Ubiquitin-conjugating enzyme UBE2N modulates proteostasis in immunoproteasome-positive acute myeloid leukemia
Chiharu Ishikawa, … , Kenneth D. Greis, Daniel T. Starczynowski
Chiharu Ishikawa, … , Kenneth D. Greis, Daniel T. Starczynowski
Published May 15, 2025
Citation Information: J Clin Invest. 2025;135(10):e184665. https://doi.org/10.1172/JCI184665.
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Research Article Hematology Oncology

Ubiquitin-conjugating enzyme UBE2N modulates proteostasis in immunoproteasome-positive acute myeloid leukemia

Abstract

Altered protein homeostasis through proteasomal degradation of ubiquitinated proteins is a hallmark of many cancers. Ubiquitination, coordinated by E1, E2, and E3 enzymes, involves up to 40 E2-conjugating enzymes in humans to specify substrates and ubiquitin linkages. In a screen for E2 dependencies in acute myeloid leukemia (AML), ubiquitin conjugating enzyme E2 N (UBE2N) emerged as the top candidate. To investigate UBE2N’s role in AML, we characterized an enzymatically defective mouse model of UBE2N, revealing UBE2N’s requirement in AML without an impact on normal hematopoiesis. Unlike other E2s, which mediate lysine-48 (K48) polyubiquitination and degradation of proteins, UBE2N primarily synthesizes K63-linked chains, stabilizing or altering protein function. Proteomic analyses and a whole-genome CRISPR-activation screen in pharmacologically and genetically UBE2N-inhibited AML cells unveiled a network of UBE2N-regulated proteins, many of which are implicated in cancer. UBE2N inhibition reduced their protein levels, leading to increased K48-linked ubiquitination and degradation through the immunoproteasome and revealing UBE2N activity is enriched in immunoproteasome-positive AML. Furthermore, an interactome screen identified tripartite motif–containing protein 21 (TRIM21) as the E3 ligase partnering with activated UBE2N in AML to modulate UBE2N-dependent proteostasis. In conclusion, UBE2N maintains proteostasis in AML by stabilizing target proteins through K63-linked ubiquitination and prevention of K48 ubiquitin–mediated degradation by the immunoproteasome. Thus, inhibition of UBE2N catalytic function suppresses leukemic cells through selective degradation of critical proteins in immunoproteasome-positive AML.

Authors

Chiharu Ishikawa, Laura Barreyro, Avery M. Sampson, Kathleen M. Hueneman, Kwangmin Choi, Sophia Y. Philbrook, Issac Choi, Lyndsey C. Bolanos, Mark Wunderlich, Andrew G. Volk, Stephanie S. Watowich, Kenneth D. Greis, Daniel T. Starczynowski

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Figure 2

A catalytic inactive mutant of UBE2N suppresses AML.

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A catalytic inactive mutant of UBE2N suppresses AML. (A) UBE2N mouse mod...
(A) UBE2N mouse model to substitute cysteine-87 to serine (C87S). WT UBE2N cDNA cassette is excised by tamoxifen and replaced by mutant (C87S) exon 2. (B) UBE2NC87S mutation inhibits the transfer of ubiquitin to its substrates. (C) DNA-Seq confirmed recombination from cysteine (TGT) to serine (TCT) in MLL-AF9 cells. (D) AML models were developed by retroviral expression of leukemia oncogenes in Ube2nWT or Ube2nC87S HSPCs. (E) Immunoblots of MLL-AF9 Ube2nWT and Ube2nC87S cells. (F) Colony formation of normal HSPCs (n = 3) or MLL-AF9– (n = 3), MN1- (n = 4), AML1-ETO9a– (n = 5), RUNX1D171N- (n = 4), and FLT3-ITD–transduced (n = 4) Ube2nWT and Ube2nC87S cells. (G) Cell proliferation of normal HSPCs or MLL-AF9 Ube2nWT and Ube2nC87S cells (n = 3). (H) Flow cytometry analysis of GFP+ BM, spleen, and peripheral blood cells. (I) Kaplan-Meier survival of Ube2nWT and Ube2nC87S MLL-AF9 cells transplanted into irradiated BoyJ mice (n = 9 per group). Tamoxifen was injected at weeks 4 and 6. Mantel-Cox test was used to determine significance. (J) Total BM transplant to assess hematopoiesis. BM cells from Ube2nWT (n = 10) or Ube2nC87S mice (n = 8) were transplanted to lethally irradiated mice. (K) Peripheral blood counts of white blood cells, red blood cells, and platelets (PLT) from Ube2nWT and Ube2nC87S mice before and 15 weeks after tamoxifen. (L and M) HSPC frequency in BM was analyzed by flow cytometry at week 15 after tamoxifen administration. LK, lineage– cKit+; LSK, lineage–Sca1+cKit+; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor, MEP, megakaryocyte erythroid progenitor. Two-way ANOVA (F, G, and K–M) or Student’s t test (unpaired, 2-tailed) (H) was used to determine significance. Error bars represent the SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.