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 6

UBE2N utilizes TRIM21 for immunoproteasome-dependent degradation of oncoproteins.

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UBE2N utilizes TRIM21 for immunoproteasome-dependent degradation of onco...
(A) Proximity assay using doxycycline-inducible UBE2N-V5-APEX2. Cells were treated with biotin phenol to activate APEX2 for biotin labeling and quenched by hydrogen peroxide. Biotinylated proteins were pulled down and analyzed by LC-MS/MS. (B) Enriched proximal proteins compared with control (DOX-untreated sample) (1.7 > fold change, P < 0.05). (C) KEGG pathway enrichment of the hits. The enrichment score and P value are shown. (D) Coimmunoprecipitation in HEK293T cells expressing empty vector, FLAG-UBE2N, or GFP-TRIM21. FLAG was immunoprecipitated and GFP was immunoblotted. (E) Immunoblots in AML and healthy CD34+ cells. (F) Colony formation of TRIM21-deleted cells (n = 3 per group). (G) Colony formation assay of TRIM21-deficient MDSL, PD-AML (patient ID 17-14) cells (n = 5 per group), and healthy CD34+ cells (n = 3 per group). (H) Kaplan-Meier survival analysis of xenografted mice (n = 7 per group). Mantel-Cox test was used to determine significance. (I) Engraftment of hCD45+hCD33+ cells in BM at time of death. (J) Ubiquitin enrichment of cells expressing shControl, shUBE2N, or shTRIM21. After selection, protein lysates were digested by trypsin, followed by enrichment of ubiquitinated peptides using K-e-GG magnetic beads and analysis by LC-MS/MS. (K) Venn diagram of significantly reduced substrates in UBE2N- and TRIM21-deficient cells from the ubiquitin-enriched analysis. (L) Coimmunoprecipitation in HEK293T cells expressing FLAG-UBE2N, TRIM21, myc-STAT3 and HA-Ub (WT, K48-specific, or K63-specific). Myc was immunoprecipitated and HA-ubiquitin was immunoblotted. (M) Immunoblots of MV4;11 and MDSL cells. (N) Colony formation of MV4;11 cells (n = 5) expressing empty vector or active STAT3 (STAT3CA) and treated with UBE2Ni (5 μM). Two-way ANOVA was used to determine significance. Student’s t test (unpaired, 2-tailed) was used to determine significance (F, G, and I). Error bars represent the SEM. *P < 0.05; **P < 0.01; ***P < 0.001.