Primary Hematopoietic Cells of Vexas Patients Are Highly Sensitive to Treatment with TAK-243 and Pevonedistat

Introduction: VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome represents a newly identified autoinflammatory disease characterized by somatically acquired mutations in the ubiquitin-activating enzyme 1 (UBA1) gene in hematopoietic cells, frequently associated with myelodysplastic neoplasms (MDS). The UBA1 protein plays a crucial role in post-translational modification processes, including ubiquitination and neddylation, which are essential for protein folding and degradation. Given the recent discovery of VEXAS, standardized treatment options are not yet established, leaving patients with high symptomatic burdens and mortality. A particularly promising approach for molecularly targeted treatment involves the ubiquitin-proteasome pathway inhibitors TAK-243 and pevonedistat, specifically targeting UBA1- and NEDD8-activating enzymes. Recently, Chiaramida et al. (Blood Adv., 2023) demonstrated this concept’s potential using a murine Uba1M41L knock-in cell line, which replicated the features of VEXAS syndrome and showed increased sensitivity to TAK-243. In our study, we conducted in vitro testing of TAK-243 and pevonedistat on primary CD34+ cells from patients with VEXAS syndrome and MDS, as well as healthy individuals, to further explore their therapeutic potential in a clinical context.

Methods: We collected primary bone marrow samples of n=4 VEXAS patients, as well as n=5 low-risk MDS patients and n=5 healthy (HY) donors. Subsequently, these samples were enriched for CD34+ cells and treated with different concentrations of TAK-243 (range: 0.24-1,000 nM) and pevonedistat (range: 0.78-3,000 nM). After 48 hours of incubation, cell viability was assessed using the CellTiter-Glo luminescence assay. The molecular characterization of DNA from the patient cells was conducted using panel sequencing and Sanger sequencing.

Results: CD34+ cells from VEXAS patients exhibited a significantly higher sensitivity to escalating doses of TAK-243 and pevonedistat as compared to samples from MDS patients and healthy controls. The greatest differences were observed with TAK-243 treatment. IC50 values for TAK-243 were 21.6 nM for VEXAS patient cells versus 133.2 nM for cells from MDS patients (p < 0.01) and 191.9 nM for cells from HY individuals (p < 0.001). For pevonedistat, the IC50 values were 552.6 nM for VEXAS, compared to 1048 nM for MDS and 1107 nM for HY CD34+ cells (p < 0.001 for both comparisons). Notably, CD34+ cells of all three groups demonstrated significantly higher sensitivity to TAK-243 than to pevonedistat, as evidenced by lower IC50 values (p < 0.0001 for all). Interestingly, MDS and HY samples only responded to higher concentrations of TAK-243 (> 100 nM) and pevonedistat (> 500 nM). A difference in sensitivity to both inhibitors between MDS and HY was only observed for TAK-243 at concentrations greater than 100 nM, but not to the same extent as in VEXAS samples. No difference was observed between MDS and HY when treated with pevonedistat.

The UBA1 mutational status in CD34+ cells of VEXAS patients was confirmed with Sanger sequencing. Two patients harbored the hot spot variant p.Met41Leu (VAF 90% and 83%), while two others carried the p.Met41Thr variant (VAF 90% and 89%). Panel sequencing of the co-mutational profile revealed the VEXAS-typical co-mutation DNMT3A p.Pro904Leu in one patient (VAF 40%).

Discussion: Complete loss of UBA1 function is lethal to cells and organisms. The mutations that cause VEXAS syndrome significantly reduce catalytic activity. However, current scientific data suggest that a minimal residual catalytic activity persists in VEXAS cells, allowing affected myeloid progenitor cells to survive and gain a selective clonal advantage. We propose two small molecule inhibitors that selectively and directly target the underlying molecular lesion of the disease. With our results we hypothesize that the application of targeted inhibitors of UBA1, such as TAK-243, or related pathway steps, such as neddylation with pevonedistat, creates a "synthetic lethality" in UBA1-mutated cells by reducing the remaining UBA1 activity to a non-viable level. Consequently, treatment with these substances selectively eradicates UBA1-mutated myeloid progenitor cells, which are the basis of the inflammatory disease and thus holds promise for effectively treating VEXAS patients.