Lipid uptake via FATP2 enhances CAR-t therapy resistance in B-cell acute lymphoblastic leukemia Free

Chimeric Antigen Receptor T cell (CAR-T) therapy is highly effective for treating relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). Critically, for those patients who initially respond to CAR-T therapy (~80–90%), approximately 50% will relapse within one year of T cell infusion (Laetsch et al., 2023; Maude et al., 2014; Pasquini et al., 2020). Short duration of CAR-T cell persistence and loss of CAR target antigen expression represent major relapse mechanisms (Shah & Fry, 2019). Notably, the current CAR-T paradigm suggests that despite prior selection for chemo-resistant leukemia cells in relapsed/refractory patients, CAR-T cells overcome chemotherapy resistance by killing antigen-positive leukemia cells via perforin and granzyme and do not rely on the genotoxic or metabolic insults elicited by chemotherapy. However, emerging clinical evidence suggests that additional leukemia-intrinsic factors contributing to chemotherapy resistance may be linked to suboptimal CAR-T responses.

Although TP53 mutations are uncommon at the time of pediatric B-ALL diagnosis—except in low-hypodiploid cases (Holmfeldt et al., 2013)—they are frequently acquired in relapsed or chemotherapy-refractory B-ALL (Hof et al., 2011) and have been linked to resistance to CD19-directed CAR-T therapy (Aldoss et al., 2025; Pan et al., 2020; Zhang et al., 2020). Therefore, there is an urgent need to understand how leukemia-intrinsic resistance mechanisms, such as TP53 mutations, contribute to CAR-T resistance. To test the impact of p53 activity in leukemia cell on CAR-T cell effectiveness, we generated multiple isogenic human CD19⁺ B-ALL cell lines harboring TP53 wild-type, a TP53 “hotspot” point mutation (p.G245D DNA-binding domain mutant), or TP53 frameshift mutations. Consistent with recent studies (Cox et al., 2025), we found TP53 mutations (point or frameshift) promote CAR-T resistance in human pre-B-ALL cell lines. Through genome-wide CRISPR/Cas9 screening of CAR-sensitive TP53 wildtype and CAR-resistant TP53-mutant CD19⁺ B-ALL cell lines, we identified Fatty Acid Transport Protein 2 (FATP2, encoded by SLC27A2) as a leukemia-intrinsic mechanism of CAR-T resistance in TP53-mutant B-ALL.

FATP2, a transmembrane protein, regulates lipid homeostasis by facilitating long-chain fatty acid (LCFA) transport and exhibits very long-chain acyl-CoA synthetase activity, modulating the metabolism of very long-chain fatty acids (Black et al., 2016). We found that CAR-T resistance in both FATP2-expressing TP53-mutant B-ALL cell lines and patient-derived TP53-mutant B-ALL xenografts is dependent on exogenous lipid uptake in vitro and in vivo. Using ¹³C-labeled LCFA tracing, we found that CAR-resistant FATP2-expressing TP53-mutant B-ALL cell lines exhibited increased levels of LCFAs, and fatty acid-derived acyl-carnitine intermediates compared to isogenic CAR-sensitive B-ALL cells (TP53 wildtype B-ALL or FATP2-deficient TP53-mutant B-ALL), consistent with elevated fatty acid oxidation (FAO). To assess whether lipid mobilization and mitochondrial import contribute to CAR resistance, we treated isogenic B-ALL cells with the lipase inhibitor Lalistat-1 (LAL1i) or the CPT1 inhibitor Etomoxir—blocking fatty acid liberation from lipid droplets or mitochondrial transport of FAO substrates, respectively—during Mock or CAR-T co-culture. In TP53 wild-type B-ALL, CAR-T treatment responses were unaffected by the addition of LAL1i or Etomoxir. In contrast, TP53-mutant B-ALL exhibited significantly enhanced CAR-T sensitivity in the presence of either inhibitor, suggesting that FAO supports resistance in TP53-mutant B-ALL.

These findings suggest a potential interaction between TP53 mutation status and fatty acid metabolism in mediating CAR-T resistance, although the precise regulatory relationship between p53 and SLC27A2/FATP2 remains unclear. Overall, our findings highlight a previously unappreciated link between lipid metabolism and CAR-T resistance in B-ALL. Future studies aim to understand the role of LCFA uptake in conventional chemotherapy responses, as well as determine whether pharmacological modulation of lipid uptake could enhance CAR-T efficacy, particularly in TP53-mutant disease.