Integrating Genomics and Functional Phenotypes to Refine Risk Assessment in Acute Myeloid Leukemia across the Age Spectrum: Insights from Singapore

Background Despite recent advances, the reasons for discrepancies between ELN risk assessment and clinical outcomes in acute myeloid leukemia (AML) remain poorly understood. This creates an opportunity to explore additional predictive biomarkers using functional phenotypic analysis. Most cancer therapies induce cell death by activating the mitochondrial apoptotic pathway. Therefore, measuring the apoptotic threshold maintained between pro-survival proteins (BCL-2, BCL-XL, and MCL-1) and pro-apoptotic proteins through BH3 profiling (BP) and dynamic BH3 profiling (DBP) can provide valuable information beyond known genetic risk factors in acute leukemia. We propose that variations in apoptosis thresholds, assessed through BP and DBP, can offer insights into the risk, therapeutic response, treatment options, and outcomes of acute leukemia. Assessing patient-specific dependencies on BCL-2 family proteins can guide the selection of BH3 mimetic and chemotherapy combinations for potential therapies. Methods We exposed blasts from bone marrow aspirates or peripheral blood to various pro-apoptotic BH3 peptides and measured mitochondrial outer membrane permeabilization using BP. We examined pro-survival BCL-2 family members (BCL-2, BCL-XL, and MCL-1) and their sensitivity to BAD, HRK, and MS-1 peptides to identify factors influencing differential chemosensitivity to BH3-mimetics in these malignancies. We evaluated 30 pediatric and 43 adult AML patients, and 70 pediatric and six adult ALL patients. The samples included 96 at diagnosis, 46 at relapse, and seven during treatment. For DBP, nine pediatric AML tumors were treated with cytarabine, daunorubicin, etoposide, and fludarabine, while 12 T-ALL samples were treated with nelarabine, vincristine, dasatinib, asparaginase, and dexamethasone. We analyzed delta priming to assess chemotherapy response in vitro and conducted bulk RNA-seq of pediatric AML samples to understand differential gene expression using DESeq2 version 1.42.0. Our analysis aimed to comprehensively understand acute leukemia. Results We present an initial analysis of functional BH3 profiling on 149 primary tumor samples (73 AML, 29 T-ALL, and 47 B-ALL cases) with complete clinical and molecular details from three cancer centers in Singapore. Pediatric favorable-risk AML demonstrated significantly higher susceptibility to apoptosis than non-favorable-risk AML (p<0.05), measured by cytochrome c release when exposed to BIM peptide (mean 60% ± 20% for favorable risk vs. 27% ± 15% for non-favorable risk). Adult AML showed higher mitochondrial priming in favorable risk group compared to intermediate/adverse risk group, indicating apoptotic sensitivities correlates with ELN risk. Pediatric AML (N=30) exhibited greater priming than adults (N=43) (p<0.001), potentially explaining functional basis of improved survival outcomes in children vs adults. Direct mitochondrial sensitivity to venetoclax in lymphoblasts was higher in T-ALL (n=29) than in B-ALL (n=47). Lymphoblasts showed greater direct mitochondrial sensitivity to navitoclax, suggesting the potential use of dual inhibition of BCL2 and BCL-XL in ALL. Ex vivo apoptotic priming was enhanced after etoposide treatment in myeloblasts of 50% of pediatric AML cases refractory to standard induction. Differential gene signatures derived from RNAseq identified pathways associated with low and high apoptotic sensitivity in pediatric AML patients. Conclusions This study demonstrates the potential of integrating mitochondrial priming phenotypes with genetic data to refine AML risk assessment. Our approach facilitates the evaluation of treatment sensitivity at the cellular level to address limitations in current ELN risk stratification where favorable-risk patients may relapse, and some adverse-risk patients achieve good outcomes. Incorporating functional BH3 profiling into diagnostic assessments could enhance contemporary risk stratification strategies for both pediatric and adult AML patients. The consistent sensitivity of lymphoblasts and myeloblasts to BCL-2 inhibition across age groups and lineages suggests the broad applicability of our approach. By integrating cellular vulnerabilities to anti-apoptotic proteins into clinical assessments, we may significantly improve treatment decision-making and risk stratification in acute leukemia.