The CXCR4 receptor (Chemokine C-X-C motif receptor 4) is highly expressed in different hematological malignancies including chronic lymphocytic leukemia (CLL). The CXCR4 ligand (CXCL12) stimulates CXCR4 promoting cell survival and proliferation, and may contribute to the tropism of leukemia cells towards lymphoid tissues. Therefore, strategies targeting CXCR4 may constitute an effective therapeutic approach for CLL. To address that question, we studied the effect of Ulocuplumab (BMS-936564), a fully human IgG4 anti-CXCR4 antibody, using a stroma – CLL cells co-culture model. We found that Ulocuplumab (BMS-936564) inhibited CXCL12 mediated CXCR4 activation-migration of CLL cells at nanomolar concentrations. This effect was comparable to AMD3100 (Plerixafor - Mozobil), a small molecule CXCR4 inhibitor. However, Ulocuplumab (BMS-936564) but not AMD3100 induced apoptosis in CLL at nanomolar concentrations in the presence or absence of stromal cell support. This pro-apoptotic effect was independent of CLL high-risk prognostic markers, was associated with production of reactive oxygen species and did not require caspase activation. Overall, these findings are evidence that Ulocuplumab (BMS-936564) has biological activity in CLL, highlight the relevance of the CXCR4-CXCL12 pathway as a therapeutic target in CLL, and provide biological rationale for ongoing clinical trials in CLL and other hematological malignancies.
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.
Expression of CXCR4 receptor (CD184) has been reported in different malignancies including Chronic Lymphocytic Leukemia (CLL) and the CXCR4-CXCL12 axis has been described to play a very important role in cancer development. Moreover, this pathway can be inhibited using CXCR4 antagonists. Previously, we have shown that BMS-936564, an anti-CXCR4 IgG4 antibody, can block in vitro the CXCR4/CXCL12 pathway in CLL overcoming stromal cell protection by inducing apoptosis, inhibiting F-actin polymerization, and migration of cells. These findings provide the rationale for an ongoing phase I clinical study in which CLL subjects received four weekly infusions of BMS-936564 antibody followed by 5 monthly cycles of bendamustine, rituximab and BMS-936564 antibody (BRB chemoimmunotherapy). Eleven (n=11) subjects with relapse / refractory disease have been enrolled and treated in this study. Here, we present correlative studies performed in three of those CLL subjects with samples collected at different time points (day 1, 2, 8, 15 and 22) during the monotherapy cycle of BMS-936564. All three subjects showed leukocytosis that was detected as early as in 4 hours following the initial BMS-936564 infusion (median increase of 64.6% above base line; range: 59.7% - 112.7%). Leukocytosis was present during the entire four weeks of monotherapy with BMS-936564. Leukocytosis in these three patients was due primarily to increase in absolute counts of CLL cells (Median increase of 129.6%; range: 95.3% - 324.8%). Interestingly, there was no evidence of increase in the absolute number of normal lymphocytes. Only one of the three patients showed an increase in neutrophil counts after infusion of BMS-936564. We observed changes in the level of CXCR4 expression after infusion with BMS-936564. All subjects showed CXCR4 down-regulation in peripheral CLL cells with a median percentage decrease in the level of expression of 106.7% (Range: 25.1% - 350.7%). We did not observe changes in CXCR4 expression in normal B cells. In contrast to our in vitro studies where we observed that all tested CLL samples underwent apoptosis after treatment with BMS-936564, only one subject showed significant increase in the percentage of apoptosis after infusion of BMS-936564 in vivo. The levels of apoptosis detected were relatively low (<18%) and this suggests a potential mechanism of rapid clearance of apoptotic cells. Importantly, associated with the mobilization of CLL cells, all three patients showed significant decrease in the lymphadenopathy size with a median decrease of 59.83% (Range: 55.6-72%). Lymphadenopathy response was assessed using stringent IWCLL response assessment criteria by an independent radiologist using CT scan measurements. All three subjects tolerated the infusions well with no evidence of side effects or toxicities. After the initial monotherapy treatment phase with BMS-936564 antibody, all three subjects received BRB chemoimmunotherapy combination and had a dramatic decrease in lymphocytosis during the first week after initiation of combination therapy. These subjects are currently finishing treatment under protocol and response assessments will be presented during the meeting. In conclusion, we found that BMS-936564 infusion in subjects with CLL induces specific leukemia cell mobilization associated with an objective decrease in the lymphadenopathy bulk and phenotypic changes associated with CXCR4 down-regulation in peripheral leukemia cells. These findings strongly suggest a CXCR4-CXCL12 dependent mobilization process of CLL cells from lymphatic tissues. Together, our study provides for the first time evidence at in vivo level, which supports inhibition of the CXCR4-CXCL12 pathway mediated by BMS-936564 in CLL patients. These findings suggest a compartmental biological distribution and recirculation of CLL cells opening the possibility to explore targeted therapies against minimal residual disease that traditionally has been refractory to conventional chemoimmunotherapy treatment and contributes to the incurable characteristics of this disease. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Kipps:Bristol-Myers Squibb: Research Funding. Cardarelli:Bristol-Myers Squibb: Employment.
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.
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