UBE2N Is a Druggable Target and an Essential Ubiquitin-Conjugating Enzyme in Myeloid Malignancies

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) originate in clonal mutant hematopoietic stem and progenitor cells, referred to as leukemic stem and progenitor cells (LSPCs). In our recent manuscript (Barreyro et al., Science Translational Medicine, 2022), we reported that UBE2N represents a key immune-related signaling node in AML and that small molecule inhibitors targeting the catalytic activity of UBE2N suppress LSPCs. UBE2N is a ubiquitin-conjugating E2 enzyme that synthesizes Lysine-63 (K63) linked-polyubiquitin chains on target proteins in conjunction with select E3 ubiquitin ligases, which leads to stabilization and/or activation of substrates.

In this study we report that UBE2N is overexpressed and activated in MDS and AML LPSCs and that UBE2N is an essential gene in a large panel of MDS/AML cell lines. The catalytic function of UBE2N is mediated by cysteine 87 (C87) within its active site. To rigorously investigate the catalytic requirement of UBE2N in myeloid malignancies, we generated a conditional knock-in mouse model in which the catalytic residue C87 is substituted to serine (C87S) upon Cre-mediated recombination. Utilizing this model, we generated AML in wild-type (WT) and UBE2N(C87S) BM HSPCs by expressing the leukemic oncogenes MLL-AF9, FLT3-ITD, and MN1. Expression of the catalytically impaired UBE2N(C87S) resulted in a significant reduction in leukemic progenitor cell function in vitro, impaired cell viability, and suppression of overt leukemia in vivo. UBE2N(C87S) expression in AML cells correlated with changes in gene expression related to impaired innate immune/inflammatory signaling (NF-kB, ERK1/2) and cell viability. In contrast, expression of UBE2N(C87S) during normal hematopoiesis had negligible effects on blood development and overall survival of the mice. Thus, the catalytic function of UBE2N is required for AML but not for normal hematopoiesis in mice.

To gain mechanistic insight into the requirement of UBE2N in AML, we first performed a proximity-labeling assay followed by a proteomic analysis and identified Tripartite motif containing 21 (TRIM21) as one of the top binding partners of UBE2N in AML cells. TRIM21, an E3 ubiquitin ligase, is overexpressed >2 fold in AML patients compared to the healthy controls and its dependency in a panel of AML cell lines correlates with UBE2N. As a functional link between UBE2N and TRIM21, we observed that inhibition or deletion of UBE2N corresponded with reduced protein expression of TRIM21. Importantly, restoration of TRIM21 rescued the functional defect of UBE2N-deficient AML cells, indicating that the UBE2N-TRIM21 axis is operational in AML. Furthermore, knockout (KO) of TRIM21 by CRISPR/Cas9 resulted in a significant reduction in AML LSPC function in vitro. Xenograft studies using TRIM21 KO AML cells extended the overall survival and reduced leukemic cell burden in recipient mice as compared to recipient mice engrafted with WT AML cells. These findings establish that TRIM21 is a critical effector of UBE2N in AML. In summary, our data reveal that inhibiting UBE2N catalytic function, such as with small molecule inhibitors, can effectively suppress LSPCs by targeting the UBE2N-TRIM21 axis.

Starczynowski:Sumitomo Pharma Oncology, Inc: Research Funding; Captor Therapeutics: Consultancy; Kurome Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Treeline Biosciences: Research Funding; Tolero Therapeutics: Research Funding; Kymera Therapeutics: Consultancy.