Defining targetable vulnerabilities in UBA1-mutated VEXAS HSCs for clonal eradication

ABSTRACT The expansion of somatically mutated clones is prevalent in hematopoiesis with profound implications for physiological aging and across a myriad of pathologies. This has been recently highlighted by the discovery of the inflammatory disorder – vacuoles E1 enzyme X-linked autoinflammatory somatic (VEXAS) syndrome. VEXAS is caused by somatic mutation in the ubiquitin-activating enzyme E1 (UBA1) gene that arises in hematopoietic stem cells (HSCs). Mutated UBA1 leads to accumulation of misfolded proteins, activation of the unfolded protein response and ultimately systemic inflammation accompanied by severe clinical manifestations. However, we currently lack the ability to target disease-initiating mutant HSCs, as the mechanisms that lead to HSC inflammation, differentiation bias and survival are unknown. This limitation stems in part from the fact that somatic clones are admixed with wild-type HSCs, limiting the ability to define mutant-specific phenotypes with either bulk or single-cell techniques. To overcome this gap, we have developed genotype-aware single-cell multi- omics techniques that are specifically designed to map somatic genotypes to phenotypes. These tools turn the limitation of admixed mutated and wild-type cells into an advantage, allowing for direct comparison in primary human samples of mutated cells to wild-type cells residing within the same bone marrow environment. Our proof- of-principle studies demonstrated that we can leverage these tools to pinpoint UBA1 mutation-specific HSC perturbation, that can be leveraged for selective targeting. Our preliminary data show that UBA1-mutant HSCs have increased upregulation of the unfolded protein response pathway through PERK activation, suggesting a compensatory mechanism for mitigating proteomic stress to promote survival in mutant cells where normal protein degradation is disrupted, which we validated in vitro. Moreover, this approach enables the characterization of how the impacts of a somatic mutation vary as a function of cell type, providing insights into mechanisms of clonal outgrowth that cannot be obtained through bulk analysis. Here, to test the hypothesis that UBA1-mutant HSCs resist proteome toxicity via dysregulated signaling pathways, we will directly analyze VEXAS primary patient samples using single-cell multi-omics to map the impact of UBA1 mutations on HSC chromatin profiles, gene expression and protein expression. Moreover, given the critical role of the bone marrow niche on HSC biology, we will apply advanced tools for spatial transcriptomic profiling to identify candidate cell-extrinsic mechanisms supporting mutant HSC survival, defining altered cell-cell interactions in VEXAS syndrome. Finally, we will use new in vitro and in vivo model systems to mechanistically interrogate and functionally validate key factors that promote disease phenotypes, representing potential targets for the development of novel precision treatments. Collectively, these efforts and new techniques will provide novel insights into VEXAS biology, and further provide a framework for deciphering the critical factors that are disrupted in disease-initiating cells and the microenvironment in somatic mosaicism and hematological disease more broadly.