Ubiquitin-conjugating enzyme UBE2N modulates proteostasis in immunoproteasome-positive acute myeloid leukemia
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
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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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 3

Proteostasis regulation in AML by UBE2N.

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Proteostasis regulation in AML by UBE2N. (A) Gene-expression analysis of...
(A) Gene-expression analysis of MLL-AF9–transduced Ube2nWT and Ube2nC87S cells. The cells were treated with 4-OHT for 48 hours and RNA was collected for sequencing. (B) Volcano plots of differentially expressed genes. (C) Pathway enrichment analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) of the significantly downregulated genes in Ube2nC87S cells (>2-fold change; P < 0.05). The enrichment score and corresponding P value is shown. (D) Ubiquitin-enriched proteomic of MV4;11 cells transduced with nontargeting shRNA (shControl) or shUBE2N. After selection, these cells were lysed and digested by trypsin, followed by the enrichment of the ubiquitin-bound peptides using K-e-GG magnetic beads and analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS). (E) Pathway enrichment analysis using KEGG of the significantly reduced ubiquitinated substrates in UBE2N-deficient condition. The enrichment score and corresponding P value are shown. (F) Total proteomics analysis of MV4;11 cells treated with UBE2Ni (UC-65, 5 μM) or vehicle for 24 hours and then lysed and digested by trypsin, followed by LC-MS/MS. (G) Pathway enrichment analysis using KEGG of the significantly reduced proteins in UBE2N-inhibited conditions. The enrichment score and corresponding P value are shown. (H) Overview of the CRISPRa screen in MOLM13 cells expressing dCas9-VP64 and lentiviral sgRNA pooled library. After selection, the cells were treated with DMSO or 2.5 μM UBE2Ni for 7 days, and deep sequencing was performed. (I) The FDR and the corresponding P value of top hits from CRISPRa screen. (J) Venn diagram of the commonly identified hits from UBE2N-dependent substrates from MS and enriched genes from CRISPRa screen.