ABT-199 IN COMBINATION WITH RITUXIMAB OR GS-9973: IMPLICATIONS FOR MACROPHAGE PHAGOCYTOSIS AND T-CELL MEDIATED ABT-199 RESISTANCE IN CLL PATIENTS

BORGE, MERCEDES; RISNIK, DENISE; POSE CABARCOS, JULIO; ALMEJÚN, MARÍA BELÉN; GAMBERALE, ROMINA; ELÍAS, ESTEBAN ENRIQUE; PODAZA, ENRIQUE; CABREJO, MARÍA; COLADO, ANA; GIORDANO, MIRTA; FERNÁNDEZ GRECCO, HORACIO; BEZARES, RAIMUNDO FERNANDO

New York

Workshop; XVII International Workshop on Chronic Lymphocytic Leukemia; 2017

Bio Ascend LLC

Introduction: Leukemic B cells from CLL patients survive and proliferate within lymphoid tissues receiving signals through the BCR and in close contact with activated T cells and myeloid cells. ABT-199, a potent and selective BCL-2 inhibitor, is highly cytotoxic against unstimulated peripheral blood CLL cells in vitro but is much less effective against CLL cells that have received survival signals from the microenvironment. Effective therapy should target both, leukemic cells and the protective microenvironment. Combination of ABT-199 with anti-CD20 MoAbs or BCR-associated kinase inhibitors appears as an attractive strategy. The aims of this study were: a) to evaluate the effect of ABT-199 on the tumor microenvironment, focusing on the activation of autologous T cell and in the capacity of macrophages to phagocyte CLL cells, and b) to evaluate the impact of ABT-199 on the survival of CLL cells that have received signals from autologous activated T cells.Methods:Peripheral blood mononuclear cells (PBMC) from CLL patients were cultured with vehicle (DMSO) or ABT-199 and cell survival was evaluated by flow cytometry comparing FSC and SSC parameters and/or by Annexin V FITC assay. PBMC were cultured on microplates coated with anti-CD3 MoAb (aCD3, 50 ng/well, 48-well plate) and the expression of CD25 and CD69 on T cells and CD86 on CLL cells was evaluated by flow cytometry. The ABT-199 induced cell death on resting and activated CLL with or without the BCR-associated kinase inhibitor GS-9973, was evaluated as mentioned above. Phagocytosis of CFSE-labeled CLL cells coated or not with anti-CD20 MoAb, Rituximab (Rx, 50 nM) was evaluated by flow cytometry. Results:PBMC from CLL patients (n=18) were cultured in the presence of ABT-199 or DMSO at 37˚C and cell survival was evaluated by flow cytometry. Apoptotic cells could be distinguished from viable cells because of their lower forward light scatter, consistent with reduction of cell size and cytoplasmic volume occurring during apoptosis (Figure 1A). We found that CLL cells were very sensitive to ABT-199 (Figure 1B) while T cells (CD3+ CD56-) or NK cells (CD56+ CD3-) were less sensitive to the drug (Figure 1C). In order to induce the activation of T cells, PBMC from CLL patients were cultured with aCD3, which induced the expected upregulation of the activation markers CD69 and CD25 on T cells at 24hs. We found that ABT-199 did not modify aCD3-induced CD69 expression on T cells (Figure 1D) and only slightly reduced CD25 expression (Figure 1E). As we and others have previously reported (Borge M, Cancer Immunol Immunother. 2013, 62:113 and Patten PE, Blood 2008, 111:5173), we confirmed that the activation of autologous T cells favored leukemic cell activation (Figure 1F). Interestingly, we observed that leukemic cells cultured in the presence of activated T cells for 48hs were clearly less sensitive to the drug compared to leukemic cells with of non-activated T cells (Figure 1G), showing that autologous T cell activation induced ABT-199 resistance in CLL patients. As we expected, GS-9973, which impairs T cell activation (Colado A, Cancer Immunol Immunother. 2016), overcomes aCD3-mediated resistance of CLL cells to ABT-199 (Figure 1H). Finally, since it was recently reported that combining ABT-199 with rituximab in CLL patients provides substantial benefit compared with ABT-199 monotherapy (Freise KJ, Hematological Oncology, 2016), we determined whether ABT-199 increases the phagocytosis of CLL cells. We found that ABT-199 increases annexin V expression on cultured cells (Figure 1I) as well as their phagocytosis by macrophages, in a dose dependent manner (Figure 1J). As expected, the presence of rituximab improved the phagocytosis CLL cells compared to uncoated-CLL cells (Fiugre 1J). Interestingly, ABT-199 allows a successful phagocytosis of rituximab-coated CLL cells which seems to be slightly enhanced by the drug even no statistically significance was reached.Conclusions:The ABT-199 resistance observed in vitro when CLL cells where cultured with autologous activated T cells suggests that leukemic cells from the supportive microenvironment might not be properly targeted by the drug. Moreover, our results encourage the combination of ABT-199 with therapeutic agents, such as GS-9973 which overcomes the resistance induced by activated-T cells or with CD20 MoAbs because the drug allows an efficient phagocytosis of coated-CLL cells.