AT-101-induced apoptosis in CLL cells is independent of cell adhesion-mediated drug resistance

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Chronic lymphocytic leukemia (CLL) is a disease with relentless accumulation of replicationally quiescent leukemic B-cells in bone marrow and peripheral blood owing to their longer survival. The survival advantage is due to over-expression of Bcl-2 family anti-apoptotic proteins in these lymphocytes. Evidence is also accumulating that interactions between CLL B cellsand accessory cells of microenvironment confer a growth advantage as well as extended cellsurvival, and cell-adhesion mediated drug resistance (CAM-DR) to these lymphocytes. Therefore,identification of agents that target anti-apoptotic proteins as well as can overcome survival of malignant B cells that is linked with the microenvironment may lead to new therapeutic avenues for CLL patients. AT-101, the [R-(-) gossypol] l-isomer, currently in clinic, binds to the BH3 motif of all major anti-apoptotic proteins with high affinity such as 230, 570, and 130 nM for Bcl-2, Bcl-xL, and Mcl-1, respectively. We hypothesized that in CLL cells, AT-101 would have inhibitory interactions with Bcl-2 family proteins, therefore diminishing the survival advantage. Here, we evaluated if AT-101 can induce apoptosis in CLL lymphocytes and simultaneously can overcome CAM-DR. CLL primary cells were incubated with AT-101 at increasing concentrations (1 - 30 µM) for 4, 8, and 24 h. There was time- and dose- dependent induction of apoptosis. Compared to time-matched controls, at 24 h with 20 µM AT-101, apoptosis was median 38% (range 7-78%, n=25). Samples from two patients did not respond (<10% apoptosis). Apoptosis induction was not related with Rai stage, WBC, prior treatment, B₂M level and ZAP-70 status. Pan-caspase inhibitor Z-VAD (50 µM) did not abrogate the AT-101-induced apoptosis (n=7), suggesting that the cell death was caspase-independent. Stroma has been shown to protect chemotherapy-induced cell death, hence we evaluated if stroma can also guard CLL cells from AT-101-mediated apoptosis. Lymphocytes from patients (n = 6) were co-cultured with M2-10B4 (the stromalcell line) and viability with or without stroma in presence or absence of AT-101 (20 µM) was determined. Endogenous apoptosis in CLL cells was comparatively low in presence of stroma (median 30% without stroma, 20% with stroma) that supports the observation of up-regulation of Bcl-2, Bcl-xL, and Mcl-1 proteins in co-cultured CLL cells. AT-101 significantly down-regulated expression of these proteins in CLL cells that correlates with cell death. Stroma cells were not able to abrogate AT-101-induced cytotoxicity (median 68% without stroma, 67% with stroma). AT-101 had minor effect on stroma cells itself (median 12% apoptosis), supporting the evidence of absence of AT-101 target, as these cells showed undetectable levels of anti-apoptotic proteins. Taken together, these data suggest that AT-101 was effective in inducing apoptosis specifically in leukemia cells, and is independent of CAM-DR.