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Minor intron retention drives clonal hematopoietic disorders and diverse cancer predisposition

Nature Genetics volume 53, pages 707–718 (2021)Cite this article

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Abstract

Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias.

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Fig. 1: Zrsr2 loss increases HSC self-renewal.
Fig. 2: Widespread minor intron retention with ZRSR2 loss.
Fig. 3: ZRSR2 RNA binding targets and features of ZRSR2-responsive introns.
Fig. 4: Impaired LZTR1 minor intron excision confers competitive advantage.
Fig. 5: Impaired LZTR1 minor intron excision promotes clonal advantage and effects of LZTR1 re-expression on Zrsr2-null hematopoietic cells.
Fig. 6: Aberrant LZTR1 minor intron retention in Noonan syndrome, schwannomatosis and diverse cancers.

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Data availability

Genome annotations for humans and mice were from NCBI GRCh37/UCSC hg19 and NCBI GRCm38/UCSC mm10, respectively. Isoform annotations were from the MISO version 2.0 database50 and were merged with genome annotations from Ensembl release 71 (ref. 51) and the UCSC knownGene track52. RNA-seq reads for the human samples reported in Madan et al.7 were downloaded from the Gene Expression Omnibus (accession number GSE63816). RNA-seq data generated by TCGA (dbGaP accession phs000178.v11.p8) and the Beat AML data (dbGaP accession phs001657.v1.p1) were downloaded from the National Cancer Institute Genomic Data Commons. Branchpoint-related data were obtained from a published study29. RNA-seq data generated as part of this study were deposited in the Gene Expression Omnibus (mouse data, accession GSE149455) and the human RNA-seq data were deposited in dbGaP (phs002212.v1.p1). Source data are provided with this paper.

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Acknowledgements

This work was supported by the Conquer Cancer Foundation of the American Society of Clinical Oncology (J.T.), the American Association for Cancer Research (J.T.), the American Society of Hematology (D.I., J.T., K.K.), the Robert Wood Johnson Foundation (J.T.), the ARCS Foundation (J.M.B.P.), NIH grants 1K08CA230319-01 (J.T.), T32 CA009657 (J.T.P.), R01 DK103854 (R.K.B), R01 HL151651 (R.K.B.), R01 HL128239 (R.K.B. and O.A.-W.), R01 CA251138 (R.K.B. and O.A.-W.), the Department of Defense Bone Marrow Failure Research Program (W81XWH-12-1-0041, R.K.B. and O.A.-W.), the Leukemia & Lymphoma Society (D.I. and O.A.-W.), the Henry and Marilyn Taub Foundation (O.A.-W.), the Edward P. Evans MDS Foundation (J.T.P., J.T., O.A.-W. and R.K.B.) and the Blood Cancer Discoveries Grant program through the Leukemia & Lymphoma Society, the Mark Foundation for Cancer Research and the Paul G. Allen Frontiers Group (8023-20, R.K.B.). D.I. is supported by AMED (JP19cm0106165 and JP20cm0106165), the Takeda Science Foundation, the MDS Foundation, the Yasuda Medical Foundation, the Kanae Foundation for the Promotion of Medical Science, the MSD Life Science Foundation, the Bristol Myers Squibb Foundation, JSPS KAKENHI (JP20H00537 and JP 20H03717) and the Leukemia Research Foundation. R.K.B. is a scholar of the Leukemia & Lymphoma Society (1344-18). The results shown here are in part based upon data generated by TCGA Research Network (https://cancergenome.nih.gov/).

Author information

Author notes

  1. These authors contributed equally: Daichi Inoue, Jacob T. Polaski, Justin Taylor.

Authors and Affiliations

  1. Department of Hematology–Oncology, Institute of Biomedical Research and Innovation, Foundation for Biomedical Research and Innovation at Kobe, Kobe, Japan

    Daichi Inoue, Susumu Kobayashi, Yasutaka Hayashi, Miki Fukumoto, Hiromi Yamazaki, Atsushi Tanaka & Chie Fukui

  2. Human Oncology and Pathogenesis Program, Memorial Sloan KetterAbsolute numbers of live mature hematopoietic cellsing Cancer Center, New York, NY, USA

    Daichi Inoue, Sisi Chen, Simon J. Hogg, Ettaib El Marabti, Caroline Erickson, Katherine Knorr, Sydney X. Lu, Benjamin H. Durham, Bo Liu, Eric Wang, Alex Penson & Omar Abdel-Wahab

  3. Public Health Sciences and Basic Sciences Divisions, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Jacob T. Polaski, Jose Mario Bello Pineda & Robert K. Bradley

  4. Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine, Miami, FL, USA

    Justin Taylor

  5. Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA

    Pau Castel & Frank McCormick

  6. Division of Cellular Therapy, The Institute of Medical Science, the University of Tokyo, Tokyo, Japan

    Susumu Kobayashi

  7. Department of Genome Sciences, University of Washington, Seattle, WA, USA

    Jose Mario Bello Pineda & Robert K. Bradley

  8. Medical Scientist Training Program, University of Washington, Seattle, WA, USA

    Jose Mario Bello Pineda

  9. Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

    Atsushi Tanaka

  10. Gene Editing & Screening Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Sanjoy Mehta, Daniel Zakheim & Ralph Garippa

  11. Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore

    Guo-Liang Chew

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  1. Daichi Inoue
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Contributions

D.I., J.T.P., J.T., R.K.B. and O.A.-W. conceived the project and wrote the paper with editorial contributions from all authors; D.I., J.T., P.C., S.C., S.K., C.F., S.J.H., Y.H., J.M.B.P., E.E.M., C.E., K.K., M.F., H.Y., A.T., C.F., S.X.L., B.H.D., B.L., E.W., S.M., D.Z. and R.G. performed experiments; D.I., J.T.P., J.T., A.P., G.-L.C. and R.K.B. analyzed data; and F.M., R.K.B. and O.A.-W. supervised the project.

Corresponding authors

Correspondence to Robert K. Bradley or Omar Abdel-Wahab.

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Competing interests

O.A.-W. has served as a consultant for H3B Biomedicine, Foundation Medicine Inc., Merck, Prelude Therapeutics and Janssen and is on the scientific advisory board of Envisagenics Inc., AIChemy and Pfizer Boulder; O.A.-W. received prior research funding from H3B Biomedicine and Loxo Oncology unrelated to the current manuscript. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Genetics thanks George Vassiliou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Generation and validation of Zrsr2 conditional knockout (cKO) mice.

a, Punnett square enumerating male and female myeloid neoplasm patients wild-type versus mutant for ZRSR2 across 2,302 patients. b, Lollipop diagram of ZRSR2 mutations from (a). c, Schematic depiction of the targeting strategy to generate Zrsr2 cKO mice. The Zrsr2 allele was deleted by targeting exon 4 in a manner that results in a frameshift following excision. Two LoxP sites flanking exon 4 and an Frt-flanked neomycin selection cassette were inserted in the downstream intron. d, Verification of correct homologous recombination using Southern blots from targeted embryonic stem cells. The experiment was repeated twice with similar results. e, Verification of the presence of Mx1-cre and Zrsr2 floxed alleles as well as excision of Zrsr2 using genomic PCR. The experiment was repeated three times with similar results. f, Full-length Western blot of Zrsr2 protein in protein lysates from bone marrow mononuclear cells from Mx1-cre control or Mx1-cre Zrsr2fl/y mice. Black arrow indicates full-length Zrsr2 protein while grey arrows indicate non-specific bands. The experiment was repeated three times with similar results. g, Zrsr2 expression (relative to 18 s rRNA) in long-term hematopoietic stem cells (DAPI- lineage-negative c-Kit+ Sca-1+ CD150+ CD48-) in Mx1-cre control, Mx1-cre Zrsr2fl/y, and Mx1-cre Zrsr2fl/fl mice 6-weeks following polyinosinic-polycytidylic acid (pIpC) administration. Mean values ± SD. P-values calculated relative to the control group by a two-sided t-test. n = 3 biologically independent experiments. h, RNA-seq coverage plots from lineage-negative c-Kit+ cells from mice in (g) illustrating excision of exon 4 of Zrsr2 following pIpC.

Source data

Extended Data Fig. 2 Zrsr2 loss enhances self-renewal of hematopoietic stem cells (HSCs).

a, FACS of cells from 5th methylcellulose plating (see Fig. 1b) for live, LSK cells. Wild-type bone marrow (BM) from 6-week old mouse (left) as a staining control. b, Number of CFU-GM, CFU-GEMM, and BFU-E colonies from initial plating of LT-HSCs (lin- LSK CD150+ CD48-) from Mx1-cre control, Mx1-cre Zrsr2fl/y, and Mx1-cre Zrsr2fl/fl mice into methylcellulose. Mean ± SD, two-sided t-test. n = 4 biologically independent experiments. c, Schematic of competitive BM transplantation. d, Number of methylcellulose colonies from LT-HSCs from Mx1-cre control, Mx1-cre Zrsr2fl/x, Mx1-cre Zrsr2fl/y, and Mx1-cre Zrsr2fl/fl. Mean value ± SD. n = 3 biologically independent experiments. e, Box-and-whisker plots of percentage of peripheral blood CD45.2+ cells in competitive transplantation pre- and post-pIpC using CD45.2+ Mx1-cre control, Mx1-cre Zrsr2fl/x, Mx1-cre Zrsr2fl/y, and Mx1-cre Zrsr2fl/fl mice. For box and whiskers plots throughout, bar indicates median, box edges first and third quartile values, and whisker edges minimum and maximum values, two-sided t-test. Experiment repeated three times with similar results in (d) and (e). f, Percentage of CD45.2+ B220+ (left), CD45.2+ CD11b+ Gr1− (middle), and CD45.2+ CD3+ cells (right) in primary competitive transplantation. Mean ± SD. P values by two-sided t-test using the values at 16 weeks after transplant. P value relative to the control group at 16 weeks by a two-sided t-test. g, FACS analysis and gating strategy of BM cells from representative primary recipient mice in competitive transplantation. h, Box-and-whisker plots of numbers of ST-HSCs, CMPs, MEPs, and pDCs in BM of primary recipient mice in competitive transplantation. For box and whiskers plots throughout, bar indicates median, box edges first and third quartile values, and whisker edges minimum and maximum values. P value relative to control by a two-sided t-test.

Source data

Extended Data Fig. 3 Characterization of Zrsr2 conditional knockout (cKO) mice.

a, Absolute number of LT-HSCs, ST-HSCs, MPPs, LSKs, CMPs, and GMPs in primary, non-transplanted 20-week-old Mx1-cre control (‘control’) and Mx1-cre Zrsr2fl/y mice. Mean ± SD. n = 5 animals. b, Absolute number of live, bone marrow (BM) mononuclear cells in 10-week old Mx1-cre control (‘control’) and Mx1-cre Zrsr2fl/y mice (‘Zrsr2 knockout’) 4-weeks following Zrsr2 excision. Mean ± SD. n = 5 animals. P-values relative to control by two-sided t-test and indicated in figures. c, Schematic of BrdU analysis of hematopoietic stem cells from mice in (b) and Fig. 1f. d, Hyposegmented, hypogranular neutrophils in peripheral blood of 10-week old Mx1-cre Zrsr2fl/y mice (yellow arrows). Bar: 10 mm. e, BM cytospins indicating hyposegmented, hypogranular neutrophils (left panel) and dysplastic erythroid progenitors (middle and right panels, yellow arrows). Bar: 10 mm. Experiment repeated three times with similar results in (d) and (e). f, Peripheral blood white blood cell counts (WBC), platelet count, hemoglobin (Hb), and mean corpuscular volume (MCV) in primary Mx1-cre control (n = 9) and Mx1-cre Zrsr2fl/y (n = 10) mice (following Zrsr2 excision at 6 weeks age). Mean± SD. g, Kaplan-Meier survival of primary control and Zrsr2 KO mice (following Zrsr2 excision at 6 weeks age). Absolute numbers of (h) bone marrow and (i) spleen B-cell subsets. j, Numbers of live, spleen (left) and thymic (right) mononuclear cells in mice from (h)-(i). Mean ± SD. Absolute numbers of live mature hematopoietic cells (k) in marrow and (l) spleen of 8-week-old Mx1-cre control (‘control’) and Mx1-cre Zrsr2fl/y (‘knockout’ or ‘KO’) mice (Zrsr2 excision at 4 weeks). m, Absolute numbers of T-cell subsets in thymus of mice from (k). Mean ± SD shown throughout. Mx1-cre control (n = 5) and Mx1-cre Zrsr2fl/y (n = 5) mice were used in (h) to (m). P-values relative to control by two-sided t-test and indicated in figures.

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Extended Data Fig. 4 Comparison of the effects of Zrsr2 loss versus Tet2 knockout or Sf3b1K700E or Srsrf2P95H mutations on hematopoietic stem and progenitor cells.

a, Schema of competitive bone marrow (BM) transplantation assays. b, Absolute number of CD45.2+ long-term HSCs (LT-HSCs), LSK, and MPPs in the bone marrow of CD45.1 recipient mice 16 weeks following pIpC (n = 8–10 each). For box and whiskers plots throughout, bar indicates median, box edges first and third quartile values, and whisker edges minimum and maximum values. c, Percentage of CD45.2+ LT-HSCs, LSK, CMP, MEP, and GMP cells in the BM of CD45.1 recipient mice 16-weeks following pIpC (n = 8–10 per each). P value was calculated relative to the control group by a two-sided t-test. d, Representative FACS plots of data in (c). e, Number of methylcellulose colonies generated from 100 sorted LT-HSCs from mice with the indicated genotype. n = 3 biologically independent experiments. Error bars, mean values +/− SEM. P-values by one-way ANOVA with Tukey’s multiple comparisons test. f, Percentage of CD45.2+ cells in the blood of recipient mice from Zrsr2 knockout/Sf3b1K700E/WT double mutant cells and relevant controls pre- and post-pIpC administration to recipient mice (n = 10 each). P-values by two-way ANOVA with Tukey’s multiple comparisons test. Data in (b), (c), and (f) are shown as box-and-whisker plots where bar indicates median, box edges first and third quartile values, and whisker edges minimum and maximum values.

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Extended Data Fig. 5 Effect of ZRSR2 loss on minor intron splicing.

a, ZRSR2 mutations in our cohort. ‘MDS 05–14’ are wild-type; ‘i-p’ are ZRSR2-mutant. VAF: variant allele frequency, fs: frameshift, ptc: premature termination codon, del: deletion, ins: insertion, ms: missense mutation. b, Comparison of U12-type intron retention in MDS samples vs. normal marrow. c, Differential splicing of U12-type introns. Each point corresponds to a single intron, illustrating percentage of mRNAs in which intron is spliced out. Blue/red dots: introns with significantly increased/decreased retention in ZRSR2-mutant vs. WT, with absolute change ≥10% or absolute log fold-change of ≥2 with p≤0.05 (two-sided Mann-Whitney U, without adjustments for multiple comparisons). d, Distribution of intron retention in samples with ZRSR2 mutations. Blue/red dashed lines: thresholds of −10% and 10% for differential retention; gold line: median change in intron retention. e, As (b), and (f) as (d), for U2-type introns. g, As (a), and (h) As (b), for Madan et al. (i) As (h), for U2-type introns. j, As (f), for Madan et al. (k) RNA-seq coverage plots of U12-type introns averaging samples with indicated genotypes. l, Splicing efficiencies of introns in (k) relative to normal marrow (median over n = 4 normal samples). P-values: two-sided Mann-Whitney U. Middle line, hinges, notches, and whiskers: median, 25th/75th percentiles, 95% confidence interval and most extreme points within 1.5x interquartile range from hinge. m, Expression of genes with retained U12-type introns between ZRSR2-mutant vs. WT. n, Immunoblot in K562 cells used for eCLIP-seq (repeated twice with similar results). o, eCLIP of ZRSR2-binding sites. Input-normalized peak signals as log2 fold-change. Purple points: eCLIP-enriched ZRSR2 peaks in biological replicates. p, Overlap of genes bound by ZRSR2 vs. differentially spliced in ZRSR2-mutant versus WT (‘ZRSR2 responsive’). P-value: Fisher’s exact test. q, U2 snRNA binding energy within ZRSR2 non-responsive and responsive minor introns.

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Extended Data Fig. 6 Consequences of ZRSR2 and LZTR1 dysregulation.

a, RNA-seq of LZTR1’s minor intron in normal and ZRSR2-mutant and WT MDS (n = 10 each) marrow. b, Ratio of intron retained (IR) to normal LZTR1 in MDS samples (n = 10 each). Mean ± SD. c, Data from (b) ±SD. P-value; two-sided t-test. d, Qualitative RT-PCR using primers amplifying exons 18-19 (‘e18-e19’) as well as specific to IR isoform. e, Splicing efficiencies of LZTR1 in patient samples (median over n = 4 normal samples; left) or mouse lineage-negative c-Kit+ cells (right). P-values: two-sided Mann-Whitney U. f, LZTR1 expression by level of minor intron retention (‘Low’: <10%; ‘Mid’: 10–20%; ‘High-retention’: >20%). P-values: one-sided Mann-Whitney U. In (e) and (f): middle line, hinges, notches, and whiskers indicate median, 25th/75th percentiles, 95% confidence interval, and most extreme points within 1.5x interquartile range from hinge. g, UPF1 immunoblot in K562 cells with mutation disrupting LZTR1s U12 sequence +/− anti-UPF1 shRNA. h, Expression of U12-retained LZTR1 following actinomycin D +/− anti-UPF1 shRNA. n = 3 biological replicates. Mean +/− SD. i, Immunoblot of K562 cells +/− ZRSR2-targeting sgRNA. j, Expression of LZTR1 (left) or CHD4 (right) isoforms in ZRSR2-null K562 cells with DMSO or NMD inhibitor (PMID 24662918). Mean +/− SD; P values: two-sided t-test. n = 3 biological replicates. k, LZTR1 minigene with mutations generated. l, RT-PCR of LZTR1 minigene and endogenous mRNA from WT or ZRSR2-KO K562 cells. m, RT-PCR of LZTR1 minigene and endogenous using native (‘N’) or mutant minigenes. n, Lztr1 minor intron with location of sgRNA, PAM site, 3’ U12 consequence (blue text), and sequence in individual Ba/F3 cell clones (red dash: deleted nucleotides). o, As (n) in K562 cells. p, Immunoblot of Lztr1 in Ba/F3 single-cell clones +/− Lztr1 protein-coding or minor intron sgRNAs. q, Median relative percentage of GFP-labeled K562 cells following Rebastinib. Experiments in (d), (g), (i), and (l) were repeated twice with similar results.

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Extended Data Fig. 7 Impaired Lztr1 minor intron splicing augments clonogenic capacity of hematopoietic precursors.

a, Schema of experiment whereby sgRNAs targeting the conserved U12 sequence in Lztr1’s minor intron are delivered to lineage-negative hematopoietic precursors from Scl-CreERT Rosa26-Lox-STOP-Lox Cas9-EGFP Zrsr2fl/y or Zrsr2 wild-type mice followed by serial replating in vitro. In this experiment, sgRNAs are encoded from an RFP657 expressing plasmid and GFP+/RFP657+ double-positive cells were purified for plating. b, Mean number of colonies following Lztr1 minor intron mutagenesis versus control sgRNA treated bone marrow cells in Zrsr2 wild-type or knockout background from (a). Bars represent standard deviation. P-values calculated relative to the control group by a two-sided t-test. n = 3 biologically independent experiments. Error bars, mean values +/− SD. c, Representative FACS plots of GFP% in cells just before transplantation and in peripheral blood of recipient transplanted mice 4 weeks after transplantation from Fig. 5g. d, Number of colonies in methylcellulose CFU assays from LT-HSCs from mice in (c). n = 3 biologically independent experiments. Error bars, mean values +/− SD. P-values by two-way ANOVA with Tukey’s multiple comparisons test. e, Relative percentage of GFP-labeled K562 cells with knockout of RIT1 and/or mutagenesis of the minor intron in LZTR1 mixed with equal proportions of unlabeled cells to the BCR-ABL inhibitor imatinib. f, Relative percentage of Ba/F3 cells treated with sgRNAs targeting Rit1 and/or the minor intron of Lztr1 following IL-3 withdrawal (median % relative to day 2 is plotted).

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Extended Data Fig. 8 LZTR1 minor intron retention in cancer predisposition syndromes.

a, Sanger sequence electropherogram of the LZTR1 intron 18 retained isoform (from a representative affected family member in Fig. 6d; corresponds to the top band in the LZTR1 RT-PCR gel in Fig. 5d) and LZTR1 normal spliced isoform from a control fibroblast sample (corresponds to the bottom band in the LZTR1 RT-PCR gel in Fig. 6d). Red arrow indicates mutant nucleotide in the affected family members. b, RNA-seq coverage plots of LZTR1 in fibroblasts from Noonan syndrome family and controls. Zoom in magnifies the minor intron of LZTR1. c, As (b), but zoomed in on the region of mutation in the father. d, As (b), but zoomed in on the region of mutation within LZTR1’s minor intron.

Extended Data Fig. 9 LZTR1 minor intron retention is pervasive in cancers.

a, Degree of major (U2-type) intron retention across normal (N) and tumor (T) samples in cancers from TCGA. Each point corresponds to a single U2-type intron and indicates the percentage of all tumor samples in which retention of that intron exceeds the maximum corresponding retention of that intron observed in normal samples. Red dot indicates the U12-type intron of LZTR1 for comparison. b, Each point illustrates the frequency of retention of a single intron of LZTR1 (see inset for key) across all TCGA cohorts with matched normal samples. Values along the x axes represents the mean difference in intron retention in tumor versus normal samples within a cancer type, while the y axes represent the fraction of tumor samples with intron retention that exceeds that of the normal sample with the most intron retention within a cancer type. Points represent the mean value computed across all cancer types, while whiskers represent the interquartile range across cancer types. c, As (b), but whiskers represent the entire range.

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Supplementary information

Supplementary Information

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Supplementary Table 1

Supplementary Tables 1–10

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Inoue, D., Polaski, J.T., Taylor, J. et al. Minor intron retention drives clonal hematopoietic disorders and diverse cancer predisposition. Nat Genet 53, 707–718 (2021). https://doi.org/10.1038/s41588-021-00828-9

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