Acute myeloid leukemia

Identification of gene targets of mutant C/EBPα reveals a critical role for MSI2 in CEBPA-mutated AML

Abstract

Mutations in the gene encoding the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) occur in 10–15% of acute myeloid leukemia (AML). Frameshifts in the CEBPA N-terminus resulting in exclusive expression of a truncated p30 isoform represent the most prevalent type of CEBPA mutations in AML. C/EBPα p30 interacts with the epigenetic machinery, but it is incompletely understood how p30-induced changes cause leukemogenesis. We hypothesized that critical effector genes in CEBPA-mutated AML are dependent on p30-mediated dysregulation of the epigenome. We mapped p30-associated regulatory elements (REs) by ATAC-seq and ChIP-seq in a myeloid progenitor cell model for p30-driven AML that enables inducible RNAi-mediated knockdown of p30. Concomitant p30-dependent changes in gene expression were measured by RNA-seq. Integrative analysis identified 117 p30-dependent REs associated with 33 strongly down-regulated genes upon p30-knockdown. CRISPR/Cas9-mediated mutational disruption of these genes revealed the RNA-binding protein MSI2 as a critical p30-target. MSI2 knockout in p30-driven murine AML cells and in the CEBPA-mutated human AML cell line KO-52 caused proliferation arrest and terminal myeloid differentiation, and delayed leukemia onset in vivo. In summary, this work presents a comprehensive dataset of p30-dependent effects on epigenetic regulation and gene expression and identifies MSI2 as an effector of the C/EBPα p30 oncoprotein.

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Fig. 1: C/EBPα p30 knockdown results in global changes in the chromatin landscape.
Fig. 2: C/EBPα p30 regulates the expression of differentiation-associated genes in the context of p30-regulated REs.
Fig. 3: CRISPR/Cas9 screening identifies MSI2 as a critical factor in C/EBPα p30-expressing AML cells.
Fig. 4: The RNA-binding protein MSI2 is critical for Cebpa-mutated AML cell proliferation.
Fig. 5: MSI2 is required for the proliferation of human CEBPA-mutated AML cells.

References

  1. 1.

    Döhner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. N Engl J Med. 2015;373(Sep):1136–52.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  2. 2.

    Zhang Y, Wang F, Chen X, Liu W, Fang J, Wang M, et al. Mutation profiling of 16 candidate genes in de novo acute myeloid leukemia patients. Front Med. 2019;13(May):229–37.

    PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Fasan A, Haferlach C, Alpermann T, Jeromin S, Grossmann V, Eder C, et al. The role of different genetic subtypes of CEBPA mutated AML. Leukemia. 2014;28:794–803. http://www.nature.com/doifinder/10.1038/leu.2013.273

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4.

    Keeshan K, Santilli G, Corradini F, Perrotti D, Calabretta B. Transcription activation function of C/EBPalpha is required for induction of granulocytic differentiation. Blood. 2003;102(Aug):1267–75.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  5. 5.

    Lin F-T, MacDougald OA, Diehl AM, Lane MD. A 30-kDa alternative translation product of the CCAAT/enhancer binding protein alpha message: transcriptional activator lacking antimitotic activity. Proc Natl Acad Sci USA. 1993;90(Oct):9606–10.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Nerlov C. C/EBPalpha mutations in acute myeloid leukaemias. Nat Rev Cancer. 2004;4(May):394–400.

    CAS  Article  Google Scholar 

  7. 7.

    Koschmieder S, Halmos B, Levantini E, Tenen DG. Dysregulation of the C/EBPalpha differentiation pathway in human cancer. J Clin Oncol. 2009;27(Mar):619–28.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8.

    Su L, Tan Y, Lin H, Liu X, Yu L, Yang Y, et al. Mutational spectrum of acute myeloid leukemia patients with double CEBPA mutations based on next-generation sequencing and its prognostic significance. Oncotarget. 2018;9(May):24970–9.

    PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Tyner JW, Tognon CE, Bottomly D, Wilmot B, Kurtz SE, Savage SL, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;1. http://www.nature.com/articles/s41586-018-0623-z. Accessed 18 Oct 2018.

  10. 10.

    Schmidt L, Heyes E, Grebien F. Gain-of-function effects of N-terminal CEBPA mutations in acute myeloid leukemia. BioEssays. 2020;42(Feb):e1900178.

    PubMed  Article  PubMed Central  Google Scholar 

  11. 11.

    Grebien F, Vedadi M, Getlik M, Giambruno R, Grover A, Avellino R, et al. Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia. Nat Chem Biol. 2015;11(Aug):571–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, Schnittger S, et al. Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nat Genet. 2001;27(Mar):263–70.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  13. 13.

    Schmidt L, Heyes E, Scheiblecker L, Eder T, Volpe G, Frampton J, et al. CEBPA-mutated leukemia is sensitive to genetic and pharmacological targeting of the MLL1 complex. Leukemia. 2019;33(Jul):1608–19.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Jakobsen JS, Laursen LG, Schuster MB, Pundhir S, Schoof E, Ge Y, et al. Mutant CEBPA directly drives the expression of the targetable tumor-promoting factor CD73 in AML. Sci Adv. 2019;5(Jul):eaaw4304 http://www.ncbi.nlm.nih.gov/pubmed/31309149

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Gröschel S, Sanders MA, Hoogenboezem R, De Wit E, Bouwman BAM, Erpelinck C, et al. A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in Leukemia. Cell. 2014;157:369–81.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  16. 16.

    Hnisz D, Abraham BJ, Lee TI, Lau A, Saint-André V, Sigova AA, et al. Super-enhancers in the control of cell identity and disease. Cell. 2013;155:934–47.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  17. 17.

    Tian Y, Wang G, Hu Q, Xiao X, Chen S. AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer. J Cell Biochem. 2018;119:3706–15.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    Loke J, Assi SA, Imperato MR, Ptasinska A, Cauchy P, Grabovska Y, et al. RUNX1-ETO and RUNX1-EVI1 differentially reprogram the chromatin landscape in t(8;21) and t(3;21) AML. Cell Rep. 2017;19:1654–68.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Corces MR, Buenrostro JD, Wu B, Greenside PG, Chan SM, Koenig JL, et al. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nat Genet. 2016;48:1193–203.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Schmidt L, Heyes E, Scheiblecker L, Eder T, Volpe G, Frampton J, et al. CEBPA-mutated leukemia is sensitive to genetic and pharmacological targeting of the MLL1 complex. Leukemia. 2019;1(Jan). http://www.nature.com/articles/s41375-019-0382-3 Accessed 25 Jun 2019.

  21. 21.

    Kirstetter P, Schuster MB, Bereshchenko O, Moore S, Dvinge H, Kurz E, et al. Modeling of C/EBPα mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells. Cancer Cell. 2008;13(Apr):299–310. http://www.ncbi.nlm.nih.gov/pubmed/18394553

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Klemm SL, Shipony Z, Greenleaf WJ. Chromatin accessibility and the regulatory epigenome. Nat Rev Genet. 2019;20(April):207–20.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    McLean CY, Bristor D, Hiller M, Clarke SL, Schaar BT, Lowe CB, et al. GREAT improves functional interpretation of cis-regulatory regions. Nat Biotechnol. 2010;28(May):495–501.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Bereshchenko O, Mancini E, Moore S, Bilbao D, Månsson R, Luc S, et al. Hematopoietic stem cell expansion precedes the generation of committed myeloid leukemia-initiating cells in C/EBPα Mutant AML. Cancer Cell. 2009;16(Nov):390–400. http://www.sciencedirect.com/science/article/pii/S1535610809003444?via%3Dihub

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. 25.

    Rao J, Ashraf S, Tan W, Van Der Ven AT, Gee HY, Braun DA, et al. Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant nephrotic syndrome. J Clin Investig. 2017;127(Dec):4257–69.

    PubMed  Article  PubMed Central  Google Scholar 

  26. 26.

    Vassar R. BACE1: The β-secreiase enzyme in Alzheimer’s disease. J Mol Neurosci. 2004;23:105–13.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27.

    Kaeda J, Ringel F, Oberender C, Mills K, Quintarelli C, Pane F, et al. Up-regulated MSI2 is associated with more aggressive chronic myeloid leukemia. Leuk Lymphoma. 2015;56:2105–13.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Minuesa G, Albanese SK, Xie W, Kazansky Y, Worroll D, Chow A, et al. Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia. Nat Commun. 2019;10:2691.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  29. 29.

    Hattori A, McSkimming D, Kannan N, Ito T. RNA binding protein MSI2 positively regulates FLT3 expression in myeloid leukemia. Leuk Res. 2017;54:47–54.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Li Z, Jin H, Mao G, Wu L, Guo Q. Msi2 plays a carcinogenic role in esophageal squamous cell carcinoma via regulation of the Wnt/β-catenin and Hedgehog signaling pathways. Exp Cell Res. 2017;361:170–7.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  31. 31.

    Wang X, Wang R, Bai S, Xiong S, Li Y, Liu M, et al. Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway. J Exp Clin Cancer Res. 2019;38(Dec):505.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Sheng W, Shi X, Lin Y, Tang J, Jia C, Cao R, et al. Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling. J Exp Clin Cancer Res. 2020;39(Jan):16.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Zhao J, Zhang Y, Liu X-S, Zhu F-M, Xie F, Jiang C-Y, et al. RNA-binding protein Musashi2 stabilizing androgen receptor drives prostate cancer progression. Cancer Sci. 2020;111(Feb):369–82.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Wang Z-L, Wang C, Liu W, Ai Z-L. Emerging roles of the long non-coding RNA 01296/microRNA-143-3p/MSI2 axis in development of thyroid cancer. Biosci Rep. 2019;39(Nov):BSR20182376.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Lan L, Xing M, Kashipathy M, Douglas J, Gao P, Battaile K, et al. Crystal and solution structures of human oncoprotein Musashi-2 N-terminal RNA recognition motif 1. Proteins. 2020;88(Apr):573–83.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  36. 36.

    Yang Z, Li J, Shi Y, Li L, Guo X. Increased Musashi 2 expression indicates a poor prognosis and promotes malignant phenotypes in gastric cancer. Oncol Lett. 2019;17(Mar):2599–606.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Kharas MG, Lengner CJ, Al-Shahrour F, Bullinger L, Ball B, Zaidi S, et al. Musashi-2 regulates normal hematopoiesis and promotes aggressive myeloid leukemia. Nat Med. 2010;16(Aug):903–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Nguyen DTT, Lu Y, Chu KL, Yang X, Park SM, Choo ZN, et al. HyperTRIBE uncovers increased MUSASHI-2 RNA binding activity and differential regulation in leukemic stem cells. Nat Commun. 2020;11(Dec):1–12.

    Google Scholar 

  39. 39.

    Byers RJ, Currie T, Tholouli E, Rodig SJ, Kutok JL. MSI2 protein expression predicts unfavorable outcome in acute myeloid leukemia. Blood. 2011;118(Sep):2857–67.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Asou H, Gombart AF, Takeuchi S, Tanaka H, Tanioka M, Matsui H, et al. Establishment of the acute myeloid leukemia cell line Kasumi-6 from a patient with a dominant-negative mutation in the DNA-binding region of the C/EBPα gene. Genes Chromosom Cancer. 2003;36(Feb):167–74.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. 41.

    DepMap, Broad (2020): DepMap 20Q1 Public. 2020.

  42. 42.

    De Kouchkovsky I, Abdul-Hay M. Acute myeloid leukemia: a comprehensive review and 2016 update. Blood Cancer J. 2016;6(Jul):e441 http://www.ncbi.nlm.nih.gov/pubmed/27367478

    PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND, et al. Genomic classification and prognosis in acute myeloid leukemia. N Engl J Med. 2016;374:2209–21. http://www.nejm.org/doi/full/10.1056/NEJMoa1516192#.V10wtXeAo1A.mendeley

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Soukup AA, Zheng Y, Mehta C, Wu J, Liu P, Cao M. et al. Single-nucleotide human disease mutation inactivates a blood-regenerative GATA2 enhancer. J Clin Investig. 2019;129(Mar):1180–92.

    PubMed  Article  PubMed Central  Google Scholar 

  45. 45.

    Mansour MR, Abraham BJ, Anders L, Berezovskaya A, Gutierrez A, Durbin AD, et al. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element. Science (80-). 2014;346:1373–7.

    CAS  Article  Google Scholar 

  46. 46.

    Nerlov C, Ziff EB. CCAAT/enhancer binding protein-alpha amino acid motifs with dual TBP and TFIIB binding ability co-operate to activate transcription in both yeast and mammalian cells. EMBO J. 1995;14(Sep):4318–28.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Pedersen TÅ, Kowenz-Leutz E, Leutz A, Nerlov C. Cooperation between C/EBPα TBP/TFIIB and SWI/SNF recruiting domains is required for adipocyte differentiation. Genes Dev. 2001;15(Dec):3208–16.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Nerlov C, Ziff EB. Three levels of functional interaction determine the activity of CCAAT/enhancer binding protein-α on the serum albumin promoter. Genes Dev. 1994;8(Feb):350–62.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  49. 49.

    Dvinge H, Kim E, Abdel-Wahab O, Bradley RK. RNA splicing factors as oncoproteins and tumour suppressors. Nat Rev Cancer. 2016;16:413–30.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Wang E, Lu SX, Pastore A, Chen X, Imig J, Chun-Wei Lee S, et al. Targeting an RNA-binding protein network in acute myeloid leukemia. Cancer Cell. 2019;35:369–384.e7.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  51. 51.

    Wang ZL, Li B, Luo YX, Lin Q, Liu SR, Zhang XQ, et al. Comprehensive genomic characterization of RNA-binding proteins across human cancers. Cell Rep. 2018;22(Jan):286–98.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  52. 52.

    Bajaj J, Hamilton M, Shima Y, Chambers K, Spinler K, Van Nostrand EL, et al. An in vivo genome-wide CRISPR screen identifies the RNA-binding protein Staufen2 as a key regulator of myeloid leukemia. Nat Cancer. 2020;1(Apr):410–22.

    Article  Google Scholar 

  53. 53.

    Thol F, Winschel C, Sonntag A-K, Damm F, Wagner K, Chaturvedi A, et al. Prognostic significance of expression levels of stem cell regulators MSI2 and NUMB in acute myeloid leukemia. Ann Hematol. 2013;92(Mar):315–23.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  54. 54.

    He L, Zhou X, Qu C, Hu L, Tang Y, Zhang Q, et al. Musashi2 predicts poor prognosis and invasion in hepatocellular carcinoma by driving epithelial-mesenchymal transition. J Cell Mol Med. 2014;18(Jan):49–58.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  55. 55.

    Aly RM, Ghazy HF. Prognostic significance of MSI2 predicts unfavorable outcome in adult B-acute lymphoblastic leukemia. Int J Lab Hematol. 2015;37(Apr):272–8.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  56. 56.

    Park SM, Gönen M, Vu L, Minuesa G, Tivnan P, Barlowe TS, et al. Musashi2 sustains the mixed-lineage leukemia’ driven stem cell regulatory program. J Clin Investig. 2015;125(Mar):1286–98.

    PubMed  Article  PubMed Central  Google Scholar 

  57. 57.

    Ghandi M, Huang FW, Jané-Valbuena J, Kryukov GV, Lo CC, McDonald ER, et al. Next-generation characterization of the cancer cell line encyclopedia. Nature. 2019;569(May):503–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

We thank the members of the Grebien laboratory for stimulating discussions and T. Weiss, E. Rzepa, and M. Piontek for technical help. We thank D. Berger and G. Stefanzl for skillful technical assistance and P. Valent for providing access to human samples. Next Generation Sequencing was performed at the VBCF NGS Unit (www.viennabiocenter.org/facilities) and at the BSF (https://cemm.at/research/facilities/). This project has received funding from the European Union’s Horizon 2020 research and innovation program (European Research Council grant agreement No 636855 and Marie Sklodowska-Curie grant agreement No 813091). LS is a recipient of the DOC Fellowship of the Austrian Academy of Sciences at the Ludwig Boltzmann Institute for Cancer Research.

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Correspondence to Florian Grebien.

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Heyes, E., Schmidt, L., Manhart, G. et al. Identification of gene targets of mutant C/EBPα reveals a critical role for MSI2 in CEBPA-mutated AML. Leukemia (2021). https://doi.org/10.1038/s41375-021-01169-6

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