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Functional screen of MSI2 interactors identifies an essential role for SYNCRIP in myeloid leukemia stem cells

Abstract

The identity of the RNA-binding proteins (RBPs) that govern cancer stem cells remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomic analysis of the MSI2-interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia. Syncrip was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP-depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation, and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. Altogether, our data identify SYNCRIP as a new RBP that controls the myeloid leukemia stem cell program. We propose that targeting these RBP complexes might provide a novel therapeutic strategy in leukemia.

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Figure 1: Mass spectrometry of the MSI2 riboproteome and an in vivo shRNA screen uncover the functionally dysregulated RBP network in leukemia.
Figure 2: SYNCRIP is required to maintain myeloid leukemia survival in vitro and in vivo.
Figure 3: SYNCRIP is required for leukemogenesis in vivo.
Figure 4: SYNCRIP is highly expressed in human AML cells, and SYNCRIP depletion results in inhibition of cell growth and apoptosis in human AML cells.
Figure 5: SYNCRIP regulates the myeloid LSC gene expression program.
Figure 6: SYNCRIP regulates HOXA9 expression post-transcriptionally.
Figure 7: HOXA9 partially rescues the survival defects of SYNCRIP-depleted cells.

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Acknowledgements

We would like to thank S. Nimer and N. Rosen for their critical advice and helpful suggestions. We would also like to thank A. Viale and the MSKCC sequencing core for all their support. We would like to thank the Lowe laboratory (Memorial Sloan Kettering Cancer Center) for their generous gift of the RN2 cells26 and the CRISPR–Cas9 constructs. We would like to thank Y. Neelamraju for technical support. M.G.K. was supported by the US National Institutes of Health National Institute of Diabetes, Digestive and Kidney Diseases Career Development Award, NIDDK NIH R01-DK101989-01A1, NCI 1R01CA193842-01, Louis V. Gerstner Young Investigator Award, American Society of Hematology Junior Scholar Award, Kimmel Scholar Award, V-Scholar Award, Geoffrey Beene Award and Alex's Lemonade Stand A Award and the Starr Foundation (M.G.K. and L.C.). L.P.V. is supported by the Damon Runyon-Sohn Pediatric Cancer Fellowship Award, E.P. was supported by grants U24 CA114737 and U10 CA180827. C.J.L. was supported by an R01 grant from the National Cancer Institute (NIH) and a fellowship from the W.W. Smith Charitable Trust. The research was funded in part through NIH/NCI Cancer Support Core Grant P30 CA08748 to M.G.K. and R.G.

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Authors and Affiliations

Authors

Contributions

M.G.K. directed the project, performed experiments, analyzed data, and wrote the manuscript. L.P.V. led the project, performed experiments, analyzed data, and wrote the manuscript. C.P., E.M.A., M.N.C.-V., T.C., A.C., G.M., T.S.B., J.T., P.T., R.P.D., L.P.C., and J.-A.K. performed experiments. J.P.-P., F.A.-S. and M.J. performed shRNA screening data analysis. S.C. and Y.L. performed RNA sequencing data analysis. C.M., A.A., M.G., C.F., M.P., E.P., M.S.T., J.G., and F.E.G.-B. provided clinical data and analysis. A.H. and R.G. provided critical reagents. S.M.P., A.M., R.L., N.H., C.J.L., S.A.A. L.C., G.S.C., D.R., J.D., C.L. and B.L.E. provided suggestions and project support.

Corresponding author

Correspondence to Michael G Kharas.

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Competing interests

There is a patent pending (M.G.K. and L.P.V.).

Integrated supplementary information

Supplementary Figure 1 MSI2 interactors are associated with the riboproteome and are functionally relevant.

(a) Coomassie blue staining of FLAG-MSI2 immunoprecipitated complexes. (b) GO analysis of MSI2-interacting proteins. (c) Proportion of total read counts sequenced per pool. cntl, control pool. (d) Scatterplot depicting the concordance of shRNA abundance (log2 normalized read counts) between each one of the replicates and its respective group (median log2 normalized read counts). (e) Diagrams showing the number of control hairpins represented with a minimum arbitrary threshold (normalized counts >100). (f) Waterfall plot depicting normalized depletion levels for all shRNAs in the spleen (SP). Control shRNAs and hairpins targeting selected candidate genes are highlighted. (g) Table showing seven candidate genes. (h) Validation of efficient knockdown of target genes in mouse MLL-AF9 leukemia cells. Cells were selected under puromycin treatment for 48 h before qRT–PCR. Actb serves as a control housekeeping gene. All data represent the means + s.e.m. of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

Supplementary Figure 2 SYNCRIP is required for survival of leukemia cells in vitro and in vivo.

(a) Efficient knockdown of SYNCRIP in mouse MLL-AF9 leukemia cells. MLL-AF9 cells were transduced with lentiviruses expressing control shRNA (directed against luciferase) or shRNAs directed against Syncrip (shRNA 1 and shRNA 2). Cells were selected under puromycin for 24 h before immunoblotting. Cells were collected and assayed 3 d after viral transduction. Actin serves as a loading control. (b) shRNA depletion of SYNCRIP promoted myeloid differentiation of leukemia cells. Myeloid differentiation status was assayed by FACS analysis of F-480 and CD115 expression. A representative FACS plot is shown of control and SYNCRIP-KD leukemia cells. (c) Quantitative summary of FACS analysis of F-480 and CD115 expression in control and SYNCRIP-KD leukemia cells 3 and 4 d after transduction. (d) Quantitative summary of FACS analysis of the percentage of c-Kithigh cells in control and SYNCRIP-KD leukemia cells 4 d after transduction. (e) Increased apoptosis was not observed in SYNCRIP-KD leukemia cells at 3 and 4 d after transduction. Quantitative summary of Annexin-V assessed by flow cytometry. (f) Annexin-V assessed by flow cytometry 5 d after transduction of control and SYNCRIP-KD leukemia cells. Representative of FACS analysis in Figure 2e. (g) qPCR showing efficient knockdown of Syncrip in mouse AML1-ETO9a-driven leukemia cells. Cells were selected under puromycin for 48 h before qRT–PCR. Actb serves as a control housekeeping gene. (h) Cells from g were plated into methylcellulose and scored for the number of colonies (average of three independent experiments). (i) Assessment of disease burden, including spleen weight and liver weight, for the recipient mice in Figure 2f. (j) Immunoblots showing SYNCRIP expression in the bone marrow of mice that succumbed to disease in i and Figure 2f. (Mice injected with Syncrip-KD1 with reduced disease burden (group 1) maintained a better Syncrip knockdown level than mice that manifested similar disease to the control group (group 2).) (k) qPCR showing efficient knockdown of Syncrip assayed by primers designed for specific gRNA targeting regions (gRNA-specific primers) in RN2 cells transduced with CRISPR–Cas9-containing Tet-inducible gRNAs specific for Syncrip (gRNA1–gRNA3) in comparison to control, Cas9-EV. (l) Quantitative summary of FACS analysis of the percentage of Gr-1+ and Mac-1+ cells in the Cas9-EV- and Syncrip-gRNA-transduced leukemia cells in Figure 2i. (m) Quantitative summary of FACS analysis of the percentage of c-Kithigh cells in the Cas9-EV- and Syncrip-gRNA-transduced leukemia cells Figure 2i. (n) Immunoblot analysis of RN2 cells overexpressing different isoforms of SYNCRIP corresponding to proteins of 527, 562 and 623 aa. The SYNCRIP 562-aa isoform was the predominantly expressed isoform in leukemia cells. Actin serves as a loading control. (o) SYNCRIP overexpression in RN2 cells promotes colony formation (average of three independent experiments). (p) qPCR showing mouse Syncrip expression as assayed by gRNA-specific primers and human SYNCRIP expression for Figure 2k. (q) Immunoblot analysis of the RN2 cells overexpressing SYNCRIP or carrying the control (empty vector) transduced with CRISPR–Cas9-containing Tet-inducible gRNA1 and gRNA3 constructs or Cas9-EV in Figure 2j. All data represent the means + s.e.m. of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

Supplementary Figure 3 Syncrip-deleted hematopoietic cells retain colony-forming and in vivo engraftment capacity.

(a) Representative PCR analysis for genotyping of Syncrip deletion in fetal liver cells. (b) Quantitative summary of FACS analysis of hematopoietic stem and progenitor cells in the WT and Syncrip-CR-KO fetal livers in Figure 3c (WT n = 7, CR-KO n = 7). (c) Quantitative summary of the number of all colony types formed by WT and Syncrip-CR-KO fetal liver cells (average of six biological samples). BFU-E, burst-forming unit–erythroid; M, macrophage; G, granulocyte; GM, granulocyte, macrophage; GEMM, granulocyte, erythroid, macrophage, megakaryocyte. (d) Assessment of disease burden, including spleen weight and liver weight, for the recipient mice in Figure 3j. *P < 0.05, two-tailed t test.

Supplementary Figure 4 SYNCRIP is expressed in human AML, and SYNCRIP depletion results in apoptosis and increased myeloid differentiation in AML cell lines.

(a) The graph shows the log2 expression of SYNCRIP from transcript profiling of bone marrow cells from healthy donors and patients with various types of hematological malignancies, including ALLs, B-ALLs, CLL, MDS, CML and subtypes of AML. Hypodiploid B-ALL, n = 40; ALL with t(1;19), n = 36; ALL with t(12;21), n = 58; B-ALL with t(8;14), n = 13; c-/pre-B-ALL without t(9;22), n = 237; c-/pre-B-ALL with t(9;22), n = 122; pro-B-ALL with t(11q23)/MLL, n = 70; CLL, n = 448; T-ALL, n = 174; MDS, n = 206; CML, n = 76; complex AML, n = 48; AML with inv(16), n = 28; AML MLL, n = 38; AML with a normal karyotype, n = 351; AML with t(15;17), n = 37; AML with t(8;21), n = 40; healthy donors, n = 73. **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test. (Hemaexplorer data for SYNCRIP probe 209024_s_at from the U133 and U133 Plus 2.0 arrays.) (b) qPCR showing SYNCRIP mRNA levels in multiple human AML cell lines and normal cord blood–derived CD34+ (CB-CD34+) cells. ACTB served as a control housekeeping gene. Relative mRNA level was normalized to the SYNCRIP mRNA level in CB-CD34+ cells. (cf) Immunoblots showing efficient SYNCRIP knockdown in the indicated human AML cell lines. (g) Quantitative summary of CD14high and CD13high cells and CD14 and CD13 MFI in MOLM13, NB4 and NOMO-1 cells transduced with control shRNA or shRNAs against SYNCRIP (shRNA 1 and shRNA 2) 4 d after transduction. (h) Representative FACS plots of the cells in Figure 4h. All data represent the means + s.e.m. of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

Supplementary Figure 5 Elevated expression of SYNCRIP correlates with expression profiles of the HOXA9 and MEIS1 target genes IKZF2 and MYC in patients with AML.

(a) Plots showing the distribution of mean normalized counts of KD1 correlating with normalized mean counts of KD2 from the RNA sequencing data in Figure 5a. (b) Normalized read counts of Syncrip, Hoxa9, Myc, Ikzf2 and Meis1 from the RNA sequencing data in Figure 5a. (cf) log2 mRNA levels of the indicated genes in patients with high versus low SYNCRIP mRNA expression in the data sets for patients with AML reported in Figure 4a (high SYNCRIP was defined as individuals with a value greater than the average + 1 s.d. while low SYNCRIP was defined as individuals with a value greater than the average – 1 s.d.). (c) MSI2 mRNA. (d) Target genes upregulated by HOXA9–MEIS1. (e) Target genes downregulated by HOXA9–MEIS1. (f) IZKF2 mRNA. (g) MYC mRNA. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

Supplementary Figure 6 SYNCRIP post-transcriptionally controls HOXA9 expression.

(a) MOLM13 human myeloid leukemia cells carrying the MLL-AF9 translocation overexpressing MSI2 were immunoprecipitated for endogenous MSI2 and SYNCRIP. (b) Immunoblots showing protein expression of HOXA9, MYC and IKZF2 upon SYNCRIP knockdown in dsRed MLL-AF9 cells 3 and 4 d after transduction. Actin serves as a loading control. (c) Immunoblots showing protein expression of HOXA9 and MYC upon SYNCRIP knockdown in MOLM13 cells after 2 d of puromycin selection (4 d after transduction). (d,e) qPCR measuring the mRNA expression of Syncrip, Hoxa9, Myc and Ikzf2 in dsRed tertiary MLL-AF9 leukemia cells 3 and 4 d after transduction. (f) Immunoblots showing protein expression of HOXA9, MYC and SYNCRIP upon MSI2 knockdown in dsRed MLL-AF9 cells at 4 d after transduction. (g) qPCR measuring mRNA expression of Syncrip, Hoxa9, Myc and Ikzf2 upon MSI2 knockdown in dsRed MLL-AF9 cells at 4 d after transduction. (h) qPCR measuring mRNA expression of Syncrip, Hoxa9, Myc and Ikzf2 in RN2 cells 24 h after induction. (i) qPCR measuring mRNA expression of SYNCRIP, HOXA9, MYC and IKZF2 in human MOLM13 leukemia cells 3 d after transduction. (j) qPCR measuring mRNA expression of SYNCRIP, HOXA9, MYC and IKZF2 in human MOLM13 leukemia cells 4 d after transduction. (k) Normalized total RNA levels in dsRed MLL-AF9, RN2 and MOLM13 cells upon SYNCRIP depletion. (l) mRNA stability of Syncrip, Hoxa9, Myc and Ikzf2 in dsRed cells transduced with control and SYNCRIP shRNAs 4 d after transduction. (m) mRNA stability of Syncrip, Hoxa9, Myc and Ikzf2 in RN2 cells expressing Cas9-EV or gRNA1 and gRNA3 targeting Syncrip after 24 h of Dox induction. Actin served as a loading control. Actb served as a control housekeeping gene. All data represent the means + s.e.m. of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

Supplementary Figure 7 HOXA9 but not MYC overexpression partially rescues the colony-forming ability of SYNCRIP-depleted cells.

(a) qPCR measuring mRNA expression showing that MSI2 overexpression increased the mRNA levels of Hoxa9, Myc and Ikzf2. (b) Immunoblots showing efficient depletion of SYNCRIP and protein expression of HOXA9 for the cells in a. (c) Colony formation was rescued in dsRed SYNCRIP-KD leukemia cells overexpressing full-length HOXA9. (d) Immunoblots showing efficient depletion of SYNCRIP and protein expression of HOXA9 for the cells in c. (e) Colony formation was not rescued in dsRed SYNCRIP-KD leukemia cells overexpressing MYC. (f) Immunoblots showing efficient depletion of SYNCRIP and protein expression of MYC for the cells in e. (g) Cell growth was not rescued in MOLM13 SYNCRIP-KD cells overexpressing MYC. (h) Immunoblots showing efficient depletion of SYNCRIP and protein expression of MYC for the cells in g. Actin served as a loading control. Actb served as a control housekeeping gene. All data represent the means + s.e.m. of at least three independent replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t test.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1347 kb)

Supplementary Table 1

Mass spectrometry list of MSI2 protein interactions. (XLSX 47 kb)

Supplementary Table 2

Top 1,000 targets bound by MSI1 in the intestine. (XLSX 185 kb)

Supplementary Table 3

List of gene sets used that are hematopoietic and MSI related. (XLSX 113 kb)

Supplementary Table 4

Prioritization matrix gene list for pool 1a. (XLSX 132 kb)

Supplementary Table 5

Prioritization matrix gene list for pool 1b. (XLSX 67 kb)

Supplementary Table 6

Prioritization matrix gene list for pool 2. (XLSX 61 kb)

Supplementary Table 7

Prioritization matrix for pool 3. (XLSX 94 kb)

Supplementary Table 8

List of genes included in the in vivo shRNA screen associated with the MSI2 interactome. (XLSX 55 kb)

Supplementary Table 9

Raw normalized read counts of all target sequences in the in vivo shRNA screen. (XLS 282 kb)

Supplementary Table 10

List of hits from the in vivo MSI2 interactome shRNA screen. (XLSX 46 kb)

Supplementary Table 11

PCR primers for Syncrip CR-KO genotyping. (XLSX 33 kb)

Supplementary Table 12

List of differentially expressed genes in Syncrip-shRNA leukemia cells with log2 (fold change) >1.5. (XLSX 28 kb)

Supplementary Table 13

Ranked list of all genes expressed in Syncrip-shRNA leukemia cells with log2 (fold change). (XLSX 488 kb)

Supplementary Table 14

GSEA analysis of enrichment for pathways that are negatively regulated by SYNCRIP. (XLSX 34 kb)

Supplementary Table 15

GSEA analysis of enrichment for pathways that are positively regulated by SYNCRIP. (XLSX 26 kb)

Supplementary Table 16

List of additional GSEA analysis for stem cell–associated pathways. (XLSX 11 kb)

Supplementary Table 17

Information on primary AML samples from patients used in Figure 7f. (XLSX 36 kb)

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Vu, L., Prieto, C., Amin, E. et al. Functional screen of MSI2 interactors identifies an essential role for SYNCRIP in myeloid leukemia stem cells. Nat Genet 49, 866–875 (2017). https://doi.org/10.1038/ng.3854

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