3025 – MITOFUSIN 2 AGONISTS REGULATES MTOR SIGNALING TO SUPPORT HUMAN HSC EXPANSION IN VITRO

Using cord blood units (CBU) for hematopoietic stem cell transplants (HSCT) improves match compatibility and reduces graft versus host disease. Limited cell numbers hinder widespread adult use. In vitro expansion overcomes dose limits, but techniques for long-term HSC expansion are underdeveloped, prompting the need for novel molecular targets. Studies in mice have implicated Mitofusin 2 (MFN2) activity as necessary to maintain potent HSCs in vitro. Recent studies have described efficacious small molecule agonists of MFN2 fusion. Thus, we hypothesized that MFN2 agonist treatment during short-term UCB cell expansion could enhance HSC repopulating function. Our study revealed phenotypic HSCs expanded with MFN2 agonists displayed a highly significant increase in long-term chimerism for both primary and secondary xenografts of MFN2 agonist HSC culture recipients compared to vehicle, revealing MFN2 fusion as necessary for human HSC function. MFN2 agonist-treated HSCs displayed upregulation of genes associated with ribosome, stress granules, and autophagy are hallmarks of HSC maintenance mechanisms. HSC cultures exposed to MFN2 agonist exhibited decreased protein translation by OP-Puro assay, heightened lysosome count and acidification using lysotracker dyes, and increased LC3B puncta, indicating heightened autophagy. Network analysis of DGEs suggested upstream regulation via MTOR signaling. HeLa cells treated with MFN2 agonist or rapamycin displayed increased TFEB nuclear levels (lysosomal biogenesis master regulator), indicating enhanced autophagy. Additionally, reduced S6 phosphorylation, a downstream target of MTOR, and direct MFN2-MTOR interaction indicated by immunoprecipitation assays, suggests MFN2 inhibits MTOR signaling. Overall, our findings elucidate a novel mechanism whereby MTOR inhibition via MFN2 induces catabolic programs to maintain HSC function in vitro.