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 2

FBXO11 facilitates ubiquitylation of NPM1 and permits its distribution into the nucleoplasm.

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FBXO11 facilitates ubiquitylation of NPM1 and permits its distribution i...
(A) Immunoblots of overexpressed FLAG-FBXO11 isoforms in HEK293T cells. Blotting was done for endogenous NPM1 and H2A. Endogenous NPM1 IPs were probed for FBXO11 and NPM1. (B) Immunoblots of co-IP FLAG-FBXO11 complexes of FBXO11 and NPM1. (C) Left: Input immunoblots for FLAG-FBXO11, NPM1, ubiquitin-GFP, and H2A in HEK293T cells. IPs of NPM1 were performed to detect ubiquitylation by FBXO11. Right: NPM1 versus IgG control IP, immunoblotted for GFP-ubiquitin. n = 3. (D) Densitometry for NPM1 poly-ubiquitin bands from C. The area quantified is 75 kDa and above. n = 3. *Q < 0.05, by Kruskal-Wallis ANOVA corrected for multiple comparisons by the Benjamini method. (E) Schematic depicting K248-Ub of NPM1 in its C-terminal core. The ubiquitin (Ub) footprint was identified by MS, Figure 1A and Supplemental Table 1. (F) HEK239T cells were transfected with NPM1-GFP or NPM1-K248R-GFP fusions. Red outlines indicate GFP-bright regions annotated in QuPath. Scale bars: 10 μm. (G and H) Annotated regions quantified for circularity (G) and aspect ratio (H). n = 2. Dots represent individual NPM1+ regions.*P < 0.05 and **P < 0.01, by 2-tailed Mann-Whitney U test. (I) Confocal images of human CD34+ cells expressing shCTRL or shFBXO11 mCherry constructs. Images are pseudocolored for FBXO11 (magenta), NPM1 (green), and DAPI (blue). Scale bars: 5 μm. (J) NPM1-bright objects per cell. *P < 0.05, by 2-tailed Mann-Whitney U test. (K) Circularity values for NPM1-bright objects in the shCTRL and shFBXO11 images. *P < 0.05, by 2-tailed Mann-Whitney U test. (L) Aspect ratio values for NPM1-bright objects in the shCTRL and shFBXO11 images. *P < 0.05, by 2-tailed Mann-Whitney U test. (M) Relative MFI values for FBXO11 signal and NPM1 signal on a per-cell basis. ****P < 0.0001, by 2-tailed t test. (N) Human CD34+ cells stained for FBXO11 (green) and NPM1 (magenta). Colocalization (white arrowheads) where green and magenta overlap. Scale bars: 5 μm. (O) Pearson correlation values for NPM1 and FBXO11 signals in the nucleolus (NPM1-bright) and nucleoplasm (NPM1-dim). Each dot represents 1 cell, with greater than 100 cells at ×63. ****P < 0.0001, by 2-tailed Mann-Whitney U test.