Abstract 5337: Tolinapant (ASTX660) enhances the anti-leukemic activity of Venetoclax and Dexamethasone in T cell acute lymphoblastic leukemia (T-ALL)
Authors: Sharma Priyanka , Piya Sujan , Ma Huaxian , Baran Natalia , Muftuoglu Muharrem , Basyal Mahesh , Ruvolo Vivian , Ward George , Smyth Tomoko , Sims Martin J. , Andreeff Michael , Borthakur Gautam 
Published: June 2022

Background: Inhibitors of apoptosis proteins (IAPs) are overexpressed in ALL leading to resistance to apoptosis and chemo-resistance. Tolinapant, a non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis (cIAP1/2 and XIAP) being evaluated in Phase 1/2 clinical trial [NCT02503423] showed single-agent activity in T-cell lymphoma. Objectives: Herein, we analyzed the preclinical activity of tolinapant in combination with ABT199 and Dexamethasone (DEX) in T-ALL (in vitro and ex-vivo patient derived xenografts (PDX).

Results: Using a panel of 8 T-ALL cell lines we analyzed the single agent activity of tolinapant. Loucy and SUP-T11 were most sensitive (IC50 = 190-309 nM). CCRF-CEM and ALL-SIL showed moderate sensitivity (IC50 = 10-18 µM) while Jurkat, MOLT16, MOLT4 and PF382 were resistant. Western blotting (WB) showed decreased levels of cIAP1and cIAP2 with no change in XIAP in response to tolinapant as a single agent. We next investigated the efficacy of tolinapant with Bcl2 inhibitor ABT199. The combination of ABT199 and ASTX660 was synergistic in Loucy cell line with combination index (CI) of 0.14. Cell death was increased to 64±3% in combination v/s 27±0.9% in ABT199. WB showed decreased cIAP2 and increased levels of cleaved caspase 7 and cleaved caspase 9 in combination compared to single agents, suggesting increased apoptosis. Treatment of PDX-derived cells with tolinapant and ABT199 was more effective than monotherapy in inducing apoptosis in CD45+ bulk (46±0.7% to 63±6%, p&lt;0.0001) and leukemia initiating cells (LICs, CD45+ CD7+ CD19- CD34+) (39±3% to 54±8%, p=0.003). Next, we checked the effect of combination of DEX with tolinapant in T-ALL cell lines. CCRF-CEM cell line is from a relapsed patient and is resistant to DEX. The combination was synergistic with CI of 0.26 and cell death of 50±4% compared to 20±3% by DEX. A triple combination of DEX, ABT199 and tolinapant increased apoptotic response to 82.9±1%, compared to DEX+ABT199 dual combination where apoptosis was induced to 52.4±2%. Strong synergy in terms of both cytoreduction as well as apoptosis induction was observed in SUP-T11 cells. Tolinapant sensitized SUP-T11 cells to DEX with ED50 value of 542 nM in combination compared to ~2 µM with DEX treatment. Simultaneous analysis of cell proliferation, stress response and DNA damage using single-cell proteomics analysis showed downregulation of proliferation (Ki-67), stress response (ATF4, LC3B) and increased levels of cleaved PARP, cleaved caspase 3 suggesting increased apoptosis in combination of tolinapant with DEX. Treatment of PDX-derived cells with tolinapant enhanced the cytotoxic effect of DEX in CD45+ bulk (58±2% to 71±0.1%, p&lt;0.0001) and LICs (62±2% to 78±0.9%, p&lt;0.0001).

Conclusion: Tolinapant synergizes with the anti-leukemic activity of ABT199 and DEX, establishing a therapeutic rationale for IAP antagonist in T-ALL.

Citation Format: Priyanka Sharma, Sujan Piya, Huaxian Ma, Natalia Baran, Muharrem Muftuoglu, Mahesh Basyal, Vivian Ruvolo, George Ward, Tomoko Smyth, Martin J. Sims, Michael Andreeff, Gautam Borthakur. Tolinapant (ASTX660) enhances the anti-leukemic activity of Venetoclax and Dexamethasone in T cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5337.