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Transcriptional control of CBX5 by the RNA-binding proteins RBMX and RBMXL1 maintains chromatin state in myeloid leukemia

Abstract

RNA-binding proteins (RBPs) are key arbiters of post-transcriptional regulation and are found to be dysregulated in hematological malignancies. Here we identify the RBP RNA-binding motif protein, X-linked (RBMX; also known as hnRNPG), and its retrogene RBMXL1 to be required for murine and human myeloid leukemogenesis. RBMX and RBMXL1 were overexpressed in individuals with acute myeloid leukemia (AML) compared to healthy individuals, and RBMX/RBMXL1 loss delayed leukemia development. RBMX/RBMXL1 loss lead to global changes in chromatin accessibility as well as chromosomal breaks and gaps. We found that RBMX and RBMXL1 directly bind to mRNAs, affect transcription of multiple loci, including CBX5 (also known as heterochromatin protein 1 alpha (HP1-α)), and control the nascent transcription of the CBX5 locus. Forced CBX5 expression rescued the RBMX/RBMXL1 depletion effects on cell growth and apoptosis. Overall, we determined that RBMX and RBMXL1 control leukemia cell survival by regulating chromatin state through the downstream target CBX5. These findings identify a mechanism for RBPs directly promoting transcription and suggest RBMX and RBMXL1, as well as CBX5, as potential therapeutic targets in myeloid malignancies.

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Fig. 1: RBMX is required for murine leukemia maintenance in vitro and in vivo.
Fig. 2: Retrogene Rbmxl1 is functionally redundant with Rbmx in leukemia maintenance in vitro and in vivo.
Fig. 3: RBMX and RBMXL1 are overexpressed in myeloid leukemia.
Fig. 4: RBMX and RBMXL1 promote human leukemogenesis.
Fig. 5: RBMX and RBMXL1 differentially regulate human HSPCs.
Fig. 6: RBMX and RBMXL1 regulate the chromatin state in leukemia cells.
Fig. 7: RBMX and RBMXL1 directly bind to CBX5 transcripts.
Fig. 8: RBMX and RBMXL1 act through direct regulation of CBX5 transcription.

Data availability

ATAC-seq and RNA-seq data that support the findings of this study have been deposited in the GEO database under the accession code GSE153637. Previously published microarray data in LSC+ versus LSC fractions that were reanalyzed here are available under accession code GSE76009. Previously published PAR-CLIP data of RBMX/RBMXL1 (HNRNPG) that were reanalyzed here are available under accession code GSE74085. Previously published TCGA data that were reanalyzed here are available at https://www.cbioportal.org/. Previously published ONCOMINE data that were reanalyzed here are available at https://www.oncomine.org/resource/login.html. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank members of the Kharas laboratory for their discussions, helpful advice and suggestions. We also thank the MSKCC Integrated Genomics Operation (IGO), Epigenetics Technology Innovation Lab and Molecular Cytogenetics Core for their technical support. We thank K. Birmingham for her technical support. M.G.K. is a Scholar of the Leukemia and Lymphoma Society and is supported by the US NIH National Institute of Diabetes Digestive and Kidney Diseases Career Development Award NIDDK NIH R01-DK101989-01A1; NCI 1R01CA193842-01, 1R01CA193842-06A1, 5R01CA186702-07, 1R01DK1010989-06A1, R01HL135564 and R01CA225231-01; NYSTEM 0266-A121-4609, the Kimmel Scholar Award; the V-Scholar Award; the Geoffrey Beene Award; the Starr Cancer Consortium; the Alex’s Lemonade Stand A Award; the LLS Translation Research Program; the Susan and Peter Solomon Fund and the Tri-Institutional Stem Cell Initiative 2016-014. C.P. is supported by an NIDDK Research Supplement to Promote Diversity in Health-Related Research (3R01DK101989-03S1). D.T.T.N. is supported by a Scholar Award from the American Society of Hematology. A.M.S. is supported by the Lauri Strauss Leukemia Foundation and AIL (Associazione Italiana contro Leucemie, Linfomi e Mielomi) through SIES (Societa’ Italiana Ematologia Sperimentale), and L.P.V. is supported by K99 CA229993 and the LLS Career Development Award.

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Authors

Contributions

C.P. led this project, designed and performed experiments, analyzed data and wrote the manuscript. D.T.T.N. provided the project strategy, designed and performed experiments, analyzed data, led the revision and wrote the manuscript. Z.L. analyzed the ATAC-seq, PAR-CLIP, RNA-seq and alternative splicing data and edited the manuscript. J.W. performed smFISH and analyzed the data. A.P. analyzed the RNA-seq data. S.G. performed experiments and analyzed data. T.C., E.B., A.V., T.R., A.C., J.T., K.H., M.D., A.S. and T.S.B. provided experimental support. A.M.S. supported the PDX experiments. L.P.V. provided help with the experimental design. C.L., U.S. and R.R. supervised the project. M.G.K. directed the project, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Michael G. Kharas.

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Competing interests

M.G.K. discloses the following relationships and financial interests: 28-7 Therapeutics (Provision of Services, uncompensated) and Accent Therapetics (Provision of Services). U.S. has received research funding from GlaxoSmithKline, Bayer Healthcare, Aileron Therapeutics and Novartis, has received compensation for consultancy services and for serving on scientific advisory boards from GlaxoSmithKline, Bayer Healthcare, Novartis, Celgene, Aileron Therapeutics, Stelexis Therapeutics, Pieris Pharmaceuticals and Vor Biopharma and has equity ownership in and is serving on the board of directors of Stelexis Therapeutics. All other authors declare no competing interests.

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Peer review information Nature Cancer thanks Catriona Jamieson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 RBMX is required for murine leukemia maintenance but is dispensable for leukemia initiation.

a, Log2 fold depletion of control shRNAs (n = 5 independent shRNAs) and Rbmx-specifc shRNAs (n = 4 independent shRNAs) in MLL-AF9 leukemic BM cells in pooled in vivo shRNA screen. b, qRT-PCR showing efficient Rbmx deletion in MLL-AF9 leukemia cells 4 days post transduction. n = 3 independent experiments. c, Representative FACS plots of Fig. 1c. d, Representative FACS plots of Fig. 1d. (e-f) qRT-PCR of Rbmx in normal c-Kit+ BM cells (control and shRNA97: n = 4, shRNA96: n = 3 independent experiments) and in MLL-AF9 leukemia cells (n = 3 independent experiments). g, qRT-PCR of Rbmx in AML1-ETO leukemia cells. n = 3 independent experiments. h, Colony formation assay of cells in h. n = 3 independent experiments. i, Diagram for generating Rbmx conditional knockout mice. j, Genotyping PCR detecting Rbmx deletion in BM cells isolated from RbmxΔ/Δ and RbmxΔ mice before leukemic transformation. Cre bands indicate the presence of Mx1-Cre allele. Representative image of 2 independent experiments with similar results. k, qRT-PCR showing Rbmx deletion of cells in j. n = 3 independent experiments. l, Experimental scheme for leukemia initiation experiments. m, Kaplan-Meier survival analysis of mice injected with female MLL-AF9-transformed Rbmxf/f and RbmxΔ/Δ cells. Rbmxf/f n = 9, RbmxΔ/Δ n = 10 mice; two-sided Mantel-Cox test. n, Spleen and liver weights from mice that succumbed to disease in m. Rbmxf/f n = 8, RbmxΔ/Δ n = 9 mice. o, Kaplan-Meier survival analysis of mice injected with male MLL-AF9 transformed Rbmxf and RbmxΔ cells. Rbmxf/f n = 10, RbmxΔ/Δ n = 12 mice; two-sided Mantel-Cox test. p, Spleen and liver weights from mice that succumbed to disease in o. Rbmxf/f n = 8, RbmxΔ/Δ n = 6 mice. q, Immunoblot analysis of MLL-AF9 Rbmxf/f Cre-ER cells expressing ectopic human RBMX and knocking down of endogenous RBMX when treated with 400nM 4-OHT. The experiment was performed 4 times with similar results. r, qRT-PCR of the cells from q. n = 3 independent experiments. Data presented in a-b, e-h, k, n, p, r as mean ± s.e.m. p value determined by two-tailed Student’s t test, unless stated otherwise.

Source data

Extended Data Fig. 2 Retrogene Rbmxl1 is functionally redundant with Rbmx in vitro and in vivo.

a, Immunoblot analysis of RBMX/L1 protein in the BM cells isolated from Rbmxf/f and Rbmxf mice and RBMXL1 from RbmxΔ/Δ and RbmxΔ before leukemic transformation. The experiment was performed once. b, qRT-PCR of Rbmx and Rbmxl1 expression by shRNAs specific to Rbmx (but not Rbmxl1) in MLL-AF9 leukemia cells. Control and shRNA96: n = 3, shRNA97: n = 4 independent experiments. c, qRT-PCR of Rbmx and Rbmxl1 expression in MLL-AF9 Rbmxf/f Cre-ER cells treated with 4-OHT for 24 hours. n = 3 independent experiments. d, Quantitative FACS analysis summary of Gr-1+Mac-1+ and CD115+F4/80+ cells in control and RBMXL1-knockdown leukemic RbmxΔ/Δ cells 4 days post transduction. Control and shRNA587: n = 6, shRNA932: n = 4 independent experiments. e, Representative FACS plots of Fig. 2j. f, Representative FACS plots of Fig. 2k. g, Spleen and liver weights from mice that succumbed to disease in Fig. 2l. Control: n = 9, shRNA587: n = 5, shRNA932: n = 4 mice. h, Immunoblot analysis of leukemic BM cells isolated from mice that succumbed to disease in Fig. 2l. The experiment was performed once. Data presented in b-d, and g as mean ± s.e.m. p value determined by two-tailed Student’s t test, unless stated otherwise.

Source data

Extended Data Fig. 3 RBMX and RBMXL1 overexpression in myeloid leukemia.

a, RBMX expression in AML patients with indicated status of NPM1 and FLT3-ITD mutation. Data are presented as mean of normalized RPKM ± s.e.m, on the basis of data from the BeatAML vizome dataset (Tyner JW et al. Nature. 2018). Double negative n = 286, FLT3-ITD positive n = 54, NPM1 mut positive n = 59, double positive n = 49 patient samples; two tailed Student’s t test with Welch’s correction. b, RBMX expression in leukemic stem cell (LSC+) and non-leukemic stem cell population (LSC) in AML patient samples on the basis of data from Ng SW et al. Nature. 2016 (GSE76009). LSC n = 89, LSC+ n = 138 patient samples; data as mean ± s.e.m, two-tailed Student’s t test. (c-d) qRT-PCRs showing RBMX and RBMXL1 mRNA levels in multiple myeloid leukemia cell lines. For RBMX CB-CD34+, THP-1, HL-60, U-937 and K562: n = 3, MOLM13, Nomo-1 and NB4: n = 5, Kasumi-1, KCL-22, KG-1 and TF-1: n = 4 independent experiments; For RBMXL1 CB-CD34+, Nomo-1, THP-1, HL-60, U-937 and TF-1: n = 3; MOLM13, Kasumi-1, K562 and KG-1: n = 4; NB4 and KCL-22: n = 5 independent experiments. Data as mean ± s.e.m, two-tailed Student’s t test. e, CRISPR score rank of RBMX, its retrogenes (RBMXL1, RBMXL2, and RBMXL3), and its paralog RBMY1A1. CRISPR score is the average log2 fold-change in the abundance of all sgRNAs targeting the gene after 14 population doublings33. f, CRISPR score of RBMX, its retrogenes, and its paralog across the 14 tested leukemia cell lines.

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Extended Data Fig. 4 RBMX and RBMXL1 are required for human myeloid leukemia cell survival.

a, Giemsa staining of control and RBMX/L1-knockdown MOLM13 cells from Fig. 4k. Original magnification 400X. Scale bar, 20μm. The experiment was performed once; control and shRNA1: 9 images, shRNA2: 16 images were collected. (b-d) Representative FACS plots of Fig. 4m, o and p, respectively. e, Immunoblot analysis of RBMX/L1 in KCL-22 transduced with EV or RBMX overexpressing vector (RBMX-R2) and shRNA control or shRNAs against RBMX/L1. The experiment was performed 3 times with similar results. f, Proliferation assay of cells in e. n = 3 independent experiments. g, Quantitative FACS analysis of apoptotic cells in control and RBMX/L1-knockdown KCL-22-EV and KCL-22-RBMX-R2 cells 5 days post transduction. n = 3 independent experiments. h, Immunoblots for RBMX/L1 in MOLM13-Cas9 cells transduced with sgRNAs targeting RBMX/L1 (sg-1 and sg-2). The experiment was performed 4 times with similar results. i, Proliferation assay of cells in h. n = 4 independent experiments. j, FACS analysis of myeloid differentiation markers CD11b and CD33 in cells from h. n = 4 independent experiments. k, Quantitative FACS analysis summary of apoptosis of cells in h, 3 days post transduction. n = 4 independent experiments. l, Immunoblots analysis of GFP+ PDX AML-1 cells. The experiment was performed once. m, qRT-PCR showing depletion of RBMX/L1 in GFP+ PDX AML-13 cells. n = 1 experiment. n, Immunoblots for RBMX/L1 upon RBMX/L1 depletion in GFP+ PDX AML-11 cells. The experiment was performed once. o, Representative FACS plots of Fig. 4t. p, Immunoblot and band densitometry of RBMX/L1 in BM cells from animals transplanted with PDX AML-1 cells that succumbed to leukemia. The experiment was performed once. (q-s) Representative FACS plots of Fig. 5c, g and h, respectively. Data presented in f-g, i-k as mean, error bars, s.e.m. p values were calculated using two-tailed Student’s t test, unless indicated otherwise.

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Extended Data Fig. 5 Loss of RBMX and RBMXL1 results in a dysregulated chromatin state in leukemia cells.

a, Immunoblot analysis of RBMX/L1, MYC, and HOXA9 in RBMX/L1 depleted MOLM13 cells. The experiment was performed 3 times with similar results. b, Heatmap of the top 1,000 peaks from ATAC-sequencing from control and RBMX/L1-knockdown MOLM13 cells. n = 3 independent experiments. c, Scatterplot showing accessibility changes at pericentric and telomeric heterochromatin upon RBMX/L1-knockdown. n = 3 independent experiments. d, Location of increased accessible and decreased accessible ATAC-sequencing peaks in RBMX/L1-knockdown MOLM13 cells. e, Location of increased accessible and decreased accessible ATAC-sequencing pericentric and telomeric heterochromatin peaks in RBMX/L1-knockdown MOLM13 cells. f, Gene expression heatmap of the top 99 upregulated and downregulated genes from RNA-sequencing analysis of MOLM13 cells upon RBMX/L1 knockdown. n = 4 independent experiments. g, Tables showing alternative splicing events and genes MOLM13 cells upon RBMX/L1 depletion. n = 4 independent experiments. h, mRNA expression of the 11 genes from the overlap in Fig. 7a. n = 4 independent experiments; data as mean ± s.e.m, two-tailed Student’s t test. i, qRT-PCR of recovered RNA in RNA-IP at 11 candidate target genes shown in Fig. 7a. n = 4 independent experiments; data as mean ± s.e.m, two-tailed Student’s t test. j, Overall survival analysis of AML patients with low versus high expression of RBMX/L1 direct regulated pathway including 8 down-regulated targets validated by PAR-CLIP, RNA-IP and RNA-sequencing (CBX5, CBS, DACH1, SEPT11, UNG, XBP1, PABPC4, and SLC38A1). Data from TCGA database.

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Extended Data Fig. 6 Lossy RBMX/L1 in MOLM13 cells leads to decreased CBX5 transcript expression.

a, Immunoblot analysis and band densitometry of RBMX/L1, DACH1, CBX5, CBS, and SEPT11 upon RBMX/L1 depletion in MOLM13 cells. The experiment was performed 3 times with similar results. b, CBX5 mRNA expression in leukemic RBMXL1 depleted RbmxΔ cells. n = 5 independent experiments. c, Immunoblots and band densitometry of RBMX/L1 and CBX5 in leukemic RBMXL1 depleted RbmxΔ cells. The experiment was performed 3 times with similar results. d, CBX5 mRNA expression upon RBMX/L1 depletion in MOLM13-Cas9 cells. n = 4 independent experiments. e, Immunoblots and band densitometry of RBMX/L1 and CBX5 upon RBMX/L1 depletion in MOLM13-Cas9 cells. Same immunoblot as Extended Data Fig. 4h with longer exposure for RBMX/L1 band. The experiment was performed 3 times with similar results. f, qRT-PCR of CBX5 mRNA expression at indicated exon and exon-exon junction. Exon 3 amplicon: n = 3, all other amplicons: n = 6 independent experiments; data as mean ± s.e.m, two-tailed Student’s t test. g, mRNA stability of RBMX and CBX5 upon RBMX/L1 depletion in MOLM13 cells. 0 min and 90 min: n = 5, 30 min and 270 min: n = 4, 150 min: n = 3 independent experiments. h, qRT-PCR of nascent mRNAs of RBMX/L1 candidate targets upon RBMX/L1 depletion. DACH1: n = 4, SEPT11 and PABPC4: n = 5, CBS control and shRNA1 n = 6 and shRNA2 n = 5, XBP1 control n = 6 for, shRNA1 and shRNA2 n = 4, UNG control and shRNA1 n = 6, shRNA2 n = 4 independent experiments. i, Quantitative summary of smFISH with CBX5-Intron1 probe (Fig. 7f). Control: n = 229, shRNA1: n = 71, shRNA2: n = 38 foci. j, qRT-PCR measuring absolute number of CBX5-Intron 1 and Renilla luciferase (Rluc) nascent mRNA transcripts in CBX5-Intron 1 luciferase reporter assay. n = 4 independent experiments. k, qRT-PCR of CBX5 and RBMX mRNA upon CBX5 depletion in MOLM13 cells. n = 3 independent experiments. l, qRT-PCR of RBMX and CBX5 mRNA upon RBMX/L1 depletion in MOLM13-EV and MOLM13-CBX5 cells. EV-shRNA2 n = 4, all other groups: n = 3 independent experiments. m, Cells from Fig. 8e were plated for proliferation assay and counted 4 days post transduction. n = 3 independent experiments. Data presented in b, d, f-m as mean ± s.e.m. p values were calculated using two-tailed Student’s t test, unless indicated otherwise.

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Prieto, C., Nguyen, D.T.T., Liu, Z. et al. Transcriptional control of CBX5 by the RNA-binding proteins RBMX and RBMXL1 maintains chromatin state in myeloid leukemia. Nat Cancer 2, 741–757 (2021). https://doi.org/10.1038/s43018-021-00220-w

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