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Acute myeloid leukemia

Drug targeting of NR4A nuclear receptors for treatment of acute myeloid leukemia

Leukemia (2018) | Download Citation

Abstract

NR4As are AML tumor suppressors that are frequently silenced in human acute myeloid leukemia (AML). Despite their potential as novel targets for therapeutic intervention, mechanisms of NR4A silencing and strategies for their reactivation remain poorly defined. Here we show that NR4A silencing in AML occurs through blockade of transcriptional elongation rather than epigenetic promoter silencing. By intersection of NR4A-regulated gene signatures captured upon acute, exogenous expression of NR4As in human AML cells with in silico chemical genomics screening, we identify several FDA-approved drugs including dihydroergotamine (DHE) that reactivate NR4A expression and regulate NR4A-dependent gene signatures. We show that DHE induces NR4A expression via recruitment of the super elongation complex to enable elongation of NR4A promoter paused RNA polymerase II. Finally, DHE exhibits AML selective NR4A-dependent anti-leukemic activity in cytogenetically distinct human AML cells in vitro and delays AML progression in mice revealing its potential as a novel therapeutic agent in AML.

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References

  1. 1.

    Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367:645–8.

  2. 2.

    Jordan CT. The leukemic stem cell. Best Pract Res Clin Haematol. 2007;20:13–8.

  3. 3.

    Kadia TM, Ravandi F, Cortes J, Kantarjian H. Toward individualized therapy in acute myeloid leukemia: a contemporary review. JAMA Oncol. 2015;1:820–8.

  4. 4.

    Cancer Genome Atlas Research Network. et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013;368:2059–74.

  5. 5.

    Goardon N, Marchi E, Atzberger A, Quek L, Schuh A, Soneji S, et al. Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia. Cancer Cell. 2011;19:138–52.

  6. 6.

    Wiseman DH, Greystoke BF, Somervaille TC. The variety of leukemic stem cells in myeloid malignancy. Oncogene. 2014;33:3091–8.

  7. 7.

    Klco JM, Spencer DH, Miller CA, Griffith M, Lamprecht TL, O’Laughlin M, et al. Functional heterogeneity of genetically defined subclones in acute myeloid leukemia. Cancer Cell. 2014;25:379–92.

  8. 8.

    Maxwell MA, Muscat GE. The NR4A subgroup: immediate early response genes with pleiotropic physiological roles. Nucl Recept Signal. 2006;4:e002.

  9. 9.

    Safe S, Jin UH, Morpurgo B, Abudayyeh A, Singh M, Tjalkens RB. Nuclear receptor 4A (NR4A) family - orphans no more. J Steroid Biochem Mol Biol. 2016;157:48–60.

  10. 10.

    Hamers AA, Hanna RN, Nowyhed H, Hedrick CC, de Vries CJ. NR4A nuclear receptors in immunity and atherosclerosis. Curr Opin Lipidol. 2013;24:381–5.

  11. 11.

    Mullican SE, Zhang S, Konopleva M, Ruvolo V, Andreeff M, Milbrandt J, et al. Abrogation of nuclear receptors Nr4a3 and Nr4a1 leads to development of acute myeloid leukemia. Nat Med. 2007;13:730–5.

  12. 12.

    Sirin O, Lukov GL, Mao R, Conneely OM, Goodell MA. The orphan nuclear receptor Nurr1 restricts the proliferation of haematopoietic stem cells. Nat Cell Biol. 2010;12:1213–9.

  13. 13.

    Cheng LE, Chan FK, Cado D, Winoto A. Functional redundancy of the Nur77 and Nor-1 orphan steroid receptors in T-cell apoptosis. EMBO J. 1997;16:1865–75.

  14. 14.

    Woronicz JD, Calnan B, Ngo V, Winoto A. Requirement for the orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas. Nature. 1994;367:277–81.

  15. 15.

    Lee SL, Wesselschmidt RL, Linette GP, Kanagawa O, Russell JH, Milbrandt J. Unimpaired thymic and peripheral T cell death in mice lacking the nuclear receptor NGFI-B (Nur77). Science. 1995;269:532–5.

  16. 16.

    Sekiya T, Kashiwagi I, Yoshida R, Fukaya T, Morita R, Kimura A, et al. Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis. Nat Immunol. 2013;14:230–7.

  17. 17.

    Sekiya T, Kondo T, Shichita T, Morita R, Ichinose H, Yoshimura A. Suppression of Th2 and Tfh immune reactions by Nr4a receptors in mature T reg cells. J Exp Med. 2015;212:1623–40.

  18. 18.

    Nowyhed HN, Huynh TR, Blatchley A, Wu R, Thomas GD, Hedrick CC. The nuclear receptor nr4a1 controls CD8 T cell development through transcriptional suppression of runx3. Sci Rep. 2015;5:9059.

  19. 19.

    Hanna RN, Carlin LM, Hubbeling HG, Nackiewicz D, Green AM, Punt JA, et al. The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes. Nat Immunol. 2011;12:778–85.

  20. 20.

    Tacke R, Hilgendorf I, Garner H, Waterborg C, Park K, Nowyhed H, et al. The transcription factor NR4A1 is essential for the development of a novel macrophage subset in the thymus. Sci Rep. 2015;5:10055.

  21. 21.

    Hanna RN, Shaked I, Hubbeling HG, Punt JA, Wu R, Herrley E, et al. NR4A1 (Nur77) deletion polarizes macrophages toward an inflammatory phenotype and increases atherosclerosis. Circ Res. 2012;110:416–27.

  22. 22.

    Ramirez-Herrick AM, Mullican SE, Sheehan AM, Conneely OM. Reduced NR4A gene dosage leads to mixed myelodysplastic/myeloproliferative neoplasms in mice. Blood. 2011;117:2681–90.

  23. 23.

    Deutsch AJ, Rinner B, Wenzl K, Pichler M, Troppan K, Steinbauer E, et al. NR4A1-mediated apoptosis suppresses lymphomagenesis and is associated with a favorable cancer-specific survival in patients with aggressive B-cell lymphomas. Blood. 2014;123:2367–77.

  24. 24.

    Wenzl K, Troppan K, Neumeister P, Deutsch AJ. The nuclear orphan receptor NR4A1 and NR4A3 as tumor suppressors in hematologic neoplasms. Curr Drug Targets. 2015;16:38–46.

  25. 25.

    Boudreaux SP, Ramirez-Herrick AM, Duren RP, Conneely OM. Genome-wide profiling reveals transcriptional repression of MYC as a core component of NR4A tumor suppression in acute myeloid leukemia. Oncogenesis. 2012;1:e19.

  26. 26.

    Pellagatti A, Cazzola M, Giagounidis AA, Malcovati L, Porta MG, Killick S, et al. Gene expression profiles of CD34+cells in myelodysplastic syndromes: involvement of interferon-stimulated genes and correlation to FAB subtype and karyotype. Blood. 2006;108:337–45.

  27. 27.

    Majeti R, Becker MW, Tian Q, Lee TL, Yan X, Liu R, et al. Dysregulated gene expression networks in human acute myelogenous leukemia stem cells. Proc Natl Acad Sci USA. 2009;106:3396–401.

  28. 28.

    Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, et al. The connectivity map: using gene-expression signatures to connect small molecules, genes, and disease. Science. 2006;313:1929–35.

  29. 29.

    Duren RP, Boudreaux SP, Conneely OM. Genome wide mapping of NR4A binding reveals cooperativity with ETS factors to promote epigenetic activation of distal enhancers in acute myeloid leukemia cells. PLoS One. 2016;11:e0150450.

  30. 30.

    Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature. 2007;448:553–60.

  31. 31.

    Guenther MG, Levine SS, Boyer LA, Jaenisch R, Young RA. A chromatin landmark and transcription initiation at most promoters in human cells. Cell. 2007;130:77–88.

  32. 32.

    Jonkers I, Lis JT. Getting up to speed with transcription elongation by RNA polymerase II. Nat Rev Mol Cell Biol. 2015;16:167–77.

  33. 33.

    Rickert P, Corden JL, Lees E. Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases. Oncogene. 1999;18:1093–102.

  34. 34.

    Akhtar MS, Heidemann M, Tietjen JR, Zhang DW, Chapman RD, Eick D, et al. TFIIH kinase places bivalent marks on the carboxy-terminal domain of RNA polymerase II. Mol Cell. 2009;34:387–93.

  35. 35.

    Donner AJ, Ebmeier CC, Taatjes DJ, Espinosa JM. CDK8 is a positive regulator of transcriptional elongation within the serum response network. Nat Struct Mol Biol. 2010;17:194–201.

  36. 36.

    Galbraith MD, Allen MA, Bensard CL, Wang X, Schwinn MK, Qin B, et al. HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia. Cell. 2013;153:1327–39.

  37. 37.

    Baylin SB, Ohm JE. Epigenetic gene silencing in cancer—a mechanism for early oncogenic pathway addiction? Nat Rev Cancer. 2006;6:107–16.

  38. 38.

    Schlesinger Y, Straussman R, Keshet I, Farkash S, Hecht M, Zimmerman J, et al. Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nat Genet. 2007;39:232–6.

  39. 39.

    Kondo Y, Shen L, Cheng AS, Ahmed S, Boumber Y, Charo C, et al. Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation. Nat Genet. 2008;40:741–50.

  40. 40.

    Issa JP. DNA methylation as a therapeutic target in cancer. Clin Cancer Res. 2007;13:1634–7.

  41. 41.

    Wagner JM, Hackanson B, Lubbert M, Jung M. Histone deacetylase (HDAC) inhibitors in recent clinical trials for cancer therapy. Clin Epigenetics. 2010;1:117–36.

  42. 42.

    Brien GL, Valerio DG, Armstrong SA. Exploiting the epigenome to control cancer-promoting gene-expression programs. Cancer Cell. 2016;29:464–76.

  43. 43.

    Gardini A, Baillat D, Cesaroni M, Hu D, Marinis JM, Wagner EJ, et al. Integrator regulates transcriptional initiation and pause release following activation. Mol Cell. 2014;56:128–39.

  44. 44.

    Chen K, Chen Z, Wu D, Zhang L, Lin X, Su J, et al. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor-suppressor genes. Nat Genet. 2015;47:1149–57.

  45. 45.

    Hargreaves DC, Horng T, Medzhitov R. Control of inducible gene expression by signal-dependent transcriptional elongation. Cell. 2009;138:129–45.

  46. 46.

    Adelman K, Kennedy MA, Nechaev S, Gilchrist DA, Muse GW, Chinenov Y, et al. Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling. Proc Natl Acad Sci USA. 2009;106:18207–12.

  47. 47.

    Brondfield S, Umesh S, Corella A, Zuber J, Rappaport AR, Gaillard C, et al. Direct and indirect targeting of MYC to treat acute myeloid leukemia. Cancer Chemother Pharmacol. 2015;76:35–46.

  48. 48.

    Dawson MA, Prinjha RK, Dittmann A, Giotopoulos G, Bantscheff M, Chan WI, et al. Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature. 2011;478:529–33.

  49. 49.

    Zuber J, Radtke I, Pardee TS, Zhao Z, Rappaport AR, Luo W, et al. Mouse models of human AML accurately predict chemotherapy response. Genes Dev. 2009;23:877–89.

  50. 50.

    Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J. 2008;22:659–61.

  51. 51.

    Silberstein SD, McCrory DC. Ergotamine and dihydroergotamine: history, pharmacology, and efficacy. Headache. 2003;43:144–66.

  52. 52.

    Mendez-Ferrer S, Battista M, Frenette PS. Cooperation of beta(2)- and beta(3)-adrenergic receptors in hematopoietic progenitor cell mobilization. Ann N Y Acad Sci. 2010;1192:139–44.

  53. 53.

    Lucas D, Bruns I, Battista M, Mendez-Ferrer S, Magnon C, Kunisaki Y, et al. Norepinephrine reuptake inhibition promotes mobilization in mice: potential impact to rescue low stem cell yields. Blood. 2012;119:3962–5.

  54. 54.

    Spiegel A, Shivtiel S, Kalinkovich A, Ludin A, Netzer N, Goichberg P, et al. Catecholaminergic neurotransmitters regulate migration and repopulation of immature human CD34+cells through Wnt signaling. Nat Immunol. 2007;8:1123–31.

  55. 55.

    Yang M, Li K, Ng PC, Chuen CK, Lau TK, Cheng YS, et al. Promoting effects of serotonin on hematopoiesis: ex vivo expansion of cord blood CD34+stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis. Stem Cells. 2007;25:1800–6.

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Acknowledgements

This work was supported by RO1 CA160747 from National Institutes of Health to OMC and by the Cell Sorting and Flow Cytometry shared resource of the Dan L. Duncan Comprehensive Cancer Center with funding from National Cancer Institute grant (P30CA125123). The authors acknowledge the joint participation by Adrienne Helis Melvin Medical Research Foundation through its direct engagement in the continuous active conduct of medical research in conjunction with Baylor College of Medicine. The authors also thank Dr. Terzah Horton and the Leukemia Research Interest Group at Texas Children’s Hospital for providing primary patient AML samples.

Statement of significance

A chemical genomics strategy identifies DHE as a novel activator of silenced NR4A tumor suppressors with repositioning potential for treatment of AML.

Author contributions

S.P.B., R.P.D., and S.G.C. carried out experiments including CMap chemical genomics integration, ChIP-experiments, bisulfite sequencing, and cell growth assays. P.R.F. did flow cytometry analysis. L.N. did transplantation assays. P.N., M.S.R., and P.R.F. were responsible for colony forming assays on primary AML samples. S.P.B. and O.M.C. conceived the strategy, designed experiments and wrote the paper.

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Author notes

    • Seth P. Boudreaux

    Present address: New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA, 70560, USA

  1. These authors contributed equally: Seth P. Boudreaux, Ryan P. Duren.

Affiliations

  1. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, 77030, USA

    • Seth P. Boudreaux
    • , Ryan P. Duren
    • , Steven G. Call
    • , Loc Nguyen
    • , Pablo R. Freire
    •  & Orla M. Conneely
  2. Program in Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, Houston, TX, 77030, USA

    • Seth P. Boudreaux
    •  & Ryan P. Duren
  3. Department of Pediatrics, Section of Hematology-Oncology, Baylor College of Medicine, Houston, TX, 77030, USA

    • Padmini Narayanan
    •  & Michele S. Redell

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The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Orla M. Conneely.

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DOI

https://doi.org/10.1038/s41375-018-0174-1

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