Independent mechanisms of myeloid bias and inflammation driven by UBA1 mutation

Somatically acquired mutations in the E1 ubiquitin-activating enzyme UBA1 within hematopoietic stem and progenitor cells (HSPCs) were recently identified as the cause of the adult-onset autoinflammatory syndrome VEXAS (vacuoles, E1 enzyme, X linked, autoinflammatory, somatic)1. Most VEXAS-associated mutations occur at Met41 in UBA1 and lead to clonal expansion within the HSPC and myeloid, but not lymphoid, compartments. Despite its severity and prevalence, the mechanisms whereby UBA1 mutations cause multiorgan autoinflammation and hematologic disease are unknown. Here, we employ mRNA-based electroporation of adenine base editing enzymes to mutate endogenous Uba1, generating canonical VEXAS-associated Met41 mutations in murine or human HSPCs and macrophages. Expression of Met41-mutant UBA1 in macrophages aberrantly triggered apoptotic and necroptotic inflammatory cell death programs upon exposure to TNF or LPS, which could be fully reversed by combined deletion of Mlkl or Ripk3 plus Casp8, or partially by genetic or pharmacologic targeting of RIPK1 kinase activity. In mice challenged with TNF or LPS, the UBA1 inhibitor TAK-243 exacerbated inflammation, which was mediated by RIPK1 kinase activity and RIPK3-Caspase-8, respectively. In contrast, Uba1-mutant HSPCs displayed an unfolded protein response signature and spontaneous myeloid skewing that were independent of RIPK3-Caspase-8, thereby propagating an inflammatory cycle driven by their mature myeloid progeny. Mechanistically, we traced the dysregulated cell death response of Uba1-mutant macrophages to a cascading, kinetic defect in Lys63/Met1 (i.e., linear) polyubiquitylation of inflammatory signaling complexes. Collectively, our results link the pathogenesis of VEXAS with that of rarer germline monogenic causes of autoinflammation; highlight specific ubiquitin-associated defects stemming from an apical mutation in the ubiquitylation cascade; and nominate the RIPK1-RIPK3-caspase-8 death axis as a therapeutic target in VEXAS.