Phosphatidylcholine Deactivates Cytotoxic CD8+ T Cells through Ufmylation Mediated By Exosomal SerpinB9 in Multiple Myeloma

Identifying novel therapeutic targets for multiple myeloma (MM), a prevalent hematological malignancy, is crucial due to its complex pathogenesis and resistance to current treatments. This study explores the role of the gut microbiome and blood metabolites in modulating the tumor microenvironment (TME), offering new strategies for MM therapy. Through comprehensive analyses, including 16S rRNA gene sequencing, metagenomics, and ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC/Q-TOF MS), we identified Lachnospiraceae and phosphatidylcholine (PC) as key differentiators between MM patients and healthy controls. Our findings reveal that PC impairs the cytotoxic activity of CD8+ T cells against MM cells by promoting the expression of SerpinB9 (Sb9) through a mechanism involving lysophosphatidic acid (LPA) and the LIN28A/B pathway. Furthermore, PC reduces granzyme B (GZMB) expression in CD8+ T cells via exosomal Sb9 derived from MM cells, thereby diminishing the cytotoxic effect of these T cells on MM cells. Additionally, Sb9 inhibits TP53 expression by preventing TP53 UFMylation, a process mediated by the competitive binding of TP53 with the ubiquitin-fold modifier conjugating enzyme 1 (UFC1) in CD8+ T cells. These insights not only enhance our understanding of MM pathogenesis but also highlight potential therapeutic targets and strategies for MM treatment, emphasizing the significance of the gut microbiome and blood metabolites in cancer therapy. Despite these advances, further research is needed to explore the underlying mechanisms of Lachnospiraceae's influence on PC levels in MM and the in vivo effects of exosomal Sb9 on CD8+ T cell cytotoxicity.

**Introduction:** Multiple myeloma (MM), the second most prevalent hematological malignancy, remains challenging to treat due to its resistance to current therapies. This study explores the gut microbiome and blood metabolites' role in influencing the tumor microenvironment (TME), aiming to identify novel therapeutic targets and strategies for MM. It focuses on distinguishing MM patients from healthy individuals by analyzing Lachnospiraceae and phosphatidylcholine (PC), investigating their potential impact on MM pathogenesis and treatment resistance.

**Methods:** Employing 16S rRNA gene sequencing and UPLC/Q-TOF MS, the study analyzed the gut microbiome and blood metabolites of MM patients compared to healthy controls. It further investigated the impact of PC on CD8+ T cell cytotoxicity towards MM cells, examining the molecular mechanisms involved.

**Results:** The study identified significant differences in the gut microbiome composition between MM patients and healthy controls, with Lachnospiraceae and PC emerging as critical differentiators. In MM patients, increased levels of PC were associated with a diminished cytotoxic response of CD8+ T cells against MM cells. This effect was mediated by the upregulation of SerpinB9 (Sb9) in MM cells, which was induced by PC through the LIN28A/B-LPA pathway. Additionally, PC was found to decrease granzyme B (GZMB) expression in CD8+ T cells via exosomal Sb9 derived from MM cells, further inhibiting the cytotoxic effect. Sb9 also played a role in reducing TP53 expression by blocking TP53 UFMylation, a process facilitated by the competitive interaction between TP53 and the ubiquitin-fold modifier conjugating enzyme 1 (UFC1) in CD8+ T cells. These findings were supported by both in vitro and in vivo experiments, demonstrating the potential of PC and Sb9 as novel targets for MM treatment.

**Conclusion:** This study underscores the significance of the gut microbiome and blood metabolites, particularly Lachnospiraceae and phosphatidylcholine, in the pathogenesis and treatment resistance of multiple myeloma. By revealing how PC influences CD8+ T cell cytotoxicity and MM cell survival through the modulation of Sb9, the research opens new avenues for therapeutic intervention in MM. These insights highlight the potential benefits of targeting the gut microbiome and blood metabolites to modulate the TME and improve outcomes for MM patients. Further investigations are warranted to explore the therapeutic implications of these findings in MM treatment strategies.

Gong:Amgen: Current equity holder in publicly-traded company; Geron: Current equity holder in publicly-traded company; Kura Oncology: Current equity holder in publicly-traded company; Revolution Medicine: Current equity holder in publicly-traded company; Syndax: Current equity holder in publicly-traded company.