ABT-199 in combination with rituximab or GS-9973: implications for macrophage phagocytosis and T-cell mediated ABT-199 resistance in CLL patients

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

New York

Workshop; XVII International Workshop on Chronic Lymphocytic Leukemia; 2017

Introduction: Leukemic B cells from CLL patients survive and proliferate withinlymphoid tissues receiving signals through the BCR and in close contact withactivated T cells and myeloid cells. ABT-199, a potent and selective BCL-2inhibitor, is highly cytotoxic against unstimulated peripheral blood CLL cells invitro but is much less effective against CLL cells that have received survivalsignals from the microenvironment. Effective therapy should target both,leukemic cells and the protective microenvironment. Combination of ABT-199with anti-CD20 MoAbs or BCR-associated kinase inhibitors appears as anattractive 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 Tcell and in the capacity of macrophages to phagocyte CLL cells, and b) to evaluatethe impact of ABT-199 on the survival of CLL cells that have received signalsfrom autologous activated T cells.Methods:Peripheral blood mononuclear cells(PBMC) from CLL patients were cultured with vehicle (DMSO) or ABT-199 andcell survival was evaluated by flow cytometry comparing FSC and SSCparameters and/or by Annexin V FITC assay. PBMC were cultured on microplatescoated with anti-CD3 MoAb (aCD3, 50 ng/well, 48-well plate) and the expressionof CD25 and CD69 on T cells and CD86 on CLL cells was evaluated by flowcytometry. The ABT-199 induced cell death on resting and activated CLL with orwithout the BCR-associated kinase inhibitor GS-9973, was evaluated asmentioned above. Phagocytosis of CFSE-labeled CLL cells coated or not withanti-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 lowerforward light scatter, consistent with reduction of cell size and cytoplasmicvolume occurring during apoptosis (Figure 1A). We found that CLL cells werevery 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 theactivation of T cells, PBMC from CLL patients were cultured with aCD3, whichinduced the expected upregulation of the activation markers CD69 and CD25 onT cells at 24hs. We found that ABT-199 did not modify aCD3-induced CD69expression on T cells (Figure 1D) and only slightly reduced CD25 expression(Figure 1E). As we and others have previously reported (Borge M, CancerImmunol Immunother. 2013, 62:113 and Patten PE, Blood 2008, 111:5173), weconfirmed that the activation of autologous T cells favored leukemic cellactivation (Figure 1F). Interestingly, we observed that leukemic cells cultured in the presence of activated T cells for 48hs were clearly less sensitive to the drugcompared to leukemic cells with of non-activated T cells (Figure 1G), showingthat autologous T cell activation induced ABT-199 resistance in CLL patients. Aswe expected, GS-9973, which impairs T cell activation (Colado A, CancerImmunol Immunother. 2016), overcomes aCD3-mediated resistance of CLL cellsto ABT-199 (Figure 1H). Finally, since it was recently reported that combiningABT-199 with rituximab in CLL patients provides substantial benefit comparedwith ABT-199 monotherapy (Freise KJ, Hematological Oncology, 2016), wedetermined whether ABT-199 increases the phagocytosis of CLL cells. We foundthat ABT-199 increases annexin V expression on cultured cells (Figure 1I) as wellas their phagocytosis by macrophages, in a dose dependent manner (Figure 1J).As expected, the presence of rituximab improved the phagocytosis CLL cellscompared to uncoated-CLL cells (Fiugre 1J). Interestingly, ABT-199 allows asuccessful phagocytosis of rituximab-coated CLL cells which seems to be slightlyenhanced by the drug even no statistically significance wasreached.Conclusions:The ABT-199 resistance observed in vitro when CLL cellswhere cultured with autologous activated T cells suggests that leukemic cells fromthe supportive microenvironment might not be properly targeted by the drug.Moreover, our results encourage the combination of ABT-199 with therapeuticagents, such as GS-9973 which overcomes the resistance induced by activated-Tcells or with CD20 MoAbs because the drug allows an efficient phagocytosis ofcoated-CLL cells