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Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells

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Abstract

Chromosomal deletions associated with human diseases, such as cancer, are common, but synteny issues complicate modeling of these deletions in mice. We use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q (del(7q)), a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). We derive del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and show that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes are rescued by spontaneous dosage correction and can be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments in a 20-Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Our approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes.

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Figure 1: Generation of del(7q)- and isogenic karyotypically normal iPSCs from patients with MDS.
Figure 2: MDS iPSCs have diminished hematopoietic differentiation potential.
Figure 3: Spontaneous compensation for chromosome 7q dosage imbalance rescues the hematopoietic defect of MDS iPSCs.
Figure 4: Engineering chr7q deletions in normal hPSCs.
Figure 5: A phenotype-rescue screen identifies candidate haploinsufficient genes in del(7q)- MDS.
Figure 6: Validation of chr7q haploinsufficient genes.

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References

  1. Beroukhim, R. et al. The landscape of somatic copy-number alteration across human cancers. Nature 463, 899–905 (2010).

    Article  CAS  Google Scholar 

  2. Alkan, C., Coe, B.P. & Eichler, E.E. Genome structural variation discovery and genotyping. Nat. Rev. Genet. 12, 363–376 (2011).

    Article  CAS  Google Scholar 

  3. Weischenfeldt, J., Symmons, O., Spitz, F. & Korbel, J.O. Phenotypic impact of genomic structural variation: insights from and for human disease. Nat. Rev. Genet. 14, 125–138 (2013).

    Article  CAS  Google Scholar 

  4. Gibson, G. Rare and common variants: twenty arguments. Nat. Rev. Genet. 13, 135–145 (2011).

    Article  Google Scholar 

  5. Li, J. et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943–1947 (1997).

    Article  CAS  Google Scholar 

  6. Berger, A.H., Knudson, A.G. & Pandolfi, P.P. A continuum model for tumour suppression. Nature 476, 163–169 (2011).

    Article  CAS  Google Scholar 

  7. Solimini, N.L. et al. Recurrent hemizygous deletions in cancers may optimize proliferative potential. Science 337, 104–109 (2012).

    Article  CAS  Google Scholar 

  8. Lindsley, R.C. & Ebert, B.L. Molecular pathophysiology of myelodysplastic syndromes. Annu. Rev. Pathol. 8, 21–47 (2013).

    Article  CAS  Google Scholar 

  9. Jerez, A. et al. Loss of heterozygosity in 7q myeloid disorders: clinical associations and genomic pathogenesis. Blood 119, 6109–6117 (2012).

    Article  CAS  Google Scholar 

  10. Soldner, F. et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146, 318–331 (2011).

    Article  CAS  Google Scholar 

  11. Grskovic, M., Javaherian, A., Strulovici, B. & Daley, G.Q. Induced pluripotent stem cells–opportunities for disease modelling and drug discovery. Nat. Rev. Drug Discov. 10, 915–929 (2011).

    Article  CAS  Google Scholar 

  12. Papapetrou, E.P. et al. Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat. Biotechnol. 29, 73–78 (2011).

    Article  CAS  Google Scholar 

  13. Papapetrou, E.P. & Sadelain, M. Generation of transgene-free human induced pluripotent stem cells with an excisable single polycistronic vector. Nat. Protoc. 6, 1251–1273 (2011).

    Article  CAS  Google Scholar 

  14. Walter, M.J. et al. Clonal architecture of secondary acute myeloid leukemia. N. Engl. J. Med. 366, 1090–1098 (2012).

    Article  CAS  Google Scholar 

  15. Sturgeon, C.M., Ditadi, A., Clarke, R.L. & Keller, G. Defining the path to hematopoietic stem cells. Nat. Biotechnol. 31, 416–418 (2013).

    Article  CAS  Google Scholar 

  16. Kennedy, M. et al. T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures. Cell Reports 2, 1722–1735 (2012).

    Article  CAS  Google Scholar 

  17. Flores-Figueroa, E., Gutierrez-Espindola, G., Guerrero-Rivera, S., Pizzuto-Chavez, J. & Mayani, H. Hematopoietic progenitor cells from patients with myelodysplastic syndromes: in vitro colony growth and long-term proliferation. Leuk. Res. 23, 385–394 (1999).

    Article  CAS  Google Scholar 

  18. Sato, T., Kim, S., Selleri, C., Young, N.S. & Maciejewski, J.P. Measurement of secondary colony formation after 5 weeks in long-term cultures in patients with myelodysplastic syndrome. Leukemia 12, 1187–1194 (1998).

    Article  CAS  Google Scholar 

  19. Amps, K. et al. Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage. Nat. Biotechnol. 29, 1132–1144 (2011).

    Article  CAS  Google Scholar 

  20. Närvä, E. et al. High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity. Nat. Biotechnol. 28, 371–377 (2010).

    Article  Google Scholar 

  21. Li, L.B. et al. Trisomy correction in down syndrome induced pluripotent stem cells. Cell Stem Cell 11, 615–619 (2012).

    Article  CAS  Google Scholar 

  22. Lewandoski, M. & Martin, G.R. Cre-mediated chromosome loss in mice. Nat. Genet. 17, 223–225 (1997).

    Article  CAS  Google Scholar 

  23. Matsumura, H. et al. Targeted chromosome elimination from ES-somatic hybrid cells. Nat. Methods 4, 23–25 (2007).

    Article  CAS  Google Scholar 

  24. Davoli, T. et al. Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome. Cell 155, 948–962 (2013).

    Article  CAS  Google Scholar 

  25. Xue, W. et al. A cluster of cooperating tumor-suppressor gene candidates in chromosomal deletions. Proc. Natl. Acad. Sci. USA 109, 8212–8217 (2012).

    Article  CAS  Google Scholar 

  26. Ebert, B.L. et al. Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature 451, 335–339 (2008).

    Article  CAS  Google Scholar 

  27. Starczynowski, D.T. et al. Identification of miR-145 and miR-146a as mediators of the 5q- syndrome phenotype. Nat. Med. 16, 49–58 (2010).

    Article  CAS  Google Scholar 

  28. Pang, W.W. et al. Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age. Proc. Natl. Acad. Sci. USA 108, 20012–20017 (2011).

    Article  CAS  Google Scholar 

  29. Nikoloski, G. et al. Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat. Genet. 42, 665–667 (2010).

    Article  CAS  Google Scholar 

  30. Lindsley, R.C. & Ebert, B.L. The biology and clinical impact of genetic lesions in myeloid malignancies. Blood 122, 3741–3748 (2013).

    Article  CAS  Google Scholar 

  31. Ernst, T. et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat. Genet. 42, 722–726 (2010).

    Article  CAS  Google Scholar 

  32. Makishima, H. et al. Novel homo- and hemizygous mutations in EZH2 in myeloid malignancies. Leukemia 24, 1799–1804 (2010).

    Article  CAS  Google Scholar 

  33. Shih, A.H., Abdel-Wahab, O., Patel, J.P. & Levine, R.L. The role of mutations in epigenetic regulators in myeloid malignancies. Nat. Rev. Cancer 12, 599–612 (2012).

    Article  CAS  Google Scholar 

  34. González, F. et al. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15, 215–226 (2014).

    Article  Google Scholar 

  35. Young, M.A. et al. Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell Stem Cell 10, 570–582 (2012).

    Article  CAS  Google Scholar 

  36. Abyzov, A. et al. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature 492, 438–442 (2012).

    Article  CAS  Google Scholar 

  37. Welch, J.S. et al. The origin and evolution of mutations in acute myeloid leukemia. Cell 150, 264–278 (2012).

    Article  CAS  Google Scholar 

  38. Le Beau, M.M. et al. Cytogenetic and molecular delineation of a region of chromosome 7 commonly deleted in malignant myeloid diseases. Blood 88, 1930–1935 (1996).

    CAS  PubMed  Google Scholar 

  39. Döhner, K. et al. Molecular cytogenetic characterization of a critical region in bands 7q35-q36 commonly deleted in malignant myeloid disorders. Blood 92, 4031–4035 (1998).

    PubMed  Google Scholar 

  40. Chen, C. et al. MLL3 is a haploinsufficient 7q tumor suppressor in acute myeloid leukemia. Cancer Cell 25, 652–665 (2014).

    Article  Google Scholar 

  41. Tang, Y.C. & Amon, A. Gene copy-number alterations: a cost-benefit analysis. Cell 152, 394–405 (2013).

    Article  CAS  Google Scholar 

  42. Carette, J.E. et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009).

    Article  CAS  Google Scholar 

  43. White, J.K. et al. Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes. Cell 154, 452–464 (2013).

    Article  CAS  Google Scholar 

  44. Bolze, A. et al. Ribosomal protein SA haploinsufficiency in humans with isolated congenital asplenia. Science 340, 976–978 (2013).

    Article  CAS  Google Scholar 

  45. Hosono, N. et al. Recurrent genetic defects on chromosome 7q in myeloid neoplasms. Leukemia 28, 1348–1351 (2014).

    Article  CAS  Google Scholar 

  46. Singh, H. et al. Putative RNA-splicing gene LUC7L2 on 7q34 represents a candidate gene in pathogenesis of myeloid malignancies. Blood Cancer 3, e117 (2013).

    Article  CAS  Google Scholar 

  47. Papapetrou, E.P. et al. Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation. Proc. Natl. Acad. Sci. USA 106, 12759–12764 (2009).

    Article  CAS  Google Scholar 

  48. Tennessen, J.A. et al. Evolution and functional impact of rare coding variation from deep sequencing of human exomes. Science 337, 64–69 (2012).

    Article  CAS  Google Scholar 

  49. Cibulskis, K. et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat. Biotechnol. 31, 213–219 (2013).

    Article  CAS  Google Scholar 

  50. Papapetrou, E.P., Korkola, J.E. & Sadelain, M. A genetic strategy for single and combinatorial analysis of miRNA function in mammalian hematopoietic stem cells. Stem Cells 28, 287–296 (2010).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institutes of Health (NIH) grants R00 DK087923 (E.P.P.), R01 HL121570 (E.P.P.), P30 CA15704 and by awards from the University of Washington Royalty Research Fund (E.P.P.), the American Society of Hematology (E.P.P.), the Sidney Kimmel Foundation for Cancer Research (E.P.P.), the Aplastic Anemia & MDS International Foundation (E.P.P.), the Ellison Medical Foundation (E.P.P.), the Damon Runyon Cancer Research Foundation (E.P.P.) and a John H. Tietze Stem Cell Scientist Award (E.P.P.). We thank D. Russell and L. Li for sharing their expertise in AAV-mediated gene targeting and T. Papayannopoulou for sharing her expertise in assessment of May-Giemsa slides and for useful discussions. We thank C. Husser, C. Sather and R. Basom for excellent technical assistance and J. Overbey for statistical advice.

Author information

Author notes

  1. Chan-Jung Chang and Ibrahim Boussaad: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Andriana G Kotini, Chan-Jung Chang & Eirini P Papapetrou

  2. The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Andriana G Kotini, Chan-Jung Chang & Eirini P Papapetrou

  3. The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Andriana G Kotini, Chan-Jung Chang & Eirini P Papapetrou

  4. Division of Hematology, Department of Medicine, University of Washington, Seattle, Washington, USA

    Ibrahim Boussaad & Eirini P Papapetrou

  5. Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA

    Ibrahim Boussaad, Abhinav B Nagulapally, Gregory A Fishbein, R David Hawkins, Charles E Murry & Eirini P Papapetrou

  6. Genomics Resource, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Jeffrey J Delrow

  7. Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, Florida, USA

    Emily K Dolezal & Stephen D Nimer

  8. Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington, USA

    Abhinav B Nagulapally & R David Hawkins

  9. Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Fabiana Perna

  10. Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA

    Gregory A Fishbein

  11. Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Virginia M Klimek

  12. Developmental Biology Program, Sloan-Kettering Institute, New York, New York, USA

    Danwei Huangfu

  13. Department of Pathology, University of Washington, Seattle, Washington, USA

    Charles E Murry & Eirini P Papapetrou

  14. Center for Cardiovascular Biology, University of Washington, Seattle, Washington, USA

    Charles E Murry

  15. Department of Bioengineering University of Washington, Seattle, Washington, USA

    Charles E Murry

  16. Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, USA

    Charles E Murry

  17. MGH Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA

    Timothy Graubert

  18. Division of Hematology and Medical Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Eirini P Papapetrou

Authors
  1. Andriana G Kotini
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  2. Chan-Jung Chang
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  3. Ibrahim Boussaad
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  4. Jeffrey J Delrow
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  5. Emily K Dolezal
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  6. Abhinav B Nagulapally
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  7. Fabiana Perna
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  8. Gregory A Fishbein
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  9. Virginia M Klimek
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  10. R David Hawkins
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  11. Danwei Huangfu
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  12. Charles E Murry
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  13. Timothy Graubert
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  14. Stephen D Nimer
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  15. Eirini P Papapetrou
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Contributions

A.G.K., C.-J.C. and I.B. performed experiments and analyzed data, J.J.D. analyzed microarray data, E.K.D., F.P., V.M.K. and S.D.N. selected and procured patient samples, A.B.N. and R.D.H. performed bioinformatics analyses, G.A.F. and C.E.M. performed histological analyses of teratomas, D.H. provided the iCas9-HUES8 cell line, T.G. analyzed whole exome sequencing data, T.G. and S.D.N. provided critical reading of the manuscript and scientific discussions, E.P.P. conceived, designed and supervised the study, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Eirini P Papapetrou.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Tables 1–3, 5–6, 9–10 and 12–14 (PDF 2137 kb)

Supplementary Table 4

Somatic variants identified by whole exome sequencing in BMMCs and iPSCs from MDS patient #2 (XLSX 59 kb)

Supplementary Table 7

Chr7q genes with reduced expression in the haploid state (XLSX 47 kb)

Supplementary Table 8

Chr7 genes included in the lentiviral library (XLSX 54 kb)

Supplementary Table 11

Gene enrichment results of all screening experiments (XLSX 50 kb)

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Kotini, A., Chang, CJ., Boussaad, I. et al. Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells. Nat Biotechnol 33, 646–655 (2015). https://doi.org/10.1038/nbt.3178

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