Inhibiting Free Fatty Acid Transport to Improve CAR-T Cell Therapy of Relapsed B-cell Acute Lymphoblastic Leukemia

Inhibiting Free Fatty Acid Transport to Improve CAR-T Cell Therapy of Relapsed B-cell Acute Lymphoblastic Leukemia Abstract Chemotherapy-resistant B-cell acute lymphoblastic leukemia (B-ALL) remains a leading cause of cancer-related death for children and young adults. While CD19-directed chimeric antigen receptor T cell (CAR-T) therapy offers promise for relapsed/refractory cases, high long-term failure rates necessitate a deeper understanding of resistance mechanisms. Our preliminary studies highlight free fatty acid (FFA) uptake inhibition as a potential metabolic vulnerability in human B-ALL to enhance CAR-T efficacy, particularly in cases harboring TP53 mutations associated with relapse and chemotherapy resistance. Using genome-wide CRISPR/Cas9 screening on TP53-wildtype and TP53-mutated CD19+ B-ALL cell lines exposed to CD19-CAR-T treatment, we identified the targeting of fatty acid transporter FATP2 (encoded by SLC27A2) as a candidate for enhancing CAR-T efficacy. Subsequent pediatric B-ALL patient cohort analyses correlated high SLC27A2 mRNA expression with worse overall survival. FATP2-expressing B-ALL lines exhibited increased long-chain FFA uptake, with in vitro studies validating a leukemia-intrinsic role for FATP2 in promoting CD19 CAR-T resistance, with reversible effects upon depleting exogenous FFA. Stable isotope FFA uptake tracing revealed a role for FATP2 in promoting succinate accumulation in TP53-mutated B-ALL cells, which may impact leukemic gene expression through α-ketoglutarate inhibition. This proposal seeks to evaluate exogenous FFA uptake inhibition via genetic and pharmacological FATP2 inhibition to overcome CAR-T resistance in human B-ALL cell lines in vivo and primary B-ALL patient biospecimens ex vivo. Through metabolomic analyses and stable isotope-labeled linoleic acid tracing, we will then elucidate the precise metabolic pathways impacted by FATP2-dependent FFA utilization in primary human B-ALL, including investigating the mechanistic impact of succinate accumulation on TP53-mutated B-ALL gene expression and its role in CAR-T responsiveness. This interdisciplinary research seeks to enhance CD19 CAR-T therapy for relapsed/refractory B-ALL by targeting FFA uptake and uncovering gene-regulatory changes. The study aims to determine whether succinate or related metabolic intermediaries mediate B-ALL cell survival during CAR-T therapy. The comprehensive approach leverages the expertise of a diverse research team, ensuring a nuanced understanding of B-ALL biology and potential avenues for improving CAR-T-treated B-ALL patient outcomes.