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U2AF1 mutations induce oncogenic IRAK4 isoforms and activate innate immune pathways in myeloid malignancies

Nature Cell Biology volume 21, pages 640–650 (2019)Cite this article

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Abstract

Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable ‘active’ IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.

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Fig. 1: Differential RNA isoform usage correlates with AML prognosis and oncogenic IRAK4 isoforms.
Fig. 2: IRAK4-L expression results in myddosome assembly and maximal activation of innate immune signalling.
Fig. 3: IRAK4-L is required for leukaemic cell function.
Fig. 4: IRAK4-L expression is associated with U2AF1 mutations in MDS and AML.
Fig. 5: U2AF1(S34F) induces expression of IRAK4-L and increased innate immune pathway activation.
Fig. 6: U2AF1(S34F) AML cells are sensitive to IRAK4 inhibitors.

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Data availability

RNA-sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession codes GSE114922 (corresponding to data in Fig. 4b), GSE58831 (corresponding to data in Supplementary Fig. 4), dbGaP: phs000159.v8 (SRX1608652, SRX1608653, SRX1608605, SRX1608609, SRX1608616 and SRX1608620) (corresponding to data in Fig. 5a), and E-MTAB-1733 (corresponding to data in Fig. 1g). The human AML data were derived from the TCGA Research Network: http://cancergenome.nih.gov. Protein isoform-isoform interaction network was generated using human protein sequences (GRCh37/hg19) from the UCSC Genome Browser’s Table Browser (https://genome.ucsc.edu/cgi-bin/hgTables, accessed February 2009); protein-domain models from PFAM v28 (ftp://ftp.ebi.ac.uk/pub/databases/Pfam/releases/Pfam28.0/), CCD v3.16 (ftp://ftp.ncbi.nih.gov/pub/mmdb/cdd), and InterPro v50 (ftp://ftp.ebi.ac.uk/pub/databases/interpro/50.0/); protein–protein interactions from HPRD v9 (http://hprd.org/download); and domain–domain interactions from DOMINE v2.0 (https://manticore.niehs.nih.gov/cgi-bin/Domine?page = download) (corresponding to data in Fig. 2i). Source data for Figs. 2–6 and Supplementary Figs. 3, 4 and 6 have been provided as Supplementary Table 5. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by Cincinnati Children’s Hospital Research Foundation, Leukemia Lymphoma Society, National Institutes of Health (R35HL135787, RO1DK102759, RO1DK113639), and Edward P. Evans Foundation grants to D.T.S. A.V. is supported by National Institutes of Health (R01HL139487 and R01DK103961), Leukemia and Lymphoma Society, and EvansMDS grants. A.P. and J.B. are supported by Bloodwise (UK, grant 13042). M.A.S. is supported by a National Institutes of Health Research Training and Career Development Grant (F31HL132420). I.D.C.F. is supported by the Intramural Research Program of NIAID. We also thank R. Booher (Curis) for suggestions. We thank M. Scott and Y.-C. Hu from the Gene Editing Core at CCHMC for their assistance.

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  1. These authors contributed equally: Molly A. Smith, Gaurav S. Choudhary.

Authors and Affiliations

  1. Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Molly A. Smith, Kwangmin Choi, Lyndsey C. Bolanos, Kakajan Komurov & Daniel T. Starczynowski

  2. Department of Cancer Biology, University of Cincinnati, Cincinnati, OH, USA

    Molly A. Smith & Daniel T. Starczynowski

  3. Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY, USA

    Gaurav S. Choudhary, Tushar D. Bhagat, Shanisha Gordon-Mitchell, Dagny Von Ahrens, Kith Pradhan, Ulrich Steidl & Amit Verma

  4. Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford and Oxford BRC Haematology Theme, Oxford, UK

    Andrea Pellagatti, Violetta Steeples & Jacqueline Boultwood

  5. Department of Medicine, Washington University, St. Louis, MO, USA

    Sanghyun Kim & Matthew Walter

  6. Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD, USA

    Iain D. C. Fraser

  7. Department of Electrical Engineering and Computer Science, University of Cincinnati, Cincinnati, OH, USA

    Aishwarya Kulkarni

  8. Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Nathan Salomonis

  9. Department of Pediatrics, University of Cincinnati, Cincinnati, OH, USA

    Nathan Salomonis, Kakajan Komurov & Daniel T. Starczynowski

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Contributions

M.A.S., G.S.C. and A.P. performed most of the experiments with assistance from L.C.B. A.P., J.B., N.S., K.K., K.C. V.S., and A.K. performed the splicing and isoform analysis. M.W., S.K., and I.D.C.F. provided essential reagents. T.D.B., S.G.-M., D.V.A., K.P., U.S. and A.V. provided patient samples and insight. M.A.S., K.K., J.B., A.V. and D.T.S. conceived the project, designed experiments, interpreted results and wrote the manuscript.

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Correspondence to Jacqueline Boultwood, Amit Verma or Daniel T. Starczynowski.

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Competing interests

A.V. has received research funding from GlaxoSmithKline, Incyte, MedPacto, Novartis, Curis and Eli Lilly and Company, has received compensation as a scientific advisor to Novartis, Stelexis Therapeutics, Acceleron Pharma and Celgene, and has equity ownership in Stelexis Therapeutics.

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Supplementary Fig. 1 Molecularly and clinically distinct subtypes of AML based on global and IRAK4 RNA isoform expression.

(a) Overview of RNA isoform switching analysis in AML with the corresponding figures and panels shown in parentheses. (b) Schematic representation of expression-level regulation versus isoform-level regulation of RNA isoforms in hypothetical samples. (c) Overall survival analysis of the 3 major groupings of AML (Group 1, blue; Group 2, red; Group 3, green) characterized by distinct patterns of differential isoform usage identified in Fig. 1b. Likelihood ratio test, Multivariate P = 0.06. (d) Exon usage of innate immune genes undergoing isoform switching in AML (Group 2) was correlated to overall survival of AML. Shown is the z-score correlation of IRAK4 exon usage to overall survival of AML patients. In red, exon 4 significantly correlates with worse overall survival of AML patients. (e) Relative expression of IRAK4-L to IRAK4-S in normal tissue (blue) relative to cancer tissue (red) for the indicated cancers analyzed in TCGA. Breast cancer (n = 1093); normal tissue (n = 112). Colon cancer (n = 285); normal tissue (n = 41). Lung cancer (n = 515); normal tissue (n = 59). The P value is indicated for each pair of samples and was determined by Welch’s t-test. (f) Sequence analysis of the IRAK4 exon 4 cassette using primers flanking exon junctions 3-4, 4-5, and 3–5 isolated from THP1 cells.

Supplementary Fig. 2 IRAK4-L and IRAK4-S activation of NF-κB and MAPK pathways.

(a) Pathway analysis of enriched genes in AML patients preferentially expressing IRAK4-L (left) or IRAK4-S (right). Shown are the significant signalling networks and relevant genes associated with IRAK4-L or IRAK4-S expression in AML. Data is adapted from Fig. 2a. (b, c) HEK293 cells transfected with empty vector (pcDNA3.1), pcDNA3.1-IRAK4-L, or pcDNA3.1-IRAK4-S and then immunoblotted for NF-κB (p-IRAK1, p-p65) and MAPK (p-JNK, p-ERK, p-p38) signalling. (Three independent experiments). (d) HL60 cells, which express primarily IRAK4-S, were transduced with empty vector (pMSCV-pGK-GFP) or IRAK4-L and then immunoblotted for the indicated proteins. (Two independent experiments). (e) TF1 and HL60 cells were immunoblotted for IRAK4 with C-terminal antibody that detects IRAK4-L and IRAK4-S after immunoprecipitation of MyD88.

Supplementary Fig. 3 Generation and characterization of THP1 cells deficient for IRAK4 isoforms.

(a) Schematic showing the exons targeted for CRISPR-Cas9 mediated editing of the human IRAK4 gene locus in THP1 cells from 2 independent clones. (b) Immunoblotting for IRAK4 (N-terminal antibody) in matched parental (THP1) and IRAK4-edited (THP1 IRAK4 KO) THP1 cells. (c) Parental and IRAK4 KO THP1 cells expressing were examined for leukaemic progenitor function in methylcellulose using independent clones. Representative images are shown on the left. Scale bar, 700 microns. Data is summarized from 3 biological replicates. Error bars represent mean +/- SD. Two-sided t-tests were used for statistical analyses. (d) Total cell counts were performed for THP1 and THP1-IRAK4 KO (clone 14) cells in liquid culture for the indicated days. (e) Viable cell counts (Trypan blue exclusion) were performed for THP1 and THP1-IRAK4 KO (clone 14) cells in liquid culture for the indicated days. (f) THP1 cells transduced with shRNAs targeting IRAK4-L (shIRAK4-L), IRAK4-L and shIRAK4-S (shIRAK4-L/S), or empty control were monitored in liquid culture by GFP expression. (g) Flow cytometric analysis of THP1 and THP1-IRAK4 KO (clone 14) cells stained with CD14-PE or CD34-APC. Dark grey histogram represents no antibody control. Representative FACS histogram from 2 independent biological replicates. Data represent the mean ± s.e.m.

Supplementary Fig. 4 IRAK4-L is required for leukaemic cell function in vitro and correlates with worse overall survival in MDS.

(a) THP1 cells were transduced with mCherry-expressing shRNAs targeting both IRAK4-L and IRAK4-S (shIRAK4-L/S), or control shRNA, sorted for mCherry expression. Isolated cells (mCherry positive) were then transduced with cDNAs expressing IRAK4-L (pMSCV-pGK-GFP), IRAK4-S, or empty vector and sorted for mCherry/GFP expression. Double positive cells were then immunoblotted with the N-terminal IRAK4 antibody (left panel) and C-terminal IRAK4 antibody (right panel). (b-c) The indicated cells from panel (A) were examined for leukaemic progenitor function in methylcellulose. Representative images are shown in (B). Data in (C) is from 3 biological replicates. Scale bar, 700 microns. Error bars represent mean +/- SEM. Two-sided t-tests were used for statistical analyses. (d) Kaplan-meier survival analysis of MDS patients stratified based on expression of high or low IRAK4-L expression. Data is adapted from Gerstung et al. (Nature Communications, 2015). Data represent the mean ± s.e.m.

Supplementary Fig. 5 Inhibition of IRAK4-L reduces signalling and results in apoptosis in U2AF1-mutant AML cells.

(a–c) K562-U2AF1-S34F cells were treated with 10 μM IRAK1/4-Inh for 1 or 2 hours and immunoblotted for NF-κB (p-IRAK1, p-IKK) and MAPK (p-ERK, p-p38, p-JNK) activation (three biological replicates).

Supplementary Fig. 6 Genetic or pharmacologic inhibition of IRAK4 results in differentiation of U2AF1-mutant MDS, but does not affect normal haematopoiesis.

(a) Immunoblot analysis of THP1 cells using the N-terminal IRAK4 antibody following transfection with a control siRNA (siCTL) or siIRAK4 after 24 hours. (b) BM cells from MDS1 following transfection with a control siRNA (siCTL) or siIRAK4 were plated in methylcellulose (for 14 days) and analyzed by flow cytometry for myeloid differentiation. (c, d) BM cells from MDS3 (C) or normal CD34+ (D) cells were treated with 10 μM CA-4948 in methylcellulose (14 days) and then analyzed by flow cytometry for erythroid differentiation. (e) NSG mice received 12.5 mg/kg of CA-4948 (n = 9 mice) or vehicle (PBS; n = 7 mice) for 5 days per week for 12 weeks. Complete blood counts were determined at the end of the study. Two-sided t-tests were used for statistical analyses. Data represent the mean ± s.e.m.

Supplementary Fig. 7

Unprocessed images of all gels and blots.

Supplementary information

Supplementary Information

Supplementary Figures 1–7, Supplementary Table titles and legends

Reporting Summary

Supplementary Table 1

Genes that undergo RNA isoform switching in AML.

Supplementary Table 2

Genes undergoing mRNA isoform switching with at least one differential exon inclusion/exclusion event that correlates with AML survival.

Supplementary Table 3

Isoforms that correlate with worst prognosis in AML.

Supplementary Table 4

Description of antibodies.

Supplementary Table 5

Statistics source data.

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Smith, M.A., Choudhary, G.S., Pellagatti, A. et al. U2AF1 mutations induce oncogenic IRAK4 isoforms and activate innate immune pathways in myeloid malignancies. Nat Cell Biol 21, 640–650 (2019). https://doi.org/10.1038/s41556-019-0314-5

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