M6A-driven control of T cell fitness via lrpprc defines a novel regulatory axis to enhance adoptive cell therapy Free

Adoptive cellular therapies (ACT) including chimeric antigen receptor (CAR)-T cells have transformed the treatment landscape for patients with relapsed/refractory hematologic malignancies. However, this therapy has not achieved similar success for patients with solid tumors due to the poor persistence and function of ACT in the immunosuppressive environment of solid cancers. Emerging data suggests that the efficacy of ACT is directly linked to the degree of representation of T memory stem cells (T_scm) in the product. T_scm are a minimally differentiated subset of T cells that are maintained over long periods in vivo and have the capacity to repopulate the full spectrum of memory and effector T cells, which makes this cell subset critical to an effective antitumor T cell response. While numerous studies highlight the therapeutic relevance and importance of this T cell population, the pathways critical to the generation of these cells and the process by which the production of T_scm is finely tuned is poorly understood.

Previously, our group demonstrated in an immunocompetent mouse model that STING (stimulator of interferon genes) agonists like cGAMP could enhance CAR T cell persistence and function through increased T_scmproduction, which was most significant using T_h/T_c17 CAR T cells. To elucidate the underlying mechanism, we performed mass spectrometry in murine T_h/T_c17 cells using biotin labeled cGAMP as bait followed by liquid chromatography-mass spectrometry. Top candidates (based on sequence coverage and Log₂ transformed LFQ intensities) were deleted via Cas9-ribonucleoproteins (RNPs) and subsequently characterized. Of those targets, we identified the leucine rich pentatricopeptide repeat containing protein (LRPPRC, also known as LRP130) as a novel downstream effector. LRPPRC is a multifunctional protein that regulates cellular metabolism and mitochondrial homeostasis where it operates as a reader of N⁶-methyladenosine (m⁶A) modified RNA helping to facilitate the appropriate processing and trafficking of RNAs that impacts their transcription. However, its function in T cells, and the immune system in general, is unknown.

Using DART-seq (deamination adjacent to RNA modification targets) we demonstrate that transcripts essential for T cell memory like Tcf7, along with relevant metabolic RNAs that promote a bioenergetic profile that supports T_scm formation like Cpt1a and Acaca, display m⁶A marks. Using RNA immunoprecipitation assays, we find that LRPPRC selectively binds to and stabilizes these transcripts in T_h/T_c17 cells. Interestingly, LRPPRC has much less association with these RNAs in conventional T_h/T_c1 cells potentially highlighting a vulnerability that could be therapeutically targeted. T_scm are known to rely more heavily on fatty acid metabolism for energy production, which is advantageous for T cells in the tumor microenvironment as it preserves ATP generation and maintains mitochondrial health to sustain an effective antitumor response. Deletion of LRPPRC with Cas9-RNPs impairs aerobic respiration as assessed by Seahorse bioanalysis, which results in bioenergetically unfit T cells that display reduced ATP production rates and mitochondrial dysfunction, thereby preventing effective T_scm generation.

Strikingly, these findings extend to human CD19-targeted CAR T cells differentiated into T_h/T_c17, where LRPPRC loss recapitulates the metabolic and transcriptional defects observed in murine cells. In an immunocompetent murine melanoma ACT model, infused T cells that lack LRPPRC are unable to effectively control tumor growth, which leads to decreased survival, emphasizing the functional significance of this pathway.

Collectively, our findings identify m⁶A post-transcriptional regulation, mediated through LRPPRC, as a novel and targetable axis for enhancing T cell stemness and metabolic fitness. This study is the first to directly implicate m⁶A-driven RNA regulation in shaping the antitumor efficacy of adoptive T cell therapies. By harnessing this pathway future ACT products may be optimized to improve outcomes for patients with hematologic malignancies.