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 1

Integrated multiomics defines NPM1 as an SCF-FBXO11 substrate.

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Integrated multiomics defines NPM1 as an SCF-FBXO11 substrate. (A) Heatm...
(A) Heatmap of log2FC values for ubiquitylated peptides in MDS-L FBXO11-KO versus WT cells. Shown are hits with a FC of greater than |0.5|. Known RNA-binding proteins are labeled in red. Data were reanalyzed from ref. 12. (B) Scatter plot of the results from co-IP and MS identification of endogenous FBXO11 complexes in F-36P cell nuclear fractions. Shown are the –log(P value) against the FC enrichment of individual proteins identified in the FBXO11 IP versus the IgG control IP. The blue box highlights proteins with a P value of less than 0.05 and a FC of greater than 1.5. The P value was derived by G test. (C) STRING network analysis of FBXO11-interacting proteins identified by MS. Only input nodes are shown, with the threshold cutoff of medium confidence at 0.7. The thickness of the lines connecting nodes indicates the relative strength of evidence supporting the protein-protein interactions. (D) Strategy for the FBXO11 substrate-focused CRISPR/Cas9 screen, in which the gRNA library encompasses all of the differentially ubiquitylated peptides identified from the ubiquitin proteomics experiment represented in A. (E) Summary table of significant hits from the MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout) analysis of the CRISPR screen in D, demonstrating selective enrichment or depletion of guides in sgCTRL or sgFBXO11 colonies. (F) Schematic overview of the integrated multiomics approach to identify relevant candidate FBXO11 substrates meeting the listed criteria, revealing NPM1 and HNRNPU. (G) FC in individual gRNA read counts for NPM1 and HNRNPU in the colony-forming assay (day 10) versus initial representation (day 0) in the CRISPR screen. The FC for individual guides was compared between sgCTRL and sgFBXO11 experimental groups, shown on the graphs. n = 6 guides per gene, in 2 independent biological replicates of the CRISPR screen. *P < 0.05, by 2-tailed, paired t test.