Targeting BH3-Domain Anti-Apoptotic Proteins with GX15-070 Significantly Increases Rituximab-Mediated Antibody Dependent Cellular Cytotoxicity (ADCC) and Complement Mediated Cytotoxicity (CMC) Against B-Cell Lymphomas.

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There is a clinical need to develop innovative targeted therapies against B-cell lymphomas with both improved anti-tumor activity and more favorable toxicity profiles. The interactions between the members of the BH3 domain family of proteins play an important role in the development, progression and prognosis of various subtypes of B-cell lymphoma. We previously reported that downregulation of Bcl-2 using anti-sense oligonucleotides improved rituximab in vitro and in vivo activity (Ramanarayanan J, et al, BJH 2004; 127:519–30). GX15-070 is novel pan-inhibitor of bcl-2 anti-apoptotic proteins, and currently is undergoing clinical testing in various cancer settings. In our hope to increase the future treatment options for B-cell lymphoma patients we studied the biological effect(s) of GX15-070 on rituximab activity using the rituximab-sensitive cell lines (RSCL) Raji and RL. Baseline expression of BH3 domain proteins Bcl-2 was studied by Western blotting. NHL cells were exposed in vitro to escalating doses of GX15-070 (0, 2, 5, 10 and 20mM) for 24 and 48 hrs. Cell viability was then determined by trypan blue staining. Once the optimal dose and time of GX15-070 exposure was determined, cell growth inhibition and immunological assays were conducted to determine the effects of BH3-domain targeting on rituximab activity. NHL cells were exposed to different concentrations of GX15-070 (2 to 20mM) in the presence of rituximab, isotype or RPMI. Standard [3H]-Thymidine incorporation assays were performed to assess changes in DNA synthesis at 24 and 48 hrs. For ADCC/CMC assays, Raji or RL cells were exposed to GX15-070 (2 and 5mM) for 24 hrs and subsequently labeled with 51Cr. Labeled cells were then exposed to rituximab (10mg/ml) or isotype (trastuzumab, 10mg/ml) with peripheral blood mononuclear cells (effector: target ration of 40:1) or human serum (1:4 dilution), respectively. 51Cr-release was measured and the percentage of lysis calculated. Statistical differences were analyzed by Chi-square test. In vitro exposure of Raji and RL cells to GX15-070 resulted in a dose-dependent decrease in DNA synthesis and increase in cell death. In addition, pre-incubation of NHL cells to GX15-070 at 2 and 5mM for 24 hrs enhanced rituximab-mediated ADCC, and to a lesser degree CMC. The mean percentage of rituximab-associated ADCC on DMSO (vehicle) pre-treated RL and Raji cells was 20.3% +/−1.14%ste and 17.6% +/−1.07%ste. Exposure of RL cells to GX15-070 (5mM) for 24 hours prior to rituximab exposure lead to a statistically significant increase in antibody-mediated ADCC [mean % lysis 58.56% +/− 3.6%ste, P= 0.007]. Similar results were seen in Raji cells [mean % lysis increased to 42.6% +/−3.03%ste, P=0.026]. Rituximab-associated CMC was also increased by GX15-070. In summary, BH3 inhibition of anti-apoptotic proteins by GX15-070 induces cell death, cell growth arrest, and renders various NHL cell lines more susceptible to rituximab-associated ADCC and CMC. Ongoing studies in lymphoma xenografts will hopefully further validate the potential utility of GX15-070 in combination with rituximab and lead to Phase I clinical studies of this novel combination.