ALBERT

Description: Abstract 2887 Chronic lymphocytic leukemia (CLL) remains incurable despite advances in the biology and treatment of this disease. Current data support the notion that resistance to therapy is promoted by a “protective” tumor microenvironment in which non-leukemia cells produce factors that enhance the resistance of CLL cells to spontaneous or drug-induced apoptosis. One such factor is the chemokine CXCL12, which interacts with its receptor CXCR4 on CLL cells to promote cancer cell survival. To examine the therapeutic potential of blocking CXCL12-CXCR4 interactions, we studied the effect of BMS-936564, a fully human IgG4 anti-CXCR4 antibody, using an in vitro co-culture model of human bone marrow derived stomal-NKter cells – leukemia cell interaction. Such stromal-NKter cells secrete CXCL12 and enhance the resistance of CLL cells to apoptosis in vitro. We observed that primary CLL cells co-cultured with stromal-NKter cells had significantly greater viability than CLL cells cultured alone (20–60% above baseline at 48 hours). Moreover, CLL cells co-cultured with stromal cells had enhanced resistance to drug-induced apoptosis. We found that BMS-936564 antibody at concentrations of 2–200nM could enhance the rate of apoptosis of CLL cells cultured alone or in the presence of stromal cells. CLL cells that expressed unmutated IgVH genes or ZAP-70 appeared equally susceptible to treatment with BMS-936564 as did CLL cells that lack these adverse prognostic markers, as did CLL cells that harbored deletions in 17p13.2 and that were resistant to chemotherapeutic agents, such a fludarabine monophosphate. BMS-936564 antibody inhibited CXCL12 mediated F-Actin polymerization in CLL cells at lower concentrations (20–200nM) compared to AMD-3100 (Mozobil), a small molecule CXCR4 inhibitor (50–150μM). In addition, AMD-3100 did not induce apoptosis in CLL cells (10–300μM). In summary, we observed that the anti-CXCR4 antibody BMS-936564 inhibited CXCL12 mediated activation of the CXCR4 receptor in CLL cells and induced apoptosis in leukemia cells. The pro-apoptotic activity of BMS-936564 was observed in cells cultured alone or together with stromal cells suggesting that this antibody had direct cytotoxic effect on leukemia cells and that it can overcome the protective tumor microenvironment. More over, the activity of BMS-936564 was independent of the presence of poor prognostic factors such as del(17p) suggesting that its mechanism of action is P53 independent. These findings show evidence that the CXCR4-CXCL12 pathway is a valid therapeutic target in CLL and provide additional biological rationale for ongoing clinical trials in CLL and other hematological malignancies using BMS-936564. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Kipps:Abbott: Consultancy, Research Funding.

Description: Abstract 3890 Alternative splicing plays a fundamental role in human biology and its relevance in cancer is rapidly emerging. Recent advances in oligonucleotide sequencing demonstrate that alternative splicing and gene mutations involved in the spliceosome can play a role in tumorigenesis and cancer progression in haematologic malignancies, including chronic lymphocytic leukemia (CLL). However, there are few, if any, well-defined molecular probes that can be used to modulate splicing events in vitro or in vivo. As such, it is uncertain whether the spliceosome system can serve as a molecular target in cancer. We have addressed these questions using FD-895 and Pladienolide B, two bioactive polyketides that target the splicing factor SF3B. Primary leukemia cells from CLL patients were incubated with these two agents and the samples were analyzed for an early evidence of mRNA intron retention (splicing inhibition) and apoptosis. We observed that primary leukemia CLL cells incubated with FD-895 and Pladienolide B (10–1,000 nM) showed evidence of mRNA intron retention as early as 4 hours after incubation with increasing ratios of unspliced/spliced mRNA in several genes tested (DnaJ homolog subfamily B member 1, DNAJB1; RIO kinase 3, RIOK3; and X-box binding protein 1, XBP1). Contrary to that, neither Bendamustine (10–100 μM) nor Fludarabine (F-Ara-A, 10–100 μM) induced accumulation of unspliced mRNAs. CLL cells incubated with FD-895 and Pladienolide B (10–1,000 nM) underwent apoptosis with evidence of changes in mitochondrial transmembrane potential detected by DiOC6. Apoptosis was observed as early as 24 hours after incubation and required 3 2 hours of incubation with FD-895 and Pladienolide B. The IC50 for both agents was in the 100 nM range. Importantly, FD-895 and Pladienolide B both were able to induce apoptosis in CLL cells that were protected from spontaneous apoptosis using co-culture conditions with stromal cell support and despite the presence of high-risk prognostic factors such as ZAP-70 expression, unmutated IGHV and Del(17p), or the presence of SF3B1 mutation. In conclusion, we have shown for the first time that FD-895 and Pladienolide B induce early mRNA intron retention (splicing inhibition) in primary leukemia cells from patients with CLL and apoptosis at nanomolar concentrations in all patient samples tested (n=20). In vitro apoptosis was observed regardless of the presence of poor prognostic factors such as Del(17p) and SF3B1 mutations. In addition, the pro-apoptotic activity of FD-895 and Pladienolide B was present in CLL-stromal cell co-culture conditions that may resemble the protective tumor microenvironment. Our data presents evidence that the spliceosome system is a valid target in CLL and provides the rationale for the development of inhibitors that target this pathway. Disclosures: Kipps: Abbott: Consultancy, Research Funding.