Single-cell genotype-phenotype mapping identifies therapeutic vulnerabilities in VEXAS syndrome

Abstract

AbstractSomatic evolution leads to the emergence of clonal diversity across tissues with broad implications for human health. A striking example of somatic evolution is the VEXAS (Vacuoles E1 enzyme X-linked Autoinflammatory Somatic) syndrome, caused by somaticUBA1mutations in hematopoietic stem cells (HSCs), inducing treatment-refractory, systemic inflammation. However, the mechanisms that lead to survival and expansion of mutant HSCs are unknown, limiting the development of effective therapies. The lack of animal or cellular models ofUBA1-mutant HSCs has hindered such mechanistic understanding, mandating analysis of primary human VEXAS samples, which harbor admixtures of wild-type andUBA1-mutant HSCs. To address these challenges, we applied single-cell multi-omics to comprehensively define mutantUBA1-induced transcriptome, chromatin accessibility and signaling pathway alterations in VEXAS patients, allowing for the direct comparison of mutant versus wild-type cells within the same environment. We confirmed the expected enrichment ofUBA1M41V/Tmutations in myeloid cells, and additionally discovered that these mutations were also prevalent in Natural Killer (NK) cells in VEXAS patients, providing new insights into disease phenotypes. Through mapping genotypes to molecular phenotypes, including transcriptome, chromatin accessibility, cell surface protein or intracellular protein profiles, in HSCs, we found thatUBA1M41V/T-mutant cells showed an increased inflammation signature (interferon alpha and gamma response pathways), as well as activation of unfolded protein response (UPR) via pro-survival, but not pro-apoptotic, mediators of the PERK pathway, compared toUBA1wild-type HSCs. Ex vivo validation experiments showed that inhibitingUBA1in normal CD34+ or usingUBA1-mutant HSCs led to PERK pathway up-regulation, increased myeloid differentiation and cell survival, which was reversed by PERK inhibition. Thus, we demonstrated that human VEXAS HSCs show cell-intrinsic inflammatory phenotypes and survive the proteomic stress caused by compromised ubiquitination through PERK-mediated activation of the UPR. Together, these analyses nominate PERK pathway inhibition as a potential new therapeutic strategy for eradicating the VEXAS-inducing clone, demonstrating the power of single-cell multi-omics profiling of primary patient sample to enable genotype-to-phenotype somatic mapping for the discovery of novel candidates for clinical intervention.