Dysregulation of protein homeostasis by mutant UBA1 in VEXAS syndrome [WES]

VEXAS is a hematopoietic disorder characterized by hyperinflammation,high mortality, and mutations at methionine 41 (M41) in the E1 ubiquitin enzyme, UBA1. Here, we developed ahuman model of VEXAS by engineering the male THP1 cell line to express the UBA1-M41V mutation. We found that UBA1-M41V cells exhibit aberrant UBA1 isoform expression, increased vacuolization, and upregulation of the unfolded protein response, recapitulating features of VEXAS.Proteomic analyses revealed dysregulated ubiquitination and proteotoxic stress in UBA1-M41V cells, with alterations in inflammatory and stress-response pathways. UBA1-M41V cells were sensitive to genetic or pharmacological inhibition of E1 enzymes. Treatment with theE1 inhibitor TAK-243 preferentially suppressed colony formation of UBA1-M41V cells. Moreover,UBA1-M41V cells exhibited greater sensitivity to TAK-243 in competition assays and showed increased apoptosis. Interestingly, TAK-243 preferentially inhibited UBA6 activity over UBA1 ,suggesting that UBA6 may compensate for UBA1 dysfunction. Targeting UBA6 using shRNA orthe UBA6-specific inhibitor phytic acid further revealed an acquired dependency on UBA6 inUBA1-M41V cells. Phytic acid impaired UBA1-M41V cells while sparing WT cells. Together, these findings establish a novel human model of VEXAS, identify key roles for UBA1 and UBA6, and demonstrate that UBA6 inhibition represents a therapeutic strategy for selectively targeting UBA1 mutant clones. Overall design: THP1 UBA1 WT and M41V mutant UBA1 cells were collected, and DNA was extracted using Qiagen Dneasy kit. Following initial sample dsDNA quality control for quantity with a Qubit fluorometer and size distribution with an Agilent Fragment Analyzer, 50 ng of gDNA is enzymatically fragmented using Twist Bioscience's Library Preparation EF Kit 2.0 (Twist Bioscience, 104207). Index adapters (Twist Bioscience, 100577) are ligated onto the DNA fragments, and then amplified with 10 cycles of PCR. Libraries are purified with Binding and Purification beads (Twist Bioscience, 100983), quantified with a Qubit fluorometer, and checked again on a Fragment Analyzer. Up to 8 libraries of like organisms are pooled together with a total mass of 1500 ng/pool. Human DNA is then hybridized with Twist's Comprehensive Exome probes (Twist Bioscience, 102032). Samples are hybridized for 30 minutes with Twist's Fast Hybridization Reagents (Twist Bioscience, 104180) before being bound to streptavidin beads and washed with Twist's Fast Hybridization and Wash Kit (Twist Bioscience, 104180). Following hybridization, hybridized pools are amplified with 8 cycles of PCR using primers and master mix from the Library Preparation Kit EF. Final libraries are purified with Twist's Binding and Purification beads, quantified with a Qubit fluorometer, and their size checked with a Fragment Analyzer. Libraries are sequenced on an Illumina NovaSeq X Plus, generating > 25 million 100 bp read pairs and analyzed using Illumina's Dragen enrichment pipeline. Data Alignment and Analysis Methods Reads were aligned using a functionally equivalent pipeline, as previously defined70. Specifically, each read pair was separately aligned to the appropriate reference (GRCh38) genome using bwa-mem version 0.7.17 with the -M option. The resulting SAM files are first sorted and converted to BAM with Picard SortSam (V2.18.22), then all resulting BAMs are merged and duplicate marked with Picard MarkDuplicates (https://broadinstitute.github.io/picard/) using the “OPTICAL_DUPLICATE_PIXEL_DISTANCE=2500” command line flag. Variants were filtered and subsetted using BCFtools (v1.9, https://samtools.github.io/bcftools/) and annotated with Variant Effect Predictor (VEP, https://useast.ensembl.org/info/docs/tools/vep/).