Characterization of E1 Ligase Dependencies in a Mutant-UBA1 Human Cell Model Reveals UBA6 As a Novel Therapeutic Target in Vexas Syndrome

VEXAS (Vacuoles, E1-ligase, X-linked, Autoinflammatory, Somatic) syndrome is a newly defined clonal hematopoietic malignancy that often co-occurs with myelodysplastic syndromes (MDS). It is characterized by hyperinflammation, bone marrow failure, and high rates of mortality. Somatic hotspot mutations at methionine 41 (M41) in the E1 ubiquitin ligase, UBA1, define the molecular feature of the disease. Humans express two major E1 ubiquitin ligases, UBA1 and UBA6 but the function of either E1 in regulation of the ubiquitinome is not fully understood. UBA1, the most abundant E1 ubiquitin ligase, is constitutively expressed as two isoforms: a long UBA1a and a short UBA1b isoform. VEXAS M41 mutations result in an isoform switch from UBA1b to a nonfunctional, truncated isoform, UBA1c. How loss of UBA1b alters the ubiquitinome to drive VEXAS syndrome pathogenesis remains unknown.

In this study, we utilized CRISPR/Cas9 to develop a human cell model of VEXAS syndrome by engineering the male monocytic cell line, THP1. We generated isogenic lines expressing either wild-type (WT) UBA1-M41M or UBA1-mutant (MUT) UBA1-M41V. Mutant UBA1 cells express aberrant UBA1c protein and have increased vacuolization, compared to WT cells, phenocopying defining features of VEXAS syndrome. Furthermore, MUT UBA1-M41V cells have significant upregulation of the ATF4-mediated unfolded protein response program compared to WT cells, as reported in VEXAS patients (Ganesan, bioRxiv, 2024).

We hypothesized that mutant UBA1 alters ubiquitination, sensitizing clones to further E1 ubiquitin ligase inhibition. To probe the ubiquitination role of each E1 ubiquitin ligase in VEXAS, we first used shRNAs to knockdown (KD) UBA1 or UBA6 in isogenic WT UBA1-M41M or MUT UBA1-M41V cells. Knockdown of either UBA1 or UBA6 alone efficiently reduced protein expression by >90% of UBA1 or UBA6, respectively, but did not result in significant changes in global ubiquitination, suggesting functional redundancy of UBA1 and UBA6. Loss of UBA1 resulted in significant growth impairment and near complete loss of colony-forming potential in both WT UBA1-M41M and MUT UBA1-M41V cells compared to a scramble shRNA control. Similarly, knockdown of UBA6 also impaired the growth in both WT and MUT cells. However, unlike the colony abrogation observed in UBA1 KD cells, knockdown of UBA6 resulted in a modest reduction in colony formation with a trend toward stronger colony inhibition in MUT-UBA1-M41V cells as compared to WT cells. These findings suggest that there is an acquired dependency on UBA6 in UBA1-mutant VEXAS cells.

To complement these shRNA studies, which resulted in almost complete abrogation of protein expression, we tested small molecules to fine-tune the levels of UBA1 or UBA6 inhibition, as some degree of UBA1 and UBA6 function are required for cellular maintenance. We utilized the UBA1-specific inhibitor, TAK-243, and phytic acid, an allosteric UBA6 inhibitor (Yuan et al, Nat Commun, 2022), to further interrogate the roles of E1 ubiquitin ligases in VEXAS syndrome. As previously reported in a mouse cell line model of VEXAS (Chiaramida, Blood Advances, 2023), MUT UBA1-M41V-expressing human cells had increased sensitivity to TAK-243 compared to WT cells in cell viability assays. However, like UBA1 KD studies, TAK-243 treatment significantly reduced cellular growth and colony formation in both WT UBA1-M41M and MUT UBA1-M41V cells. In contrast, treatment with the UBA6 inhibitor phytic acid led to a marked reduction in growth of MUT UBA1-M41V cells compared to WT cells. Furthermore, pretreatment with phytic acid specifically impaired the colony formation potential of MUT UBA1-M41V cells while no reduction in the colony formation was observed in WT UBA1-M41M cells. Taken together, these studies suggest that UBA6 activity is required to maintain UBA1 mutant cells and represents a novel therapeutic targeting strategy for the treatment of VEXAS syndrome.