Somatic loss-of-function mutations of major E1 ubiquitin activating enzyme gene UBA1 were identified in VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) and myelodysplastic syndromes (MDS) recently, suggesting that impairment of UBA1 activity contributes to pathogenesis of these diseases that have systemic inflammatory disorders. We sought to determine whether abnormal expression of UBA1 also occurs in MDS and contributes to development of disease. We analyzed RNA sequencing results obtained from the CD34+ bone marrow hematopoietic stem and progenitor cells (BM HSPC) in a cohort containing treatment naive MDS (N=55), chronic myelomonocytic leukemia (CMML, N=22), and heathy donors (N=12). Since UBA1 is an X-chromosome linked gene that escapes X-chromosomal silencing in females, all donors in this cohort were males. Results indicated that the level of UBA1 RNA was significantly downregulated in MDS but not in CMML. In the subtypes of MDS-low blasts (N=23, including MDS with ring sideroblasts, multilineage dysplasia, and single lineage dysplasia), forty-eight percent (N=11) had an over 1.5-fold downregulation of UBA1 RNA expression. Based on gene set enrichment assay, the UBA1 downregulation in MDS-low blasts was associated with altered autophagy and innate immune signaling such as complement, IL6, and TNF signals. Moreover, we also identified that the UBA1 downregulation in BM CD34+ cells of MDS-low blasts co-occurred with SF3B1 mutations detected by targeted next generation sequencing in BM mononuclear cells: Among the 11 MDS-low blasts patients with downregulated UBA1 expression, six patients (55%) carried mutant SF3B1, whereas none of the 12 patients with normal UBA1 expression had SF3B1 mutation. In another word, all SF3B1 mutant patients with MDS-low blasts (N=6) presented decreased UBA1 RNA.

To characterize whether impairment of UBA1 affects hematopoietic functions of BM HSPC, we applied TAK-243, an UBA1 specific inhibitor, to BM CD34+ cells isolated from healthy donors (N=5, all males) and cultured in vitro. Colony formation unit (CFU) assays were performed to evaluate hematopoietic repopulating activity. Results indicated the CFU in CD34+ cells treated by TAK-243 was significantly reduced compared with cells treated by vehicle control (P<0.05). Moreover, when hematopoietic colonies formed by CD34+ cells were collected from methylcellulose plates and characterized by flow cytometry analysis, the frequency of CD71-/ CD235+ erythroid cells in TAK-243 treated plates was significantly lower than that in control plates (P<0.05). These results implicated that impairment of UBA1 activity perturbed hematopoietic functions of BM HSPC, particularly the erythropoietic function.

We next evaluated in vivo hematopoietic impact of UBA1 impairment by applying TAK-243 to wildtype male B6 mice. Compared to vehicle control treated mice (N=4), mice treated by TAK-243 (N=4) exhibited significant decreases in hemoglobin and white cell counts (P<0.05) in peripheral blood (PB) and splenomegaly. In BM compartment, TAK-243 treated mice had decreased CD71+ and Ter119+ erythroid populations and increased Gr1+/ CD11b+ myeloid population (P<0.05). TAK-243 treatment also altered BM HSPCs, including reduced megakaryocyte-erythroid progenitor (MEP) and increased granulocyte-monocyte progenitor (GMP) in BM, as well as reduced CD150+/ CD48- stem cells in the BM Lin-/ Sca1+/ cKit+ (LSK) population. Moreover, BM LSK isolated from TAK-243 treated mice (N=4) formed significantly reduced number of colonies compared to vehicle control treated mice (N=4) in CFU assay. Consistently, in competitive transplantation assay, repopulating activity of BM cells of TAK-243 treated mice was significantly impaired, as measured by the donor-derived CD45.2 chimerism in PB of the CD45.1 recipients. Moreover, we performed bulk RNA-Seq in BM LSK of drug treated mice, which revealed that the ribosomal function and cell cycle regulation signals in TAK-243 treated mice were significantly affected.

In summary, our study indicated that, in addition to somatic mutations, there was also a significant downregulation of UBA1 expression in BM HSPC of MDS. Compromised UBA1 function impairs hematopoietic function of BM HSPC, which could be an important mechanism for pathogenesis of MDS.

Kanagal-Shamanna:Amgen: Consultancy; Novartis: Consultancy; Aptitude Health: Speakers Bureau; Physicians Education Resource: Speakers Bureau. Garcia-Manero:Gilead Sciences: Research Funding; AbbVie: Honoraria, Research Funding; Astex: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Aprea: Honoraria; Novartis: Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Curis: Honoraria, Research Funding; Acceleron Pharma: Consultancy.

Author notes


Asterisk with author names denotes non-ASH members.

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