Pharmacologic enhancement of UBA1 activity in models of VEXAS syndrome

SUMMARY VEXAS syndrome is a newly discovered adult-onset autoinflammatory disease that is refractory to treatment and

often fatal. It is caused by somatic missense mutations of the X-linked E1 ubiquitin-like modifier-activating enzyme 1 gene

(uba1) in hematopoietic stem cells. UBA1 protein catalyzes the initiation of most ubiquitination processes in cells. Mutant UBA1 proteins have reduced enzymatic activity. Uba1 mutant myeloid cells exhibit decreased ubiquitination and activated innate immune and stress pathways. Thus, enhancing UBA1 activity could be an effective treatment strategy for VEXAS

syndrome. In preliminary studies, we found that auranofin, a clinical drug for rheumatoid arthritis, binds to UBA1, enhances UBA1 interactions with E2 ubiquitin-conjugating enzymes. As a result, auranofin has a general activity in promoting E3

ubiquitin ligase activities and accelerates the degradation of proteasomal substrate proteins. Significantly, auranofin also markedly promotes the activity of the major VEXAS-causing mutant UBA1 protein. This novel activity of auranofin

requires concentrations about 5 to 18 times lower than the maximum serum concentration (Cmax) achieved by the therapeutic

dose for rheumatoid arthritis. Thus, auranofin, as a UBA1 activity enhancer, may have tremendous therapeutic potential for VEXAS syndrome. In this proposal, our goals are to investigate 1) the mechanism by which auranofin enhances UBA1

activity in ubiquitination and 2) the potential of auranofin as a much-needed drug for VEXAS syndrome. To facilitate the investigation, we have created U937 monocyte-like cell lines that harbor the primary VEXAS-causing mutation (p.Met41 codon mutation) in uba1 (U937Duba1b) by CRISPR/cas9 technology. Our preliminary studies showed that U937Duba1b

cells exhibit the similar pro-inflammatory and stress responses to that reported in patient-derived uba1 mutant monocytes, particularly the highly activated inflammatory signatures, including TNFa, IL-6, and IFN-g, and ER stress. We have obtained the zebrafish model (Duba1b) of VEXAS syndrome from Dr. David Beck (NIH) to assess the therapeutic effects

of auranofin. Three specific Aims are proposed to accomplish our goals. Aim 1: To define the mechanism of how auranofin enhances UBA1 activity in ubiquitination. We will test the hypothesis that auranofin binding changes UBA1 conformation

and enhances UBA1 interaction with E2s, resulting in increased ubiquitin charging to E2s and increased activities of E3s.

We will use ubiquitination of misfolded proteins in the endoplasmic reticulum as an example to validate the mechanism in cells. Aim 2: To determine whether auranofin inhibits the major pro-inflammatory signaling (TNFa via NFkB, IL-6, and IFN-g) identified in patient-derived uba1 mutant monocytes. U937Duba1b cells will be used as a cellular model of VEXAS

syndrome to determine whether auranofin inhibits the pro-inflammatory cytokine expression and their signaling. Aim 3: To profile ubiquitination reduction and proteostasis disturbance in U937Duba1b cells and determine whether auranofin alleviates the changes. Decreased ubiquitination (ubiquitinome) is likely the cause for VEXAS syndrome. Therefore, we

will use a combination of quantitative mass spectrometry-based proteomics and bioinformatics to profile the changes in ubiquitinome and proteome in U937Duba1b cells. We will assess whether auranofin reverses the changes and inhibits pro- inflammatory responses and evaluate the therapeutic potential of auranofin in the zebrafish model of VEXAS.