Abstract In order to identify novel targets and clinically exploitable vulnerabilities, we performed high-throughput RNA-interference (RNAi) synthetic lethality screening using myeloid cell lines TF-1 and ML-2 in combination with the hypomethylating agent 5-Azacytidine. A custom small interfering RNA (siRNA) library targeting nearly 1000 cancer-associated genes, including the human kinome, was utilized. Bcl-XL emerged as the strongest 5-Azacytidine sensitizing target in the erythroid cell line TF-1 but not in ML-2 cells. Additional siRNA sequences targeting Bcl-XL were transfected into a panel of myeloid cell lines and tested in drug dose response experiments using a ten-point serial dilution of 5-Azacytidine. The erythroid differentiated myeloid cell lines TF-1 and HEL both exhibited a 3 to 4-fold reduction in the IC50 of 5-Azacytidine upon knockdown of Bcl-XL, whereas IC50 values for non-erythroid, myeloid cell lines ML-2, THP-1 and MDS-L were unchanged. Furthermore, we tested lentiviral delivery of short-hairpin RNA (shRNA) targeting Bcl-XL in additional cell lines. Lentiviral knockdown of Bcl-XL confirmed 2 to 3-fold 5-Azacytidine sensitization in TF-1 and HEL cells while U937, HL-60, ML-2, THP-1 and MDS-L were not sensitized by Bcl-XL knockdown. Interestingly, while Bcl-XL knockdown with shRNA potentiated the effects of 5-Azacytidine in TF-1 and HEL, Bcl-XL knockdown alone resulted in considerable reductions in cell viability in TF-1 and HEL cells. These results demonstrate erythroid lineage-specific responses to Bcl-XL knockdown and suggest that Bcl-XL may represent an erythroid lineage-specific therapeutic vulnerability. To begin to explore this possibility we examined the putative BH3 mimetics Gossypol, Obatoclax and ABT-737 with 5-Azacytidine drug dose response experiments in myeloid cell lines. Neither Gossypol nor Obatoclax potentiated the effects of 5-Azacytdine. In striking contrast, ABT-737 resulted in a 2 to 4-fold reduction in the IC50 of 5-Azacytidine in all cell lines tested to date (TF-1, HEL, ML-2 and THP-1). ABT-737 sensitization to 5-Azacytidine was dose dependent. Furthermore, Combination Index (CI) values calculated with Calcusyn software demonstrate synergy between ABT-737 and 5-Azacytidine at clinically relevant concentrations. These results suggest that inhibition of anti-apoptotic Bcl-2 family members with a BH3 mimetic may represent an effective strategy for 5-Azacytidine combination therapy that can be directly translated into design of combination clinical trials in patients with myeloid leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-128.
Identification of a potent natural triterpenoid inhibitor of proteosome chymotrypsin-like activity and NF-κB with antimyeloma activity in vitro and in vivo
AbstractAs multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, pristimerin. Strikingly, the early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-κB activation via inhibition of IKKα or IKKβ, whereas proteosome inhibitors instead suppress NF-κB function by impairing degradation of ubiquitinated IκB. By inhibiting both IKK and the proteosome, pristimerin causes overt suppression of constitutive NF-κB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-κB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC50 < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib—providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-κB inhibitors as therapeutics for human multiple myeloma and related malignancies.