similar to the published German experience with BR (50% grade 3 or 4 hematological toxicities compared to 44% in N1087) in relapsed CLL patients (Fischer, JCO 2011). The median number of cycles received was 6 (range 1-6). Using an intent to treat analysis, two patients achieved complete response (CR, n= 2, 22%), one patient achieved nodular partial response (nPR, n=1, 11%) and five patients achieved a partial response (PR, n=5, 56%). The overall response rate (ORR) is 89%. Conclusion The Akt inhibitor MK2206 in combination with BR is well tolerated in patients with relapsed/refractory CLL. Single dose of MK2206 inhibits Akt phosphorylation, mobilizes leukemic cells and decreases plasma cytokines involved in BCR signals. The preliminary efficacy of this combination demonstrated promising results with a 89% ORR and 22% CR albeit in the small phase 1 cohort. These results compare favorably to BR alone (9% CR and 59% ORR) in relapsed/refractory CLL patients. Phase 2 testing of this combination is now underway. Acknowledgment This work was supported by the funding from NCI grant K23CA160345 (WD), NCI grant CA95241 (NEK) and Alliance for Clinical Trials in Oncology (CA025224 and CA33601). Disclosures: Off Label Use: MK2206, used in a phase 1 trial of relapsed CLL disease. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Zent:Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Reeder:Celgene: Research Funding; Novartis: Research Funding; Millenium: Research Funding.

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 Purine nucleoside combination therapy, with and without immunotherapy has become the standard treatment approach to CLL. Past approaches, other than stem cell transplantation, have not been successful in curing the disease. Inherent resistance of CLL precursors, impaired immune surveillance (T-cells, NK cells) and microenvironmental abnormalities may all play a role in the ability to effectively eradicate leukemia. PCR has been successfully employed for previously treated (Lamanna 2006) and previously untreated (Kay 2007) patients with CLL. In addition, CA has been added to FCR (Wierda 2006) to treat relapsed/refractory CLL disease. We designed a study to evaluate PCR followed by CA for relapsed/refractory CLL. This ECOG study includes, in addition to response criteria, molecular correlates (FISH anomalies and CpG karyotype), risk parameters analysis, including CD38, ZAP-70 and IgVH mutational status. We assessed the baseline and sequential changes in the angiogenic status of patients by measuring plasma VEGF, bFGF, thrombospondin and their immune status via T-cell repertoire. From September 2004 to September 2006, 32 patients with relapsed/refractory CLL were enrolled in the protocol. The protocol combined 2 regimens (PCR and CA). Step 1, PCR, was planned for 6 monthly cycles. Steps 2 and 3 consisted of short term CA (4 weeks) or standard CA (18 weeks) depending upon Step 1 response. There were 3 ineligible, 1 not evaluated, and 28 evaluable patients. FISH anomalies revealed that 8 patients had a homozygous or heterozygous 13q-defect, 9 had complex FISH defects (more than one FISH anomaly), 5 patients had FISH anomalies that included del(17p13.1), 4 patients had FISH anomalies that included 6q-and 3 had FISH anomalies that included 11q-. CD38 was positive in 10 patients, 11 were ZAP-70 positive and 12 of 19 patients tested had unmutated type clones. The planned 6 cycles of Step 1 (PCR) was administered to 14/32 patients (44%), the others receiving 0–5 cycles. All responses were considered partial responses (11/29; 37.9%), with 2 patients (6.9%) demonstrating progressive disease. However, patients receiving the planned 6 cycles of PCR, achieved a partial response of 71% (10/14) with 2 cases of tumor lysis syndrome. Toxicity (Step 1-PCR) was predominately myelosuppressive (Grade 1–4). A case of chronic idiopathic polyneuropathy and a case of “paraneoplastic pemphigus” were reported. There were 4 deaths. CMV reactivation was diagnosed in 2 patients, neither of which was fatal. During Step 2 (CA), short-term administration in one patient led to grade 3 skin toxicity, which limited administration. During long-term administration of CA (PR, PD, SD) (n=7), infectious complications occurred in 6 patients all of whom developed grade 4 lymphopenia. The median survival for the entire group was 15 months. In conclusion, a significant number of heavily pre-treated and poor prognosis patients with CLL will respond to PCR. Further analysis will determine whether a significant number of patients will achieve a higher level of response and longer survival after CA administration. Studies of association of outcome with the risk parameters are ongoing and will be presented with the clinical response data.

Abstract Abstract 3872 Background: Mesenchymal stromal cells are one of the components in the CLL microenvironment known to support leukemic cells survival and growth. Crosstalk between CLL B-cells and marrow-derived mesenchymal stromal cells (MSC) can modulate the activation of both cell types. Indeed we have shown that soluble factors secreted from CLL B cells can activate MSC with increased production of angiogenic cytokines (Blood. 2010. 116:2984). We hypothesize that MSC derived from CLL patients are modified by their interaction with leukemic cells in their gene expression profiles (GEP) and cytokine production (CP) capabilities. Given this, intrinsic differences should be present amongst MSC derived from CLL patients versus normal subjects. Method: MSC collected from CLL patients with Rai stage 1–4 before any therapy or who have not had therapies for at least 2 years as well as normal MSC from healthy controls were cultured and expanded one time in vitro. Subsequently, MSC after one in vitro passage were sorted with CD45 negative selection to eliminate any contaminating non-stromal cells. Approximately 100,000 to 300,000 cells were then stored at −70C and used for RNA purification. Nine CLL MSC and five normal MSC cell samples had RNA purified from each sample and then the RNA preparations were used for comparative analysis of GEP using affymetrix U133A plus B chip. Data were transformed using two different methods: MAS5 or Plier and only probes that were present in at least 50% of cases were included in the analysis. We have recently observed that MSC derived from bone marrow of CLL patients promotes Th17 cell differentiation (Blood. 2010. 116: Abstract 2414). Since IL-17 is the signature cytokine produced by TH17 cells, we decided to test if IL-17 could alter the cytokine production from CLL and normal MSC. Cytokine production from both CLL MSC and normal MSC as secreted into culture medium (CM) spontaneously or with recombinant IL-17 during a 72h-culture were tested using a commercial cytokine array (R&D). Result: Both MAS5 and Plier method of transformation yielded similar results for GEPs of the MSC. The preliminary data from these analyses showed that multiple transcription factors including PITX2, HOXB7, SIM1, ZFY, EMX2, HOXB3, HOXB4, and IRF6 were significantly up-regulated (more than 2 fold) in CLL MSC compared to normal MSC. In contrast, the genes up-regulated in normal MSC compared to CLL MSC include genes involved in the immune response (IGH, IGK, IGL, BST2, PPBP, RGS1, etc.), antigen processing and presentation (HLA genes, IFI30, etc.) and inflammatory response (CD163, LYZ, TNFAIP6, etc.). We are currently in the process of performing further analysis using additional MSC samples to validate these findings with further GEP assays and also by validating the dysregulated genes detected by our GEP studies using real-time PCR analysis. We performed the analysis of multiple cytokines produced by CLL (n=16) and normal MSC (n=12) with and without stimulation with recombinant IL-17 (8 ng/ml) over a 72 hour culture period. Without stimulation, there is approximately two-fold increase of MCP-1 production in CLL MSC compared to normal MSC (CLL vs. normal, 164.2 ± 26.1 vs. 82.7 ± 15.4 pg/ml, p = 0.02). After IL-17 stimulation, an approximately four-fold increase of MCP-1 production (CLL vs. normal, 1200.1 ± 258.6 vs. 334.1 ± 68.2 pg/ml, p = 0.005), three-fold increase of IL-8 production (CLL vs. normal, 2073.1 ± 504.1 vs. 708.7 ± 218.9 pg/ml, p = 0.02), two-fold increase of IL-6 production (CLL vs. normal, 1400.6 ± 214.0 vs. 705.6 ± 98.2 pg/ml, p = 0.008) were detected in CLL MSC compared to normal MSC. Conclusion: We have detected the presence of intrinsic mRNA expression differences for both transcription factors and immune response genes between CLL and normal MSC. The expression differences of these genes could be facilitating the differential cytokine responses we observed in CLL MSC compared to normal MSC. Given these findings it is possible that the dynamic interactions between the CLL leukemic cell and MSC influence the transcription profiles and cytokine production from MSC. This is of biologic and clinical interest in that the GEP dysregulation in CLL MSC may be a primary mechanism for the differential cytokine production seen in CLL MSC. This outcome in turn has the potential to modify the immune and non-immune environment in favor of tumor proliferation and progression. 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 Background:Recurrent mutations in ATM, JAK1/3 and STAT5B genes have been identified in T-cell prolymphocytic leukemia (T-PLL), a mature T cell leukemia with poor survival. However, much remains to be understood regarding the dysregulated pathways involved in the pathogenesis of this aggressive disease. Here, we integrated the analyses of whole exome sequencing (WES) and transcriptomes of a cohort of 12 T-PLL samples to gain further insights into molecular mechanisms of this malignancy. Methods: Leukemic cells were isolated from peripheral blood or bone marrow samples of T-PLL patients. Tumor DNA and RNA, and germline DNA were extracted from purified leukemic T cells and bone marrow derived stromal cells, respectively. WES (n=12) and transcriptome analysis (n=10) were performed and data analyzed with published standard methods (Robinson et al. 2010 and DePristo et al. 2011) Results: 7 paired tumor and germline DNA and 5 tumor-only DNA samples were used for WES analysis. Mutations in ATM and TET2 were detected in 4 (33%) and 1 (8%) patients, respectively. JAK3 (p.M511I, p.Q501H and p.Q503H) or STAT5B (2 p.T628S and 1 p.N642H) mutations each occurred in 3 (25%) cases and were mutually exclusive, consistent with prior reports. Importantly, we found several novel mutations in genes with regulatory roles in the NOTCH signaling pathway. Specifically, 2 somatic mutations p.A3V and p.A3S in NOTCH2 and 2 other mutations (p.S244L and p.A1414V) in NOTCH4 were identified in 4 (33%) patients. One mutation was in the EGF- like and another one in the NODP domain critical for NOTCH-mediated cellular differentiation. Two (16.7%) novel missense mutations (p.G174D and p.V836I) were found in MAML2. Three missense mutations (2 p.S583N and 1 p.R190H) occurred in MAML1. Both MAML2 and MAML1 are essential cofactors of the NOTCH signaling pathway. In addition, a somatic mutation in the NOTCH signal regulator DTX2 (p.G268D) was detected. Altogether 7 (58.3%) patients harbored mutations in genes with a regulatory role in the NOTCH pathway. Among the epigenetic modifiers, we found deletions and point mutations in KDM6B and KDM6A in 4 (30%) patients including 2 codon deletions in KDM6B (p.X751Val) and 1 missense mutation each in KDM6B (p.R1016Q) and KDM6A (p.V510G). KDM6A and KDM6B are histone H3 lysine 27 demethylases known to be involved in regulation of DNA repair and play an important role in embryonic differentiation. Transcriptome analyses of 10 T-PLL cases versus normal CD3+ T cells from 5 healthy donors revealed 706 up-regulated and 655 down-regulated genes. Consistent with prior data, TCL1A and TCL1B were significantly overexpressed (23.7 and 13.1 fold, respectively). Using Ingenuity Pathway Analysis, we found PTEN Signaling, glutamate receptor, and axonal guidance signaling to be among the top 3 upregulated pathways. The top upstream regulators implicated for the up-regulated signals were FGF, Wnt/β-catenin and TNF. The top 3 down-regulated pathways were granulocyte adhesion and diapedesis, IL-17A in psoriasis and IL-10 signaling. The top regulators for the down-regulated pathways were LPS, TNF and IFNg. Wnt/β-catenin and Human embryonic stem cell pluripotency signaling were among the top 20 up-regulated pathways. Many genes involved in the regulation of Wnt signal pathway were included in the above pathways, including Wnt ligands (Wnt9A, Wnt4), transmembrane receptors (CDH1, LRP5, FZD6, FZD7, SMO), cytoplasmic factor (AXIN2), and nuclear factors (MYC, SOX12, SOX8, SOX18, CCND1). Summary: Our study identified recurrent mutations in the NOTCH signaling pathway as well as dysregulated embryonic developmental pathway, e.g. Wnt/NOTCH as newly implicated molecular mechanisms in T-PLL pathogenesis for further functional studies. Additionally, novel (KDM6A and KDM6B) and known mutations (ATM, TET2) in chromatin regulatory genes underscore the importance of epigenetic dysregulation in the tumorigenesis of T-PLL. Disclosures Parikh: Pharmacyclics: Honoraria, Research Funding. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Ding:Merck: Research Funding.