FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome
Madeline Niederkorn, Lavanya Bezavada, Anitria Cotton, Lance E. Palmer, Lahiri Konada, Trent Hall, Vishwajeeth R. Pagala, Jinbin Zhai, Zuo-Fei Yuan, Yingxue Fu, Jacob A. Steele, Shilpa Narina, Andrew Schild, Chengzhou Wu, Sarah Aminov, Michael Schieber, Erin McGovern, Aaron B. Taylor, Sandeep Gurbuxani, Peng Xu, Peng Ji, Laura J. Janke, Anthony A. High, Guolian Kang, Shondra M. Pruett-Miller, Mitchell Weiss, Amit Verma, Raajit K. Rampal, John D. Crispino
Madeline Niederkorn, Lavanya Bezavada, Anitria Cotton, Lance E. Palmer, Lahiri Konada, Trent Hall, Vishwajeeth R. Pagala, Jinbin Zhai, Zuo-Fei Yuan, Yingxue Fu, Jacob A. Steele, Shilpa Narina, Andrew Schild, Chengzhou Wu, Sarah Aminov, Michael Schieber, Erin McGovern, Aaron B. Taylor, Sandeep Gurbuxani, Peng Xu, Peng Ji, Laura J. Janke, Anthony A. High, Guolian Kang, Shondra M. Pruett-Miller, Mitchell Weiss, Amit Verma, Raajit K. Rampal, John D. Crispino
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Research Article Hematology Oncology

FBXO11 suppression rewires an NPM1-centered interactome influencing the progression of myelodysplastic syndrome

Abstract

Myelodysplastic syndromes (MDSs) are malignant hematopoietic stem and progenitor cell (HSPC) disorders that lead to ineffective blood production with poor outcomes. We previously showed that F-box only protein 11 (FBXO11) is downregulated in MDS, and here we report how this event contributes to disease progression. Integration of multiomics data revealed that the SCF-FBXO11 complex regulates spliceosome and ribosome components in a nucleophosmin 1 (NPM1)-centric network. FBXO11 facilitates the ubiquitylation of NPM1, whereby deletion of FBXO11 results in the reorganization of NPM1 and a de-repression of alternative splicing. Label-free total quantitative proteomics demonstrated that the FBXO11-NPM1 interactome was markedly downregulated in cells from patients with CD34+ MDS. In addition, we discovered that MYC was evicted from the FBXO11 promoter by TLR2 activation, revealing that it was a MYC target gene and explaining why FBXO11 expression was decreased in MDS. In MDS mouse models, genetic ablation of Fbxo11 exacerbated neutropenia concomitant with a profound decrease in NPM1 protein levels. Finally, we discovered rare mutations in FBXO11, which mapped to a previously unstudied functional intrinsically disordered region (IDR) in the N-terminus responsible for binding NPM1. These data support a model in which FBXO11 rewires RNA binding and ribosomal subnetworks through ubiquitylation of NPM1, ultimately restricting MDS progression.

Authors

Madeline Niederkorn, Lavanya Bezavada, Anitria Cotton, Lance E. Palmer, Lahiri Konada, Trent Hall, Vishwajeeth R. Pagala, Jinbin Zhai, Zuo-Fei Yuan, Yingxue Fu, Jacob A. Steele, Shilpa Narina, Andrew Schild, Chengzhou Wu, Sarah Aminov, Michael Schieber, Erin McGovern, Aaron B. Taylor, Sandeep Gurbuxani, Peng Xu, Peng Ji, Laura J. Janke, Anthony A. High, Guolian Kang, Shondra M. Pruett-Miller, Mitchell Weiss, Amit Verma, Raajit K. Rampal, John D. Crispino

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Figure 6

Genetic deletion of Fbxo11 expands MDS progenitors in vitro and worsens murine MDS in vivo.

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Genetic deletion of Fbxo11 expands MDS progenitors in vitro and worsens ...
(A) Immunoblots of FBXO11, NPM1, and H3 with densitometry. Each sample was normalized to H3, and then to controls. (B) Densitometric values of FBXO11 and NPM1 from A. Pearson correlation for all pairs of values and line of best fit with a 95% CI. (C) Cell fitness assay using primary CD34+ cells. FC in indel percentages at the end of the assay versus the initial read. For double knockout, the indel percentage tracks NPM1 edits. *Q < 0.05 and **Q < 0.01, by 2-group specific longitudinal mixed-effects analysis corrected for multiple comparisons. (D) Number of colonies. *Q < 0.05, **Q < 0.01, and ***Q < 0.001, by multiple t tests corrected for multiple comparisons. n = 3. (E) Number of colonies. n = 62 with 3 wells per assay. *Q < 0.05 and **Q < 0.01, by t tests on pooled replicates corrected for multiple comparisons. (F) Growth curve of F-36P GFP+ vector or FBXO11-overexpressing cells. n = 9 wells per group. ****P < 0.0001, by t test from the linear mixed-effects model with wells as a random effect. (G) Growth curve of MDS92 GFP+ vector or FBXO11-overexpressing cells. n = 9 wells per group. ****P < 0.0001, by t test from the linear mixed-effects model with wells as a random effect. (H) Schematic of the RUNX1-driven MDS mouse model on an inducible Mx1-Cre+ Fbxo11+/+ or Fbxo11+/– background. (I) Percentage of GFP+ peripheral blood cells isolated from Fbxo11+/+ RUNX1-GFP or Fbxo11+/– RUNX1-GFP transplants. n = 7–10 mice per group. ****P < 0.0001, by fixed-effects (type III) analysis. (J) Percentage of GFP+ mononuclear cells isolated from BM aspirates of transplant recipients at 11 weeks. n = 8–9 mice per group. *P-linear < 0.05, by 2-tailed t test. (K) Percentage contribution to the LSK, Lin-/Sca1+/Kit+/SLAM+ (signaling lymphocyte activation molecules) (LSK-SLAM+) gate of immunophenotypically defined HSPCs in surviving RUNX1 transplant recipients. n = 6 mice per group. LT-HSC, long-term HSC; ST-HSC, short-term HSC; MPP-GM, granulocyte-monocyte biased multipotent progenitors; MPP-MkE, megakaryocyte-erythroid biased multipotent progenitors; MPP Ly, lymphoid-biased multipotent progenitor. Q > 0.05, by t tests corrected for multiple comparisons (no significant differences were detected). (L) Percentage contribution of RUNX1-GFP+ common myeloid progenitor (CMP), GMP, and megakaryocyte-erythrocyte progenitor (MEPs) in the Lin–c-kit+ HSPC compartments. n = 6 mice per group. *P < 0.05, by multiple unpaired tests, t test for groups with normal distribution, or Mann-Whitney U test. (M) Differential CBCs in mice 16 weeks after pIpC. *P < 0.05, by unpaired, 2-tailed t for groups with normal distribution or Mann-Whitney U test. (N) Strategy of Nup98-Hoxd13-driven MDS mouse model with shCTRL or shFbxo11 vectors. n = 6–10 mice per group. (O) Western blot for FBXO11, NPM1, and ACTIN in c-kit+ cells from Nup98-Hoxd13+ mice, transduced with lentiviral shCTRL or shFbxo11. (P) CBC in Nup98-Hoxd13 transplants at 2 months. shFbxo11 groups were pooled. *P < 0.05 and **P < 0.005, by unpaired, 2-tailed t test for groups with normal distribution or Mann-Whitney U test. (Q) BM cellularity of Nup98-Hoxd13 mice. shFbxo11 groups were pooled. *P < 0.05 and **P < 0.01, by Mann-Whitney U test. (R) Representative H&E-stained femur cells from Q. Original magnification, ×10. Scale bar: 200 μm.