Reduction In Mitochondrial Priming Drives Resistance To Targeted Therapy In Acute Myeloid Leukemia.

Abstract Relapse is the leading cause of treatment failure in acute myeloid leukemia (AML) patients. FDA approval of 8 targeted therapies in the past two years has drastically altered the landscape of AML treatment. Despite this success, the duration of clinical response is limited by the frequent development of acquired drug resistance to targeted therapy. To identify mechanism of resistance and to search for therapies that overcome resistance, we adopted broadly applicable functional approach to precision medicine called “dynamic BH3 profiling” (DBP) and coupled it with RNA-seq and targeted exome sequencing technology. DBP measures drug induced early death signaling using BH3 peptides that mimic pro-apoptotic BH3 proteins. To first validate the utility of DBP as precision medicine tool we created landscapes of pharmacologic sensitivity of 17 patient-derived xenograft (PDX) AML models to a panel of 40 clinically relevant agents, together with genomic and transcriptomic profiles. Aggregated across the panel, unsupervised clustering of drug-induced apoptotic signaling using DBP by itself could segregated PDXs according to prior treatment status (PDXs from treatment naïve patients clustered distinctly from R/R PDXs). While genomic mutations and transcriptomic signature profiles between R/R and treatment naïve PDXs did not show significantly distinct clustering patterns. Next we show that DBP could predict in vivo responses of drugs of widely varying mechanism of action, including a FLT-3 inhibitor, BCL-2 and MCL-1 inhibitors (BH3 mimetics), SMAC mimetic, and BRD4 inhibitor, in 6 AML PDX models (AUC of ROC 0.8731, p<0.005). Next, we created resistant PDX models to single agents including, quizartinib, birinapant, venetoclax, S63845 and JQ-1. After selecting for in vivo acquired resistance to drugs with distinct mechanism, a common mechanism of resistance was identified for all - a reduction in mitochondrial apoptotic priming. Apart from PDX models, paired pretreatment and post relapse myeloblasts of patients who had complete response followed by a relapse on venetoclax plus azacytidine therapy (NCT02203773) also showed selection for decreased mitochondrial apoptotic priming in relapsed myeloblasts using promiscuously interacting BIM (P=0.0075) and PUMA peptides (P=0.0078). Using targeted exome sequencing for recurrently mutated leukemia genes, we found that although there was acquisition of new mutations between paired clinical samples, there was no consistent molecular signature defining relapsed phenotype. We report that loss in apoptotic priming in BH3 mimetics resistant PDXs can be explained by alterations in BCL-2 family proteins levels and interaction patterns at outer mitochondrial membrane that vary among cases. However, there was absence of gene signature depicting reduction in pro-apoptotic genes and upregulation in anti-apoptotic genes, measured by unbiased RNA-seq (Bhatt et al. Cancer cell, In press). Enrichment for pro-survival pathways, including JAK-STAT, MAPK, and PI3K-AKT was observed using RNA-seq of resistant PDXs compared to matched parental counterpart in BH3-mimetics and FLT-3 inhibitor resistant models but not SMAC-mimetics and BRD-4 resistant models. To identify the agents that are effective in the resistant settings, we compared DBP profiles of 40 targeted agents in myeloblasts of pre and post resistant models. We found that reduction in overall priming led to broad chemoresistance even to mechanistically distinct agents in resistant myeloblasts, yet there was maintenance of persistent sensitivity to selected agents. For instance, in venetoclax and S63845 resistant PDXs, mitochondrial priming measured by DBP on resistant myeloblasts identified in vivo activity of FLT-3 inhibitors and SMAC mimetics while in quizartinib resistant settings, SMAC mimetics, BH3 mimetics and MAPK inhibitors showed anti-leukemic effects. This suggested that while common modes of mechanism do exist, drugs that enhance mitochondrial apoptotic sensitivity can overcome resistance to a particular agents. Finally we applied this approach to humans, showing that the pretreatment mitochondrial apoptotic priming determined by DBP identifies responders to single agent FLT-3 inhibitor gilteritinib in ADMIRAL trial and responders to lenalidomide (LEN) and MEC combination therapy in LEN-MEC Phase I trial (NCT01442714) R/R AML (Garcia* and Bhatt* et al. American Journal of hematology, 2020). In summary, our results suggest that acquired resistance to targeted therapy in AML is accompanied by common mechanism of reduction in mitochondrial priming along with drug-specific resistance mechanisms. Hence measurements of apoptotic priming using dynamic BH3 profiling may serve as a broadly applicable precision medicine tool in guiding therapy for relapsed leukemia. Citation Format: Shruti Bhatt, Marissa S. Piosos, Elyse A. Olesinski, Binyam G. Yilma, Jeremy A. Ryan, Thelma Mashaka, Buon Leutz, Sophia Adamia, David M. Weinstock, Jacqueline S. Garcia, Anthony Letai. Reduction in mitochondrial priming drives resistance to targeted therapy in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG14.