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Biomolecular condensation of NUP98 fusion proteins drives leukemogenic gene expression

Nature Structural & Molecular Biology volume 28, pages 190–201 (2021)Cite this article

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Abstract

NUP98 fusion proteins cause leukemia via unknown molecular mechanisms. All NUP98 fusion proteins share an intrinsically disordered region (IDR) in the NUP98 N terminus, featuring repeats of phenylalanine-glycine (FG), and C-terminal fusion partners often function in gene control. We investigated whether mechanisms of oncogenic transformation by NUP98 fusion proteins are hardwired in their protein interactomes. Affinity purification coupled to mass spectrometry (MS) and confocal imaging of five NUP98 fusion proteins expressed in human leukemia cells revealed that shared interactors were enriched for proteins involved in biomolecular condensation and that they colocalized with NUP98 fusion proteins in nuclear puncta. We developed biotinylated isoxazole-mediated condensome MS (biCon-MS) to show that NUP98 fusion proteins alter the global composition of biomolecular condensates. An artificial FG-repeat-containing fusion protein phenocopied the nuclear localization patterns of NUP98 fusion proteins and their capability to drive oncogenic gene expression programs. Thus, we propose that IDR-containing fusion proteins combine biomolecular condensation with transcriptional control to induce cancer.

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Fig. 1: NUP98−KDM5A does not operate in the context of the nuclear pore complex.
Fig. 2: Functional proteomic identification of conserved interactors of diverse NUP98 fusion proteins.
Fig. 3: The NUP98 fusion protein interactome is enriched for proteins with roles in biomolecular condensation.
Fig. 4: biCon-MS globally charts the cellular condensome.
Fig. 5: Expression of NUP98 fusion proteins dynamically alters the cellular condensome.
Fig. 6: An artificial IDR-containing KDM5A fusion protein phenocopies NUP98-fusion-induced changes in the condensome.
Fig. 7: Artificial FG-containing fusion proteins induce leukemogenic gene expression programs in hematopoietic progenitor cells.

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Data availability

LC–MS/MS data have been deposited into the PRIDE database under the accession number PXD022518.

RNA-seq data have been deposited into the GEO under the accession number GSE159037. Source data are provided with this paper.

Code availability

R script implemented for group size–dependent testing statistic (GSDTS) is available at https://github.com/Edert/R-scripts

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Acknowledgements

We thank all members of the Grebien laboratory for stimulating discussions and A. Orlova for help with QUANT-seq experiments. A. Spittler, G. Hofbauer (Core Facility Flow Cytometry, Medical University of Vienna), S. Fajmann and P. Jodl (Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna) are acknowledged for cell sorting. This research was supported using resources of the VetCore Facility (Imaging) of the University of Veterinary Medicine Vienna. Next-generation sequencing was performed at the VBCF NGS Unit (https://www.viennabiocenter.org/facilities/). In addition, we thank all members of the Proteomics and Metabolomics Facility (CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences) for the proteomics analysis. J.S. is a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the University of Veterinary Medicine Vienna. This work was supported by a grant from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 636855/StG) to F.G. E.M.T. was supported by a fellowship of the Austrian Science Fund (FWF, Elise Richter Fellowship V506-B28).

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Authors and Affiliations

  1. Institute for Medical Biochemistry, University of Veterinary Medicine, Vienna, Austria

    Stefan Terlecki-Zaniewicz, Theresa Humer, Thomas Eder, Johannes Schmoellerl, Elizabeth Heyes, Gabriele Manhart, Fabio G. Liberante & Florian Grebien

  2. Children’s Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria

    Stefan Terlecki-Zaniewicz & Eleni M. Tomazou

  3. Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria

    Natalie Kuchynka

  4. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

    Katja Parapatics & André C. Müller

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  1. Stefan Terlecki-Zaniewicz
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Contributions

S.T.-Z, F.G.L. and F.G. designed the study; S.T.-Z, J.S., F.G.L and F.G. developed the methodology; S.T.-Z., T.H., T.E. and A.C.M. performed data analysis; S.T.-Z., T.H., J.S., N.K., E.H., G.M. and K.P. conducted experiments and collected data; T.E. performed bioinformatics analysis and developed software; S.T.-Z. and F.G. wrote the initial draft; S.T.-Z., T.H., T.E., N.K. and F.G. generated visualization of the data; F.G. supervised the project; S.T-Z. and F.G. coordinated responsibility for the research activity; E.M.T. and F.G. acquired funding; all authors revised the final manuscript prior to submission.

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Correspondence to Florian Grebien.

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The authors declare no competing interests.

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Peer review information Nature Structural & Molecular Biology thanks Christopher Slape and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Immunoprecipitation of endogenous NUP98 and affinity purification of NUP98-KDM5A coupled to LC-MS/MS, Related to Fig. 1.

a, mRNA expression of NUP98-KDM5A in HL-60 cells and mouse AML cells expressing NUP98-KDM5A (paired, two-sided t-test, p-value: 0.33, three biological replicates). ddCt values were calculated using GAPDH/Gapdh expression for HL-60 cells and mouse leukemia cells, respectively. Graph shows individual data points, mean and s.d. n = 3 (biological replicates of the same experiment) (b) Western blot analysis of mock-transduced- and NUP98-KDM5A-expressing HL-60 cells. Control lysates were incubated with NUP98 antibody conjugated to magnetic beads and NUP98-KDM5A lysates were incubated with magnetic Strep-Tactin beads for 1 hour. Input, supernatant (sup) and pull down fractions were loaded and membranes were probed with anti-NUP98, anti-HA and anti-Tubulin antibodies. A representative blot of three independent experiments is shown. Uncropped images are available in Supplementary Fig. 1. c, String database networks of individual subcomplexes identified by Gene Ontology for Biological Processes (Fig. 1d). Identified interactors for respective GO terms were clustered using String database (cutoff 0.4). Individual proteins are highlighted (yellow to green) according to abundance in MS datasets. d, Confocal microscopy images of N-NUP98-expressing HL-60 cells stained with DAPI (green in merge) and anti-HA antibody (white in merge) for exogenous fusion proteins. Scale bar: 5 µm. Six independent experiments were performed with similar results. e, Venn diagram of identified proteins from three different affinity purifications in HL-60 cells. Protein lists from immuno-affinity purification experiments with an anti-NUP98 antibody in mock transduced cells or N-NUP98-expressing cells were intersected with each other and with proteins identified in Strep-Tactin purifications from N-NUP98-expressing cells. The overlap shows N-terminal NUP98 interactors that are conserved between different pull-down approaches.

Source data

Extended Data Fig. 2 AP-MS analysis of NUP98-fusion proteins and validation of selected interaction partners, Related to Fig. 2.

a, Schematic representation of the experimental strategy. HL-60 cells were transduced with retroviral constructs encoding NUP98 fusion proteins and protein complex pulldowns were performed using Strep-Tactin followed by LC-MS/MS analysis. b, Immunofluorescence of mock transduced (top) HL-60 cells or cells expressing NUP98-KDM5A (bottom) stained with DAPI, anti-HA (background-fluorescence corrected) and anti-RAE1. Co-localization (co-loc) was determined with the ImageJ plugin ‘Colocalization’ Six independent experiments were performed with similar results. (c) Manders’ coefficient showing the co-occurrence of NUP98-KDM5A with RAE1. **** p-value < 0.0001, n = 12 cells examined over 2 independent experiments (6 mock vs. 6 NUP98-KDM5A) Two sided, paired t-test, t = -10.277, df = 5, **** p-value = 0.0001499. The box plot centre line defines the median, the box limits indicate upper and lower quartiles, whiskers indicate minima and maxima among all data points. Data behind graph are available as Source Data. d, Western blot analysis of protein lysates from control HEK293T cells or cells transfected with NUP98-KDM5A after Strep-Tactin affinity purification. Blot is representative of three independent experiments. Uncropped images are available in Supplementary Fig. 1. (e) CORUM analysis of 157 NUP98-fusion protein core interactors. Complexes are illustrated as a network highlighting top terms ranked by p-value. The network was generated with ClueGo v3.5.4. f, GO analysis (Biological Process) was performed for 157 NUP98-fusion protein core interactors. GO terms were ranked by combined score using Enrichr.

Source data

Extended Data Fig. 3 PScore analysis of different protein lists, Related to Fig. 3.

a, GSDTS was performed for the Gene Ontology list ‘Nuclear Membrane’. The mean PScore was compared to a set of lists of equal length that were randomly subsampled from the human proteome. b, GSDTS was performed for the human proteome. p-values were calculated using the Kolmogorov–Smirnov test. c, 100 µM b-isox precipitation of HEK293T cells transfected with mock, flag-tagged NUP98-KDM5A or untagged NUP98-KDM5A. Endogenous NUP98 and NUP98-KDM5A proteins were detected with anti-NUP98 antibodies in input, supernatant (sup) and precipitated (b-isox) fractions. Blot is representative of three independent experiments. Uncropped images are available in Supplementary Fig. 1.

Source data

Extended Data Fig. 4 b-isox precipitation of N-NUP98, related to Fig. 4.

a, Western blot analysis of protein lysates from HEK293T cells expressing N-NUP98 treated with 11 µM, 33 µM or 100 µM b-isox. N-NUP98 was detected using anti-HA antibodies. Total input, supernatant and b-isox fractions (pellet) are shown. Blot is representative of three independent experiments. Uncropped images are available in Supplementary Fig. 1.

Extended Data Fig. 5 biCon-MS for NUP98-KDM5A and NUP98-NSD1, Related to Fig. 5.

a, Normalized and scaled protein abundances for selected proteins previously implicated in the formation of biomolecular condensates identified by biCon-MS from lysates of HL-60 cells expressing NUP98-KDM5A. Graph shows individual data points, mean and s.d. for n = 4 (2 biological, 2 technical replicates). b, Normalized and scaled protein abundances for selected proteins previously implicated in the formation of biomolecular condensates identified by biCon-MS within lysates of HL-60 cells expressing NUP98-NSD1. Graph shows individual data points, mean and s.d. for n = 4 (2 biological, 2 technical replicates). c, Western blot analysis of NUP98-KDM5A-expressing NIH-3T3 cell lysates treated with 11 µM, 33 µM or 100 µM b-isox. Dose-dependent precipitation was investigated for NUP98-KDM5A and HSC70 as loading control. One representative blot of three independent experiments is shown. d, Western blot analysis of NIH-3T3 cell lysates treated with 11 µM, 33 µM or 100 µM b-isox. Dose-dependent precipitation was investigated for HSC70. One representative blot of three independent experiments is shown. Blot is representative of three independent experiments. Uncropped images for panels c and d are available in Supplementary Fig. 1. e, Schematic illustration of enriched/depleted proteins identified in fusion protein biCon-MS compared to mock-transduced HL-60 cells. f, Enrichment of proteins that exhibit dose-dependent precipitation upon expression of NUP98-KDM5A and NUP98-NSD1 as compared to mock-transduced HL-60 cells based on abundances in biCon-MS analysis. Enriched/depleted proteins in NUP98-fusion protein condensates are illustrated as nodes and are colored according to calculated fold change values. Depletion cutoff: log2(fc) < −1.5 and p-value < 0.01 Enrichment cutoff: log2(fc) > 1.5 and p-value < 0.01. P-value was calculated using a two-sided ANOVA test. g, Normalized and scaled abundances of significantly enriched (MED31) or depleted (MED15) proteins in NUP98-fusion protein condensomes as identified by biCon-MS. Graph shows individual data points, mean and s.d. for n = 4 (2 biological, 2 technical replicates).

Source data

Extended Data Fig. 6 biCon-MS for artAA-KDM5A and artFG-KDM5A, Related to Fig. 6.

Volcano plot of (a) artAA-KDM5A and (b) artFG-KDM5A for 11 µM and 33 µM condensomes, generated from normalized protein abundances obtained by biCon-MS. The significance cutoff was log2(fc) < −1 or > 1 and p-value < 0.01. (c) GO (Molecular Function) analysis for 237 proteins precipitated by b-isox in artFG-KDM5A- vs. artAA-KDM5A-expressing cells. (d) Normalized and scaled abundances of selected enriched proteins in the artFG-KDM5A-induced condensome as identified by biCon-MS. Graph shows individual data points, mean and s.d. for n = 4 (2 biological and 2 technical replicates).

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Extended Data Fig. 7 RNA-seq of mouse fetal liver cells, Related to Fig. 7.

a, Principal component analysis based on normalized expression profiles of RNA-seq from murine wild type fetal liver cells and cells expressing NUP98-KDM5A, artAA-KDM5A or artFG-KDM5A. b, 761 genes that are differentially regulated upon expression of NUP98-KDM5A and artFG-KDM5A were grouped using DisGeNET according to related diseases. Most significant disease terms are illustrated as hexagons sized according to their p-value. Corresponding disease classes are shown as diamonds connected by edges to diseases, defined by DiGeNET. c, The mean PScore for all proteins involved in cancer gene fusions listed in COSMIC was compared to a list of 10,000 randomly subsampled lists of the human proteome with the same size. The p-value was calculated using the two-sided, two-sample Kolmogorov-Smirnov test, D = 0.26114, p-value < 2.2e-16.

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Supplementary information

Supplementary Information

Supplementary Table 3, Supplementary Note, Supplementary Fig. 1.

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Supplementary Table 1

Mass spectrometry of endogenous NUP98 and NUP98–KDM5A.

Supplementary Table 2

Mass spectrometry of NUP98–HOXA9, NUP98–PSIP1, NUP98–DDX10 and NUP98–NSD1 and spectrum counts for core interactors.

Supplementary Table 4

Summary of Kolmogorov–Smirnov test analyses of PScore enrichment for different protein lists.

Supplementary Table 5

Mass spectrometry of biCon-MS for HL-60 cells (mock, NUP98–KDM5A and NUP98–NSD1).

Supplementary Table 6

Mass spectrometry of biCon-MS for HL-60 cells (artAA-KDM5A and artFG-KDM5A)

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Terlecki-Zaniewicz, S., Humer, T., Eder, T. et al. Biomolecular condensation of NUP98 fusion proteins drives leukemogenic gene expression. Nat Struct Mol Biol 28, 190–201 (2021). https://doi.org/10.1038/s41594-020-00550-w

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