Genotype-Phenotype Mapping Via Single-Cell Multi-Omics Identifies Therapeutic Vulnerabilities in Vexas Syndrome

VEXAS (Vacuoles E1 enzyme X-linked Autoinflammatory Somatic) syndrome originates from hematopoietic stem cells (HSCs) carrying a somatic UBA1^p.M41 mutation. UBA1 ^p.M41 mutation leads to systemic inflammation and bone marrow failure, with poor survival. Other than allogenic stem cell transplantation, no curative therapy is available for VEXAS. This arises from the challenge of defining the phenotypic effects of the UBA1^p.M41 mutation in primary human samples, confounded due to the mixture of wildtype and mutated cells. Thus, it is essential to resolve the admixture of wildtype and UBA1^p.M41 mutated cells to understand the effect of the UBA1^p.M41 mutation in the disease-initiating HSCs. By doing so, we can identify potential mutant-specific therapeutic vulnerabilities to specifically eliminate UBA1^p.M41 HSCs.

To map genotype-phenotype relationship in human VEXAS samples, we deployed and developed single-cell multi-omics including Genotyping of Transcriptomes (GoT) and Genotyping of Targeted loci with Chromatin Accessibility (GoT-ChA), allowing the capture of UBA1 genotype, cell identity, transcriptome, chromatin accessibility and signaling pathways at single cell resolution (Fig.1a). We applied GoT to CD34+ enriched bone marrow mononuclear cells (BMMNCs) from VEXAS primary human samples (n = 31,406 cells across 6 patients). Integrating UBA1 genotyping with gene expression data showed an enrichment of the mutation in myeloid-committed cells and depletion in B and T cells. Interestingly, this mutation is also enriched in natural killer (NK) cells and plasmacytoid dendritic cells (both originating from lymphoid progenitors), which have not been previously reported. This suggests that VEXAS HSPCs display a skewed differentiation towards myeloid, NK, and plasmacytoid dendritic cells, which may contribute to inflammation in VEXAS.

High-resolution mapping of UBA1^p.M41 vs. UBA1^wt HSCs revealed myeloid priming of mutated HSCs. UBA1^p.M41 HSCs showed an increased inflammation signature (Interferon alpha and gamma response pathways) along with increased unfolded protein response (UPR), glycolysis and decreased autophagy pathways. We validated UPR pathway activity by showing the expected increase in XBP1 splicing in UBA1^p.M41 HSPCs using targeted capture of the unconventional splice site. Interestingly, transcriptome data also showed a global translational downregulation accompanied by specific ATF4 target gene enrichment (Fig.1b). These results suggest a preferential induction of the PERK arm of the UPR. Surface protein expression analysis supported the transcriptomic data, showing upregulation of CD38, CD13 and HLA-DR, denoting myeloid bias and increased inflammatory response in UBA1^p.M41 HSCs.

Next, we performed GoT-ChA on CD34⁺ cells from VEXAS patients (n = 27,894 cells across 3 patients) to study mutant-specific chromatin accessibility. Complementary to the transcriptome data, UBA1^p.M41 HSCs showed increased motif accessibility for TFs associated with myelopoiesis, indicative of myeloid priming. We also observed enrichment for ATF4 and CEPBD TF motifs - downstream of PERK pathway of UPR (Fig.1b). To validate these findings, we developed GoT-ChA integrated with intracellular protein capture and demonstrated an increased expression of phosphorylated eIF2 and ATF4 proteins in the UBA1^p.M41 HSCs.

Finally, to functionally validate the specific role of the PERK pathway on the fitness of UBA1^p.M41 HSCs, we performed assays using mobilized CD34⁺ cells in-vitro. UBA1 Inhibition in normal CD34⁺ cells led to ATF4 up-regulation, which correlated with cell survival. The addition of PERK inhibition reduced ATF4 expression and resulted in markedly reduced CD34+ viability. In contrast, inhibiting IRE1 pathway along with UBA1 inhibition did not affect HSC viability.

Altogether, we demonstrated that human VEXAS HSCs show a cell-intrinsic inflammatory phenotype and are myeloid biased. Moreover, mutated HSCs tolerate proteome toxicity through activation of the PERK pathway as a part of UPR, providing a fitness advantage to the UBA1^p.M41 mutated HSCs. Targeting the PERK pathway may thus represent a new strategy for overcoming VEXAS syndrome via precision targeting of UBA1^p.M41 mutated HSCs.