Efficacy of ALK5 inhibition in myelofibrosis

Lanzhu Yue

Matthias Bartenstein

Wanke Zhao

Wanting Ho

Xuefeng Wang+11 authors

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^{Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.}

^{Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China.}

^{Department of Oncology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, USA.}

^{Department of Pathology, Peggy and Stephenson Cancer Center, Oklahoma University Health Sciences Center, Oklahoma City, Oklahoma, USA.}

^{Department of Biotherapy, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Immunology and Biotherapy, Key Laboratory of Cancer Prevention and Therapy, Tianjin, PR China.}

^{Translational Science, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.}

^{Department of Hematopathology and Laboratory Medicine,}

^{Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, USA.}

^{Leukemia Center, Memorial Sloan Kettering Cancer Center, New York City, New York, USA.}

Address correspondence to: Pearlie K. Epling-Burnette, Department of Immunology, Malignant Hematology Division, SRB 23033, H. Lee Moffitt Cancer Center, Tampa, Florida 33612, USA. Phone: 813.745.6177; E-mail: gro.tiffom@ettenruB.eilraeP. Or to: Amit Verma, Department of Medicine and Department of Developmental & Molecular Biology, Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Chanin Building, Room 302B, New York, New York 10461, USA. Phone: 718.430.8761; E-mail: ude.uy.nietsnie@amrev.tima. Or to: Zhizhuang Joe Zhao, University of Oklahoma Health Sciences Center, Department of Pathology, 940 Stanton L. Young Blvd, BMSB 451, Oklahoma City, Oklahoma 73104, USA. Phone: 405.271.9344; E-mail: ude.cshuo@oahZ-eoJ.

Authorship note: L. Yue, M. Bartenstein, and W. Zhao contributed equally to this work.

Lanzhu Yue: moc.liamtoh@0111naleuy; Matthias Bartenstein: ude.uy.nietsnie.dhp@nietsnetrab.saihttaM; Wanke Zhao: ude.cshuo@oahz-eknaw; Ying Han: moc.361@93509121631; Cem Murdun: gro.ttiffom@nudruM.meC; Ling Zhang: gro.ttiffom@gnahZ.gniL; Xuefeng Wang: gro.ttiffom@gnaW.gnefeuX; Anjali Budhathoki: gro.eroifetnom@htahduba; Kith Pradhan: ude.uy.nietsnie@nahdarp.htik; Franck Rapaport: gro.ccksm.oibc@tropapar; Huaquan Wang: moc.621@gniolf; Zonghong Shao: moc.anis@gnohgnozoahs; Xiubao Ren: moc.hcumjt@oabuixner; Ulrich Steidl: ude.uy.nietsnie@ldiets.hcirlu; Ross L. Levine: gro.ccksm@renivel; Amit Verma: ude.uy.nietsnie@amrev.timaAddress correspondence to: Pearlie K. Epling-Burnette, Department of Immunology, Malignant Hematology Division, SRB 23033, H. Lee Moffitt Cancer Center, Tampa, Florida 33612, USA. Phone: 813.745.6177; E-mail: gro.tiffom@ettenruB.eilraeP. Or to: Amit Verma, Department of Medicine and Department of Developmental & Molecular Biology, Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Chanin Building, Room 302B, New York, New York 10461, USA. Phone: 718.430.8761; E-mail: ude.uy.nietsnie@amrev.tima. Or to: Zhizhuang Joe Zhao, University of Oklahoma Health Sciences Center, Department of Pathology, 940 Stanton L. Young Blvd, BMSB 451, Oklahoma City, Oklahoma 73104, USA. Phone: 405.271.9344; E-mail: ude.cshuo@oahZ-eoJ.Authorship note: L. Yue, M. Bartenstein, and W. Zhao contributed equally to this work.

Received 2016 Oct 4; Accepted 2017 Feb 1.

Abstract

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPL^W515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPL^W515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPL^W515L and JAK2^V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

Abstract

Acknowledgments

We would like to thank Gary W. Reuther for critical assistance in retrovirus production. We also thank the Microscopy Core and Flow Cytometry Core at the H. Lee Moffitt Cancer Center for assistance with experiments. This work has been supported in part by the Biostatistics Core Facility at the H. Lee Moffitt Cancer Center and Research Institute (P30-CA076292). The studies were supported by the MPN Foundation and Leukemia Lymphoma Society. Amit Verma was supported by the Gottesman Stem Institute.

Acknowledgments

Footnotes

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information:JCI Insight. 2017;2(6):e90932. https://doi.org/10.1172/jci.insight.90932.

Footnotes

References

1. Tefferi A, Vardiman JWClassification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia. 2008;22(1):14–22.[PubMed][Google Scholar]
2. Kralovics R, et al A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005;352(17):1779–1790. doi: 10.1056/NEJMoa051113.] [[PubMed][Google Scholar]
3. Tefferi ANovel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia. 2010;24(6):1128–1138.[Google Scholar]
4. Klampfl T, et al Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369(25):2379–2390. doi: 10.1056/NEJMoa1311347.] [[PubMed][Google Scholar]
5. Tefferi APathogenesis of myelofibrosis with myeloid metaplasia. J Clin Oncol. 2005;23(33):8520–8530. doi: 10.1200/JCO.2004.00.9316.] [[PubMed][Google Scholar]
6. Schmitt-Graeff AH, Nitschke R, Zeiser RThe hematopoietic niche in myeloproliferative neoplasms. Mediators Inflamm. 2015;2015:347270. [Google Scholar]
7. Tefferi A, et al One thousand patients with primary myelofibrosis: the mayo clinic experience. Mayo Clin Proc. 2012;87(1):25–33. doi: 10.1016/j.mayocp.2011.11.001.] [[Google Scholar]
8. Cerquozzi S, Farhadfar N, Tefferi ATreatment of myelofibrosis: a moving target. Cancer J. 2016;22(1):51–61. doi: 10.1097/PPO.0000000000000169.] [[PubMed][Google Scholar]
9. Lataillade JJ, et al Does primary myelofibrosis involve a defective stem cell niche? From concept to evidence. Blood. 2008;112(8):3026–3035. doi: 10.1182/blood-2008-06-158386.] [[PubMed][Google Scholar]
10. Walkley CR, Shea JM, Sims NA, Purton LE, Orkin SHRb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment. Cell. 2007;129(6):1081–1095. doi: 10.1016/j.cell.2007.03.055.] [[Google Scholar]
11. Schepers K, et al Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche. Cell Stem Cell. 2013;13(3):285–299. doi: 10.1016/j.stem.2013.06.009.] [[Google Scholar]
12. Kreipe H, Büsche G, Bock O, Hussein KMyelofibrosis: molecular and cell biological aspects. Fibrogenesis Tissue Repair. 2012;5(Suppl 1):S21. [Google Scholar]
13. Varricchio L, Mancini A, Migliaccio ARPathological interactions between hematopoietic stem cells and their niche revealed by mouse models of primary myelofibrosis. Expert Rev Hematol. 2009;2(3):315–334. doi: 10.1586/ehm.09.17.] [[Google Scholar]
14. Mailloux AW, et al Fibrosis and subsequent cytopenias are associated with basic fibroblast growth factor-deficient pluripotent mesenchymal stromal cells in large granular lymphocyte leukemia. J Immunol. 2013;191(7):3578–3593. doi: 10.4049/jimmunol.1203424.] [[Google Scholar]
15. Ponce CC, de Lourdes Lopes Ferrari Chauffaille M, Ihara SS, Silva MRIncreased angiogenesis in primary myelofibrosis: latent transforming growth factor-β as a possible angiogenic factor. Rev Bras Hematol Hemoter. 2014;36(5):322–328. doi: 10.1016/j.bjhh.2014.07.010.] [[Google Scholar]
16. Chagraoui H, Komura E, Tulliez M, Giraudier S, Vainchenker W, Wendling FProminent role of TGF-beta 1 in thrombopoietin-induced myelofibrosis in mice. Blood. 2002;100(10):3495–3503. doi: 10.1182/blood-2002-04-1133.] [[PubMed][Google Scholar]
17. Zingariello M, et al Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis. Blood. 2013;121(17):3345–3363. doi: 10.1182/blood-2012-06-439661.] [[Google Scholar]
18. Ceglia I, et al Preclinical rationale for TGF-β inhibition as a therapeutic target for the treatment of myelofibrosis. Exp Hematol. 2016;44(12):1138–1155.e4. doi: 10.1016/j.exphem.2016.08.007.] [[Google Scholar]
19. Leask A, Abraham DJTGF-beta signaling and the fibrotic response. FASEB J. 2004;18(7):816–827. doi: 10.1096/fj.03-1273rev.] [[PubMed][Google Scholar]
20. Pohlers D, et al TGF-beta and fibrosis in different organs - molecular pathway imprints. Biochim Biophys Acta. 2009;1792(8):746–756.[PubMed][Google Scholar]
21. Meindl-Beinker NM, Matsuzaki K, Dooley STGF-β signaling in onset and progression of hepatocellular carcinoma. Dig Dis. 2012;30(5):514–523. doi: 10.1159/000341704.] [[PubMed][Google Scholar]
22. Vannucchi AM, et al A pathobiologic pathway linking thrombopoietin, GATA-1, and TGF-beta1 in the development of myelofibrosis. Blood. 2005;105(9):3493–3501. doi: 10.1182/blood-2004-04-1320.] [[PubMed][Google Scholar]
23. Le Bousse-Kerdilès MC, Martyré MC, French INSERM research network on Idiopathic Myelofibrosis Involvement of the fibrogenic cytokines, TGF-beta and bFGF, in the pathogenesis of idiopathic myelofibrosis. Pathol Biol. 2001;49(2):153–157. doi: 10.1016/S0369-8114(00)00021-3.] [[PubMed]
24. Ciurea SO, et al Pivotal contributions of megakaryocytes to the biology of idiopathic myelofibrosis. Blood. 2007;110(3):986–993. doi: 10.1182/blood-2006-12-064626.] [[Google Scholar]
25. Schmitt A, Jouault H, Guichard J, Wendling F, Drouin A, Cramer EMPathologic interaction between megakaryocytes and polymorphonuclear leukocytes in myelofibrosis. Blood. 2000;96(4):1342–1347.[PubMed][Google Scholar]
26. Herbertz S, et al Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway. Drug Des Devel Ther. 2015;9:4479–4499.[Google Scholar]
27. Rampal R, et al Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis. Blood. 2014;123(22):e123–e133. doi: 10.1182/blood-2014-02-554634.] [[Google Scholar]
28. Rodon J, et al First-in-human dose study of the novel transforming growth factor-β receptor I kinase inhibitor LY2157299 monohydrate in patients with advanced cancer and glioma. Clin Cancer Res. 2015;21(3):553–560. doi: 10.1158/1078-0432.CCR-14-1380.] [[Google Scholar]
29. Bhola NE, et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer. J Clin Invest. 2013;123(3):1348–1358. doi: 10.1172/JCI65416.] [[Google Scholar]
30. Zhou L, et al Reduced SMAD7 leads to overactivation of TGF-beta signaling in MDS that can be reversed by a specific inhibitor of TGF-beta receptor I kinase. Cancer Res. 2011;71(3):955–963. doi: 10.1158/0008-5472.CAN-10-2933.] [[Google Scholar]
31. Li C, et al Noncanonical STAT3 activation regulates excess TGF-β1 and collagen I expression in muscle of stricturing Crohn’s disease. J Immunol. 2015;194(7):3422–3431. doi: 10.4049/jimmunol.1401779.] [[Google Scholar]
32. Laklai H, et al Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression. Nat Med. 2016;22(5):497–505. doi: 10.1038/nm.4082.] [[Google Scholar]
33. Prêle CM, Yao E, O’Donoghue RJ, Mutsaers SE, Knight DASTAT3: a central mediator of pulmonary fibrosis? Proc Am Thorac Soc. 2012;9(3):177–182. doi: 10.1513/pats.201201-007AW.] [[PubMed][Google Scholar]
34. Thiele J, Kvasnicka HM, Facchetti F, Franco V, van der Walt J, Orazi AEuropean consensus on grading bone marrow fibrosis and assessment of cellularity. Haematologica. 2005;90(8):1128–1132.[PubMed][Google Scholar]
35. Zhao W, Ho WT, Zhao ZJQuantitative analyses of myelofibrosis by determining hydroxyproline. Stem Cell Investig. 2015;2:2. [Google Scholar]
36. Xing S, et al Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice. Blood. 2008;111(10):5109–5117. doi: 10.1182/blood-2007-05-091579.] [[Google Scholar]
37. Wen QJ, et al Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition. Nat Med. 2015;21(12):1473–1480. doi: 10.1038/nm.3995.] [[Google Scholar]
38. Agarwal A, Morrone K, Bartenstein M, Zhao ZJ, Verma A, Goel SBone marrow fibrosis in primary myelofibrosis: pathogenic mechanisms and the role of TGF-β Stem Cell Investig. 2016;3:5. [Google Scholar]
39. Spangrude GJ, et al P-selectin sustains extramedullary hematopoiesis in the Gata1 low model of myelofibrosis. Stem Cells. 2016;34(1):67–82. doi: 10.1002/stem.2229.] [[PubMed][Google Scholar]
40. Zingariello M, et al A novel interaction between megakaryocytes and activated fibrocytes increases TGF-β bioavailability in the Gata1 (low) mouse model of myelofibrosis. Am J Blood Res. 2015;5(2):34–61.[Google Scholar]
41. Hong SH, et al Rescue of a primary myelofibrosis model by retinoid-antagonist therapy. Proc Natl Acad Sci USA. 2013;110(47):18820–18825. doi: 10.1073/pnas.1318974110.] [[Google Scholar]
42. Ponce CC, de Lourdes F Chauffaille M, Ihara SS, Silva MRThe relationship of the active and latent forms of TGF-β1 with marrow fibrosis in essential thrombocythemia and primary myelofibrosis. Med Oncol. 2012;29(4):2337–2344. doi: 10.1007/s12032-011-0144-1.] [[PubMed][Google Scholar]
43. Shehata M, Schwarzmeier JD, Hilgarth M, Hubmann R, Duechler M, Gisslinger HTGF-beta1 induces bone marrow reticulin fibrosis in hairy cell leukemia. J Clin Invest. 2004;113(5):676–685. doi: 10.1172/JCI19540.] [[Google Scholar]
44. Frenette PS, Pinho S, Lucas D, Scheiermann CMesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine. Annu Rev Immunol. 2013;31:285–316. doi: 10.1146/annurev-immunol-032712-095919.] [[PubMed][Google Scholar]
45. Uccelli A, Moretta L, Pistoia VMesenchymal stem cells in health and disease. Nat Rev Immunol. 2008;8(9):726–736. doi: 10.1038/nri2395.] [[PubMed][Google Scholar]
46. Keating AMesenchymal stromal cells: new directions. Cell Stem Cell. 2012;10(6):709–716. doi: 10.1016/j.stem.2012.05.015.] [[PubMed][Google Scholar]
47. Méndez-Ferrer S, et al Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature. 2010;466(7308):829–834. doi: 10.1038/nature09262.] [[Google Scholar]
48. García-García A, de Castillejo CL, Méndez-Ferrer SBMSCs and hematopoiesis. Immunol Lett. 2015;168(2):129–135. doi: 10.1016/j.imlet.2015.06.020.] [[PubMed][Google Scholar]
49. Shen Y, Nilsson SKBone, microenvironment and hematopoiesis. Curr Opin Hematol. 2012;19(4):250–255. doi: 10.1097/MOH.0b013e328353c714.] [[PubMed][Google Scholar]
50. Schneider RK, et al Activated fibronectin-secretory phenotype of mesenchymal stromal cells in pre-fibrotic myeloproliferative neoplasms. J Hematol Oncol. 2014;7:92. [Google Scholar]
51. Avanzini MA, et al Functional and genetic aberrations of in vitro-cultured marrow-derived mesenchymal stromal cells of patients with classical Philadelphia-negative myeloproliferative neoplasms. Leukemia. 2014;28(8):1742–1745. doi: 10.1038/leu.2014.97.] [[PubMed][Google Scholar]
52. Martinaud C, et al Osteogenic potential of mesenchymal stromal cells contributes to primary myelofibrosis. Cancer Res. 2015;75(22):4753–4765. doi: 10.1158/0008-5472.CAN-14-3696.] [[PubMed][Google Scholar]
53. Han Y, et al Mesenchymal cell reprogramming in experimental MPLW515L mouse model of myelofibrosis. PLoS One. 2017;12(1):e0166014. doi: 10.1371/journal.pone.0166014.] [[Google Scholar]
54. Verstovsek S, et al Role of neoplastic monocyte-derived fibrocytes in primary myelofibrosis. J Exp Med. 2016;213(9):1723–1740. doi: 10.1084/jem.20160283.] [[Google Scholar]
55. Biernacka A, Dobaczewski M, Frangogiannis NGTGF-β signaling in fibrosis. Growth Factors. 2011;29(5):196–202. doi: 10.3109/08977194.2011.595714.] [[Google Scholar]
56. Flanders KCSmad3 as a mediator of the fibrotic response. Int J Exp Pathol. 2004;85(2):47–64. doi: 10.1111/j.0959-9673.2004.00377.x.] [[Google Scholar]
57. Schnabl B, Kweon YO, Frederick JP, Wang XF, Rippe RA, Brenner DAThe role of Smad3 in mediating mouse hepatic stellate cell activation. Hepatology. 2001;34(1):89–100. doi: 10.1053/jhep.2001.25349.] [[PubMed][Google Scholar]
58. Zhao J, et al Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol. 2002;282(3):L585–L593. doi: 10.1152/ajplung.00151.2001.] [[PubMed][Google Scholar]
59. Flanders KC, et al Mice lacking Smad3 are protected against cutaneous injury induced by ionizing radiation. Am J Pathol. 2002;160(3):1057–1068. doi: 10.1016/S0002-9440(10)64926-7.] [[Google Scholar]
60. Dobaczewski M, et al Smad3 signaling critically regulates fibroblast phenotype and function in healing myocardial infarction. Circ Res. 2010;107(3):418–428. doi: 10.1161/CIRCRESAHA.109.216101.] [[Google Scholar]
61. Bujak M, et al Essential role of Smad3 in infarct healing and in the pathogenesis of cardiac remodeling. Circulation. 2007;116(19):2127–2138. doi: 10.1161/CIRCULATIONAHA.107.704197.] [[PubMed][Google Scholar]
62. Saitoh M, et al STAT3 integrates cooperative Ras and TGF-β signals that induce Snail expression. Oncogene. 2016;35(8):1049–1057. doi: 10.1038/onc.2015.161.] [[PubMed][Google Scholar]
63. Liu RY, et al JAK/STAT3 signaling is required for TGF-β-induced epithelial-mesenchymal transition in lung cancer cells. Int J Oncol. 2014;44(5):1643–1651.[PubMed][Google Scholar]
64. Liu M, et al Immunomodulation by mesenchymal stem cells in treating human autoimmune disease-associated lung fibrosis. Stem Cell Res Ther. 2016;7(1):63. doi: 10.1186/s13287-016-0319-y.] [[Google Scholar]
65. Gastinne T, et al Adenoviral-mediated TGF-beta1 inhibition in a mouse model of myelofibrosis inhibit bone marrow fibrosis development. Exp Hematol. 2007;35(1):64–74. doi: 10.1016/j.exphem.2006.08.016.] [[PubMed][Google Scholar]
66. Mascarenhas J, et al Anti-transforming growth factor-β therapy in patients with myelofibrosis. Leuk Lymphoma. 2014;55(2):450–452. doi: 10.3109/10428194.2013.805329.] [[PubMed][Google Scholar]
67. Manshouri T, et al Bone marrow stroma-secreted cytokines protect JAK2 (V617F)-mutated cells from the effects of a JAK2 inhibitor. Cancer Res. 2011;71(11):3831–3840. doi: 10.1158/0008-5472.CAN-10-4002.] [[Google Scholar]
68. Pikman Y, et al MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med. 2006;3(7):e270. doi: 10.1371/journal.pmed.0030270.] [[Google Scholar]
69. Lennon DP, Caplan AIIsolation of human marrow-derived mesenchymal stem cells. Exp Hematol. 2006;34(11):1604–1605. doi: 10.1016/j.exphem.2006.07.014.] [[PubMed][Google Scholar]
70. Santos AR, Duarte CBValidation of internal control genes for expression studies: effects of the neurotrophin BDNF on hippocampal neurons. J Neurosci Res. 2008;86(16):3684–3692. doi: 10.1002/jnr.21796.] [[PubMed][Google Scholar]