The XIAP Inhibitor BMT-062789 Displays Anti-Tumor Activity in Cell Line Models of De Novo and Rituximab Relapse/Refractory Lymphoma

The addition of rituximab to front line therapy for aggressive lymphomas has improved clinical outcomes, but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for rituximab associated chemotherapy cross-resistance our group developed several rituximab resistance cell lines (Raji 4RH and RL 4RH), which also display significant resistance to a wide range of chemotherapy agents. These rituximab resistant cell lines (RRCL)s exhibit multiple deregulations in the BCL-2 and inhibitor of apoptosis (IAP) protein families, including loss of the pro-apoptotic proteins Bax and Bak. We previously demonstrated that the X linked inhibitor of apoptosis protein (XIAP) is critically required for chemotherapy resistance in the RRCLs, and that an shRNA knockdown of XIAP increased chemotherapy response in both in vitro and in vivo models of rituximab resistant lymphoma. BMT-062789 is a heterodimeric mimetic of the second mitochondrial activator of caspases (SMAC) developed by Bristol-Myers Squibb, which can inhibit both the caspase 9 and caspase 3/7 binding domains of XIAP. BMT-062789 demonstrated dose and time dependent single agent anti-tumor effect (as measured by the Cell TiterGlo luminescent viability assay) in a panel of lymphoma cell lines, with IC₅₀ values of less than 5uM for all cell lines tested except the rituximab resistant cell line Raji 4RH. The Burkitt's lymphoma cell line Daudi and diffuse large B-cell lymphoma cell line U2932 were particularly sensitive to BMT-062789 with IC₅₀ values of 0.91uM and 0.76uM respectively. To investigate if the observed anti-tumor effect of BMT-062789 was due to increased apoptosis we exposed rituximab sensitive (Raji, RL) and rituximab resistant (Raji 4RH, RL 4RH) cells to escalating doses of BMT-062789 with or without the addition of 20uM etoposide for 48 hours. The induction of apoptosis was measured by flow cytometry with an Annexin-V:PE-Cy7 conjugate and Sytox blue (a DNA stain). 2uM BMT-062789 alone triggered apoptosis in 75% of Raji cells and 65% of RL cells. It is also worth adding that 2uM BMT-062789 induced higher rates of apoptosis than 20uM etoposide alone in both cell lines. The combination of 2uM BMT-062789 and 20uM etoposide together triggered apoptosis in 80% of Raji cells and 85% of RL cells, indicating that BMT-062789 may be able to augment the anti-tumor activity of conventional chemotherapy. More importantly, the combination of BMT-062789 and etoposide was also able to induce apoptosis in the rituximab resistant cell line models Raji 4RH and RL 4RH. 3uM BMT-062789 in combination with 20uM etoposide triggered apoptosis in 90% of Raji 4RH cells and 55% of the RL 4RH cells, which is a substantial improvement compared to 15% apoptosis with 20uM etoposide alone in each cell line. Additional studies are in progress to evaluate the anti-tumor effect of BMT-062789 in ex vivo samples from lymphoma patients with de novo and relapse/refractory disease. In summary, the novel heterodimeric XIAP inhibitor BMT-062789 has anti-tumor effect at low micromolar concentrations in lymphoma cell line models, including models of rituximab resistant disease. These results support earlier studies by our group indicating that XIAP is critical for survival in models of rituximab resistant lymphoma, and establish that XIAP inhibitors may have potential clinical value for the treatment of both de novo, and rituximab relapse/refractory lymphomas.