Ubiquitin-dependent cell-fate decisions during human development and disease

National Institute Of Dental & Craniofacial Research

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Abstract

To determine the functions of spatially regulated E1 activity and ubiquitin activation during hematopoietic cell-fate decisions In collaboration with the labs of Dr. Peter Grayson (NIAMS) and Dr. Daniel Kastner (NGHRI) we have previously discovered VEXAS (Vacuoles, E1 ubiquitin activating enzyme, X-linked, Autoinflammatory, Somatic) syndrome, which is an overlapping hematologic and inflammatory disease arising from somatic mutations in UBA1 in a hematopoietic stem cell (Beck et al. NEJM 2020). Disease-defining mutations were found primarily at p.Met41, the translation start site for the cytoplasmic isoform of this enzyme. However, recent reports, including ours (Poulter et al, Blood 2021), suggest that missense mutations at other residues in UBA1 can also cause VEXAS syndrome. Since our initial discovery 2.5 years ago, there have been over 500 reports on this clinically heterogenous and often-fatal disease and we have recently determined that VEXAS syndrome is present in 1:4000 men over the age of 50 years (Beck et al, JAMA, 2023). This makes VEXAS syndrome the most common genetic autoinflammatory disease known to date. Despite extensive clinical investigations and this high prevalence, treatment options for VEXAS syndrome have remained limiting. Therefore, mechanistic details of how different missense mutations in UBA1 lead to disease are urgently needed.The main goal of this project is to dissect how loss of cytoplasmic UBA1 function leads to autoinflammation and how regulated ubiquitin activation may drive cell-fate decisions during blood and embryonic development. a) Identification and characterization of pathogenic mechanisms of UBA1 mutations across VEXAS syndrome, lung cancer, and X-linked spinal muscular atrophy (Collins et al., EMBO J 2024): We identify novel pathogenic UBA1 mutations and provide the first comprehensive mechanistic dissection of VEXAS-causing mutations. Our study reveals that while canonical VEXAS mutations (p.Met41) reduce expression of the cytoplasmic UBA1 isoform, non-canonical VEXAS mutations (non-p.Met41) reduce catalytic activity in both nucleus and cytoplasm. This occurs by multiple modes of UBA1 inactivation, including aberrant formation of oxyesters, that most prominently affect the E2 transthiolation step. Intriguingly, we observe a similar E2 transfer bottleneck with a subset of UBA1 mutations driving lung cancer in never smokers, but not with UBA1 mutations causing spinal muscular atrophy, which instead, render UBA1 activity thermolabile. Taken together, our systematic screening of disease-causing UBA1 mutations identify loss of ubiquitin conjugation to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes. In addition, our results define distinct and shared mechanisms of UBA1 inactivation across different disease states (i.e. autoinflammation, lung cancer, and X-linked spinal muscular atrophy). These molecular insights provide a framework for studying regulatory principles of ubiquitylation at the E1-E2 level, for determining cellular downstream consequences of loss of UBA1 activity, and for developing therapeutic strategies to treat VEXAS syndrome and UBA1-related diseases in the future.