PROJECT SUMMARY Elucidation of a newly characterized clonal bone marrow and autoinflammatory disease, VEXAS Syndrome, prompts re-examination of the master regulator of ubiquitylation, UBA1. VEXAS stands for Vacuoles, E1, X- linked, Autoinflammatory, Somatic syndrome and presents with disparate inflammatory (e.g. relapsing polychondritis, vasculitides) and hematologic (e.g. pancytopenia, myelodysplastic syndrome) conditions. The disease is present in 1:4000 men over the age of 50 and has a 40% mortality rate. Current therapeutic treatment is limited to high dose corticosteroids. In affected patients, acquired or somatic variants in the UBA1 gene are present in hematopoietic stem cells (HPSCs) and become lineage restricted to myeloid cells. VEXAS presents primarily in elderly males as UBA1 is located on the X-chromosome. UBA1 encodes the primary ubiquitin (Ub) activating enzyme (E1), accounting for over 97% of all downstream ubiquitylation. Protein ubiquitylation occurs via a tightly regulated enzymatic cascade where sequential activation of E1 to Ub-conjugating enzyme (E2) to Ub-ligase (E3) enzymes ultimately leads to substrate ubiquitylation. Prior studies have predominantly focused on the role of E3 ligases in disease progression due to their protein-specific targeting in ubiquitylation. Far less is known regarding the role of UBA1 in disease pathogenesis. Notably, VEXAS highlights the importance of UBA1 in maintaining immune homeostasis and driving clonal blood disease. Through transcriptomic and cytokine profiling approaches, our group has identified mutant myeloid cells as the primary drivers of inflammation although the underlying mechanism remains unclear. Here I propose to determine the mechanism of VEXAS using unbiased approaches. Despite UBA1 mutation present in HSPCs, the mutation is lineage restricted to myeloid cells and absent in lymphocytes. Our preliminary results utilizing a temperature sensitive cell line (Chinese Hamster Ovary [CHO] line ts20) suggests VEXAS mutant UBA1 fails to charge the cognate E2s of the endoplasmic reticulum associated degradation pathway (ERAD), UBE2J1 and UBE2G2, potentially upregulating the unfolded protein response (UPR). These findings suggest that inappropriate UBA1 expression may both drive disease in VEXAS and allow selective clonal expansion of a pro-inflammatory myeloid lineage as potentiated by specific downstream effectors possibly linked to ERAD and UPR. In this study I propose to 1) perform an unbiased delineation of novel effectors driving UBA1-dependent inflammation pathways and 2) functionally validate effectors identified through model systems and unbiased approaches through a combination of small molecule, biologic, and transgenic approaches. My combined studies will not only be important for elucidating the mechanism of disease and treatment in VEXAS but may also apply to a wider understanding of key cellular ubiquitylation processes, autoinflammatory disease, and bone marrow-derived dis...