UBA1a dependency leads to synthetic lethality in vacuoles, E1-ligase, x-linked, auto-inflammatory, somatic (VEXAS) syndrome

Abstract VEXAS is a recently described, monogenic auto-inflammatory condition affecting approximately 1:4000 men over 50. It is characterized by severe, multi-system inflammation, cytopenias, and increased risk of myelodysplastic syndrome (MDS) and plasma cell disorders. VEXAS typically follows a progressive course with high morbidity and ~50% 5-year mortality. No clear disease-modifying therapies exist, and potentially curative allogeneic transplantation is highly toxic; better treatments are therefore urgently needed. VEXAS is caused by somatic mutations in UBA1 on the X chromosome. UBA1 encodes the primary ubiquitin E1 ligase, which catalyzes the first step in the ubiquitin-proteasome pathway. A single mRNA transcript produces 2 isoforms: a longer nuclear form (UBA1a) and a shorter cytoplasmic form (UBA1b), the latter initiated at Met41. Most cases involve Met41 mutations, resulting in reduced UBA1b and the emergence of a truncated form with reduced catalytic activity (UBA1c). This leads to impaired global ubiquitylation (Ub) and subsequent induction of the unfolded protein response (UPR) and autophagy. Interestingly, UBA1 Met41 mutations are variably tolerated throughout hematopoiesis, enriched in myeloid cells and depleted in lymphocytes. Low variant allele frequency (VAF) UBA1 Met41 mutations have been reported in asymptomatic individuals, suggesting a threshold for disease. We therefore hypothesized that even a partial reduction of the VEXAS clone might improve symptoms. Thus, our goal was to develop an ex vivo gene therapy to correct UBA1 Met41 mutations in patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs). Sinceprimary VEXAS specimens are rare, we first sought to install and correct pathogenic UBA1 Met41 mutations in healthy male HSPCs via base editing (BE). We efficiently installed the canonical Met41Thr and Met41Val mutations (avg. VAFs approx. 90% and 30%, resp.). However, due to a lack of strong PAMs, the need to use less efficient cytidine base editors, and consequential bystander edits, direct correction via BE proved impractical. Surprisingly, despite efficient installation of Met41 mutations and the expected isoform shifts, we did not observe the reported downstream phenotype (e.g., decreased Ub, UPR activation, autophagy) in the engineered VEXAS HSPCs. Closer examination revealed an increased ratio of UBA1a:UBA1b protein compared to what has been published for primary VEXAS monocytes (which uniformly display VEXAS-associated pathology). We then reasoned that the relative increase in UBA1a might buffer the loss of UBA1b and mitigate the phenotype in HSPCs. To assess this possibility, we first designed CRISPR sgRNAs to knock out UBA1a alone or in combination with UBA1b. Loss of UBA1a alone had no apparent effect on Ub or the cells' behavior in culture or clonogenic potential. In contrast, loss of UBA1a and UBA1b resulted in decreased Ub, rapid negative selection, and marked up-regulation of p53, possibly due to impaired Ub by MDM2. These results suggest that UBA1a is dispensable in wild-type (WT) HSPCs but becomes essential in the setting of UBA1b deficiency – opening the possibility of synthetic lethality in VEXAS. Specifically, we hypothesized that suppression of UBA1a will selectively kill UBA1b-deficient VEXAS cells without affecting WT hematopoiesis. Our data also provide insight into VEXAS pathogenesis: dependence on UBA1a may explain lineage-specific tolerance of Met41 mutations, while p53 activation may contribute to MDS risk. Because pathogenic Met41 mutations still produce low levels of UBA1b, complete UBA1 KO does not accurately model the disease. To specifically interrogate the interaction between UBA1a and Met41 mutations, we next suppressed UBA1a using BE to alter its start codon (Met1Thr). Like our CRISPR KO strategy, Met1Thr-mediated loss of UBA1a was tolerated by HSPCs with no apparent effect on their biology, while in combination with Met41-mediated UBA1b loss, cells demonstrated decreased Ub and underwent rapid negative selection. Collectively, these results suggest that loss of UBA1a in VEXAS may be an efficient and unique opportunity to use synthetic lethality to eliminate diseased HSPCs without affecting residual WT hematopoiesis.