BH3 Profiling in AML to Measure Apoptotic Signaling Kinetics of Approved Drugs and Identify Integrated Stress Response Apoptotic Susceptibilities - Acute myeloid leukemia (AML) is a devastating hematologic malignancy with poor outcomes and is characterized by uncontrolled proliferation of immature myeloid blasts, cytopenia, infections, and bone marrow failure. Although AML can present in individuals of all ages, AML is more frequently diagnosed in elderly individuals where the median age at diagnosis is 68. Intensive “7+3” combination chemotherapy (cytarabine + daunorubicin) was adopted five decades ago and remains the standard of care. Cytotoxic therapies induce apoptotic signaling that converges on mitochondrial-localized BCL-2 family proteins that constitute pro-apoptotic (e.g., Bax, Bak, BH3 only proteins) and anti-apoptotic (e.g., BCL-2, MCL-1, Bcl-xL) members. The balance between pro-apoptotic and anti-apoptotic BCL-2 family proteins controls mitochondrial outer membrane permeabilization and apoptosis. Leveraging the unique biology surrounding programmed mitochondrial apoptotic cell death, we developed dynamic BH3 profiling (DBP) to measure mitochondrial apoptotic priming and the extent anti-cancer drugs induce apoptotic signaling. Using DBP, we previously identified a targetable BCL-2 dependency in AML and demonstrated the potent anti-leukemic properties of the BCL-2 inhibitor venetoclax. These findings supported the initiation of successful clinical trials that led to venetoclax approval in combination with low-dose cytarabine or hypomethylating agents (azacitidine or decitabine). However, incomplete responses and resistance due in part to reduced apoptotic priming is a clinical challenge. Thus, an improved understanding of apoptotic signaling is necessary to inform treatment strategies and identify actionable therapeutic vulnerabilities. Although combination therapies are the backbone of AML induction therapy, no study has systematically evaluated apoptotic signaling kinetics to inform the timing of approved AML drugs. Preliminary data demonstrated that different anti-cancer drugs induce variable apoptotic priming kinetics. As apoptotic priming predicts in vivo therapeutic efficacy of cancer drugs, knowledge of priming kinetics may enhance the efficacy of existing drugs. Moreover, I showed in the F99 phase that the integrated stress response (ISR) modulates venetoclax cytotoxicity in AML. The ISR is an evolutionarily conserved process used by cells to regulate protein synthesis and adapt to environmental or pathogenic stressors. ISR activation can paradoxically serve pro- survival or pro-death functions in a context-dependent manner. I demonstrated that ISR inhibition with ISRIB blunted venetoclax cytotoxicity in AML cells. These data suggest an interplay between the ISR and apoptotic signaling which remains incompletely understood. Thus, in the K00 phase I will use DBP to 1) measure the apoptotic signaling kinetics of approved AML drugs to inform optimal combination timing, and 2) evaluate the relationship between apoptotic signaling and the ISR in AML. Our overarching hypothesis is that elucidating apoptotic priming kinetics of approved AML drugs and understanding the interface between apoptotic signaling and the ISR will enhance existing drug combinations and identify ISR-linked vulnerabilities.
