Abstract 4690: Novel Ring1B-Bmi1 small molecule inhibitors target leukemia stem cells

Abstract

Abstract Leukemia is a clonal neoplastic disorder which possesses a rare population of cells, termed leukemia stem cells (LSCs), capable of limitless self-renewal and are necessary to initiate, sustain or regenerate the disease. Targeting LSCs self-renewal not only would offer substantial therapeutic benefit but also likely to be essential and sufficient for selective eradication of LSCs. BMI1, polycomb group protein, has been identified and shown to be required for self-renewal of LSCs. Bmi1 alongwith Ring1B constitute enzymatic active unit of Poly-comb Repressive Complex 1(PRC1) and execute gene silencing through histone H2A lysine119 ubiquitination (H2AK119ub). Considering Bmi1 as a key regulator of self-renewal, targeting Bmi1 activity could impair LSCs self-renewal ability and may represent a new therapeutic approach for leukemia. Thus, in this study we aimed to develop and characterize novel small molecule inhibitors for Ring1B - Bmi1. Using NMR fragment-based screening and extensive medicinal chemistry optimization we developed WY332 that binds to Ring1B-Bmi1 with low micromolar affinity. Further characterization of the WY332 demonstrated selective reduction in Ring1B-Bmi1 mediated H2AK119ub levels in in vitro and in leukemic cells. Contrary, structurally similar and less potent compound displayed similar effect at much higher concentrations establishes that higher binding affinity of potent inhibitor is associated with blocking Ring1B-Bmi1 E3 ligase activity. Further evaluation of lead inhibitor, WY332, in colony formation assay using leukemic cells demonstrated significant decrease in the number and size of the colonies indicated that interference with Ring1B- Bmi1 activity alter the frequency of LSCs colony forming potential. Moreover, expression analysis of CD11b and CD34, cell surface markers associated with myeloid differentiation and AML LSCs phenotype, in WY332 treated TEX cells revealed marked increase in CD11b expression and significantly decreased CD34 expression establishes the specific effect of our Ring1B-Bmi1 small molecule inhibitors on induction of differentiation in leukemic cells. Interestingly, gene expression analysis of TEX cells treated with WY332 revealed significant increase in expression of genes suppressed in LSCs. Overall, these compounds represent the novel small molecule inhibitors of Ring1B-Bmi1 which inhibit E3 ubiquitin ligase activity and impair leukemic stem cell self-renewal capacity. Citation Format: Shirish Shukla, Felicia Gray, Weijang Ying, Hyoje Cho, Qingjie Zhao, Hongzhi Miao, Trupta Purohit, Jolanta Grembecka, Tomasz Cierpicki. Novel Ring1B-Bmi1 small molecule inhibitors target leukemia stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4690.