Abstract 5505: Synergistic anticancer activity of clinical stage, non-genotoxic apoptosis inducing agents RG7388 (MDM2 antagonist) and ABT-199 (GDC-0199, BCL2 inhibitor) in p53 wild-type AML tumor models

Abstract

Abstract MDM2, the most prominent natural inhibitor of the p53 tumor suppressor pathway, and BCL2, an anti-apoptotic regulator and important player in preventing induction of apoptosis in tumor cells, both play a pivotal role in leukemia cell survival and response to therapy in the clinic. The principal objectives of this study were to assess a synergistic antitumor effect by combining the clinical stage compounds RG7388, a MDM2 antagonist reactivating p53, and the BCL2 selective inhibitor GDC-0199 and to elucidate the underlying mechanism of action. Further insight into the biological rationale for this potential therapeutic strategy may pave the path for clinical combination studies in AML patients. We first investigated the effect of co-treatment with RG7388 and GDC-0199 on the viability and induction of apoptosis in AML tumor cell lines followed by high-resolution cell cycle kinetic analysis using BrdU-Hoechst quenching technology in-vitro. Cell viability assays demonstrated synergistic potency of combined RG7388 and GDC-0199 treatment with low nM IC50 values. Subsequent annexin binding assays confirmed the efficacy and revealed different kinetics of apoptosis induction. To further explain the mode of action, high-resolution cell cycle compartment analysis of consecutive cell cycles was performed. Mechanistically, RG7388 induces G1 arrest and preferentially causes nuclear fragmentation in G1 phase of the second cycle. The BCL2 inhibitor GDC-0199 mainly causes apoptosis in G1 compartments and hence cells transiently protected from apoptosis by RG7388 induced G1 arrest are hit by GDC-0199. In addition we analyzed changes in expression of pro- and anti-apoptotic genes and proteins upon combined treatment using qRT-PCR, Western Blot and Luminex technology. We identified down-modulation of MCL-1 levels, a known GDC-0199 resistance factor, as one potential mechanism for the synergistic efficacy and confirmed this finding by shRNA experiments in-vitro. Furthermore, the efficacy of the compounds was assessed in subcutaneous and orthotopic AML tumor cell line based xenograft models in-vivo, confirming the synergy observed in-vitro. Taken together, our study demonstrates that combined treatment with RG7388 and GDC-0199 shows a synergistic anticancer effect on AML cells. This indicates that activating the p53 tumor suppressor pathway and inhibiting a key anti-apoptosis target simultaneously induces a more than additive inhibitory effect by modulating the levels of key pro-survival proteins, such as Mcl-1, thereby further lowering the apoptotic threshold level for tumor cell death. Based on the observations from our study, the combined administration of RG7388 and GDC-0199 might offer a promising new therapeutic regimen for treating AML patients. Citation Format: Markus Dangl, Yuchen Chien, Christian Lehmann, Thomas Friess. Synergistic anticancer activity of clinical stage, non-genotoxic apoptosis inducing agents RG7388 (MDM2 antagonist) and ABT-199 (GDC-0199, BCL2 inhibitor) in p53 wild-type AML tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5505. doi:10.1158/1538-7445.AM2014-5505