Targeting BH3-Domain Anti-Apoptotic Proteins with ABT-737 Decreases DNA Synthesis, Induces Cell Death and Sensitizes Rituximab-Sensitive and Rituximab-Resistant Non-Hodgkin’s Lymphoma Cell Lines to the Anti-Tumor Activity of Chemotherapy Agents.

Abstract ABT-737 is a potent pan-bcl-2 inhibitor with known activity against chronic lymphocytic leukemia (CLL) cells and Mantle cell lymphoma (MCL) cell lines, currently undergoing phase I testing. Bcl-2 over-expression is associated with chemotherapy resistance and correlates with a poor clinical outcome in diffuse large B-cell lymphoma (DLBCL). Recently, we demonstrated that rituximab resistance is associated with deregulation of BH3-domain pro and anti-apoptotic proteins leading to concomitant resistance to several chemotherapy agents. Targeting BH3-domain anti-apoptotic proteins with ABT-737 is an attractive strategy to circumvent/overcome both acquired antibody and chemotherapy resistance. To this end, we studied the effects of ABT-737 in combination with rituximab or chemotherapy agents in a panel of rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL). NHL cell lines were exposed to escalating doses of ABT-737(01, 0.01, 0.1, 1, 10, 20, 50 and 100 mM); changes in mitochondrial potential and ATP production were determined by alamar blue reduction or celltiter-glo® luminescent cell viability assays at different time periods, respectively. Once the optimal dose and time of ABT-737 exposure was determined, cell growth inhibition and immunological assays were conducted to determine the effects of BH3-domain targeting on rituximab activity utilizing standardized immunological assays evaluating changes in DNA synthesis, as well as rituximab-mediated complement mediated cytotoxicity (CMC) and antibody dependent cellular cytotoxicity (ADCC). In addition, lymphoma cells were exposed in vitro to ABT-737 (0 to 100mM) with or without CDDP (0 to 10mM) or Doxorubicin (0 to 1mM). Following a 24 and a 48 hour-period of drug exposure, induction of apoptosis was determined by caspase-3 activity assays. ABT-737 induced a dose-dependent cell death in various NHL cell lines. Anti-tumor activity correlated directly with baseline Bcl-2 and inversely to Mcl-1 levels. Up to 75% of cell death was observed in all cell lines exposed to 10–100mM of ABT-737. Pre-incubation of NHL cells to ABT-737 at 10mM enhanced rituximab-mediated CMC. The mean percentage of rituximab-associated CMC on vehicle pre-treated Raji cells was 18% versus 30% for Raji cells exposed to ABT-737. Rituximab-associated ADCC was not affected by ABT-737. In addition, in vitro exposure of NHL cells to ABT-737 in combination with either CDDP or Doxorubicin induced a three fold increase in caspase-3 activity as compared to that achieved by each chemotherapy agent alone. In summary, our data strongly suggests that ABT-737 is active against various RSCL and RRCL and augments the anti-tumor activity of rituximab and chemotherapy agents. Ongoing studies in lymphoma xenografts and fresh primary lymphoma patient samples will further validate the use of ABT-737 in combination with rituximab and/or chemotherapy and give further support for their use in clinical trials.