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 5

UBE2N dependency is predominant in immunoproteasome-positive AML.

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UBE2N dependency is predominant in immunoproteasome-positive AML. (A) Pr...
(A) Proposed model. (B) Immunoblots of MV4;11 cells cotreated with DMSO or UBE2Ni (5 μM) and the constitutive proteasome inhibitor MG132 (5 μM, 6 hours) or the lysosomal inhibitor bafilomycin A (100 nM, 20 hours). (C) Immunoblots of MV4;11 cells treated with DMSO or UBE2Ni (5 μM) and the immunoproteasome inhibitor ONX-0914 (10 nM or 200 nM) for 24 hours. (D) Immunoblots of Ube2nWT or Ube2nC87S MLL-AF9 cells cotreated with 4-OHT (0.5 μM) and ONX-0914 (10, 100, or 200 nM) for 24 hours. (E) Coimmunoprecipitation in MV4;11 cells treated with DMSO or UBE2Ni (5 μM) and ONX-0914 (200 nM) for 24 hours. NPM1 and STAT3 were immunoprecipitated and immunoblotted for K48-linked polyubiquitination. Densitometric values were calculated based on the expression of K48-ubiquitinated NPM1 (left) or STAT3 (right) relative to immunoprecipitated NPM1 or STAT3. (F) Cell viability of MV4;11 cells (n = 3 per group) 48 hours after treatment with UBE2Ni (2.5 μM) and ONX-0914 (100 nM and 200 nM). Two-way ANOVA was used to determine significance. (G) Overview of experiments using PD-AML. PD-AML cells were (a) treated with UBE2Ni and MTS assay was conducted or (b) analyzed by RNA-Seq. (H) Heatmap of percentage of viability of UBE2Ni-treated PD-AML cells determined by MTS assay. PD-AMLs were classified as UBE2Ni resistant or sensitive. (I) The mRNA expression levels of immunoproteasome genes in UBE2Ni-sensitive (n = 5) and -resistant (n = 6) PD-AML. (J) Immunoproteasome activity was measured in UBE2Ni-sensitive and -resistant PD-AML. Student’s t test (unpaired, 2-tailed) was used to determine significance (I and J). Error bars represent the SEM. *P < 0.05; **P < 0.01.