Advertisement

Autophagy inhibition potentiates ruxolitinib-induced apoptosis in JAK2V617F cells

  • João Agostinho Machado-Neto
  • Juan Luiz Coelho-Silva
  • Fábio Pires de Souza Santos
  • Priscila Santos Scheucher
  • Paulo Vidal Campregher
  • Nelson Hamerschlak
  • Eduardo Magalhães Rego
  • Fabiola TrainaEmail author
PRECLINICAL STUDIES
  • 19 Downloads

Summary

JAK2V617F can mimic growth factor signaling, leading to PI3K/AKT/mTOR activation and inhibition of autophagy. We hypothesized that selective inhibition of JAK1/2 by ruxolitinib could induce autophagy and limit drug efficacy in myeloproliferative neoplasms (MPN). Therefore, we investigated the effects of ruxolitinib treatment on autophagy-related genes and cellular processes, to determine the potential benefit of autophagy inhibitors plus ruxolitinib in JAK2V617F cells, and to verify the frequency and clinical impact of autophagy-related gene mutations in patients with MPNs. In SET2 JAK2V617F cells, ruxolitinib treatment induced autophagy and modulated 26 out of 79 autophagy-related genes. Ruxolitinib treatment reduced the expressions of important autophagy regulators, including mTOR/p70S6K/4EBP1 and the STAT/BCL2 axis, in a dose- and time-dependent manner. Pharmacological inhibition of autophagy was able to significantly suppress ruxolitinib-induced autophagy and increased ruxolitinib-induced apoptosis. Mutations in autophagy-related genes were found in 15.5% of MPN patients and were associated with increased age and a trend towards worse survival. In conclusion, ruxolitinib induces autophagy in JAK2V617F cells, potentially by modulation of mTOR-, STAT- and BCL2-mediated signaling. This may lead to inhibition of apoptosis. Our results suggest that the combination of ruxolitinib with pharmacological inhibitors of autophagy, such as chloroquine, may be a promising strategy to treat patients with JAK2V617F-mutated MPNs.

Keywords

Autophagy Ruxolitinib Myeloproliferative neoplasms Chloroquine Apoptosis 

Notes

Acknowledgments

The authors would like to thank Dr. Nicola Conran for English revision.

Funding

This study was supported by grants #2014/23092–0, #2017/19864–6, #2014/50947–7, and #2013/08135–2, São Paulo Research Foundation (FAPESP) and grant #402587/2016–2, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Compliance with ethical standards

Ethical approval

Informed consent was obtained from all individual participants included in the study prior to sample collection and the study was approved by the Institutional Review Board.

Competing interests

The authors declare that they have no competing interests.

Supplementary material

10637_2019_812_MOESM1_ESM.pdf (3.8 mb)
ESM 1 Supplementary Fig. 1. Whole gel images of Western blotting analysis. Western blot analysis for protein phosphorylation and expression in total cell extracts from SET2 upon treatment with ruxolitinib and/or 3-methyladenine (3-MA), Bafilomycin A1 (Baf-A1), and chloroquine, as indicated; membranes were reprobed with the antibody for the detection of the respective total protein or actin, and developed with the SuperSignal™ West Dura Extended Duration Substrate system and a Gel Doc XR+ imaging system. Antibodies, merged and unmerged images are indicated. (PDF 3883 kb)
10637_2019_812_MOESM2_ESM.pdf (425 kb)
ESM 2 (PDF 425 kb)

References

  1. 1.
    Helgason GV, Karvela M, Holyoake TL (2011) Kill one bird with two stones: potential efficacy of BCR-ABL and autophagy inhibition in CML. Blood 118:2035–2043CrossRefGoogle Scholar
  2. 2.
    Galluzzi L, Pietrocola F, Bravo-San Pedro JM, Amaravadi RK, Baehrecke EH, Cecconi F, Codogno P, Debnath J, Gewirtz DA, Karantza V et al (2015) Autophagy in malignant transformation and cancer progression. EMBO J 34:856–880CrossRefGoogle Scholar
  3. 3.
    Bellodi C, Lidonnici MR, Hamilton A, Helgason GV, Soliera AR, Ronchetti M, Galavotti S, Young KW, Selmi T, Yacobi R et al (2009) Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells. J Clin Invest 119:1109–1123CrossRefGoogle Scholar
  4. 4.
    Thoennissen NH, Krug UO, Lee DH, Kawamata N, Iwanski GB, Lasho T, Weiss T, Nowak D, Koren-Michowitz M, Kato M et al (2010) Prevalence and prognostic impact of allelic imbalances associated with leukemic transformation of Philadelphia chromosome-negative myeloproliferative neoplasms. Blood 115:2882–2890CrossRefGoogle Scholar
  5. 5.
    Visconte V, Przychodzen B, Han Y, Nawrocki ST, Thota S, Kelly KR, Patel BJ, Hirsch C, Advani AS, Carraway HE et al (2017) Complete mutational spectrum of the autophagy interactome: a novel class of tumor suppressor genes in myeloid neoplasms. Leukemia 31:505–510CrossRefGoogle Scholar
  6. 6.
    Pardanani A, Vannucchi AM, Passamonti F, Cervantes F, Barbui T, Tefferi A (2011) JAK inhibitor therapy for myelofibrosis: critical assessment of value and limitations. Leukemia 25:218–225CrossRefGoogle Scholar
  7. 7.
    Harrison C, Kiladjian JJ, Al-Ali HK, Gisslinger H, Waltzman R, Stalbovskaya V, McQuitty M, Hunter DS, Levy R, Knoops L et al (2012) JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N Engl J Med 366:787–798CrossRefGoogle Scholar
  8. 8.
    Verstovsek S, Mesa RA, Gotlib J, Levy RS, Gupta V, DiPersio JF, Catalano JV, Deininger M, Miller C, Silver RT et al (2012) A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med 366:799–807CrossRefGoogle Scholar
  9. 9.
    Vannucchi AM, Kiladjian JJ, Griesshammer M, Masszi T, Durrant S, Passamonti F, Harrison CN, Pane F, Zachee P, Mesa R et al (2015) Ruxolitinib versus standard therapy for the treatment of polycythemia vera. N Engl J Med 372:426–435CrossRefGoogle Scholar
  10. 10.
    Meyer SC, Keller MD, Chiu S, Koppikar P, Guryanova OA, Rapaport F, Xu K, Manova K, Pankov D, O'Reilly RJ et al (2015) CHZ868, a Type II JAK2 inhibitor, reverses Type I JAK inhibitor persistence and demonstrates efficacy in myeloproliferative neoplasms. Cancer Cell 28:15–28CrossRefGoogle Scholar
  11. 11.
    Thome MP, Filippi-Chiela EC, Villodre ES, Migliavaca CB, Onzi GR, Felipe KB, Lenz G (2016) Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy. J Cell Sci 129:4622–4632CrossRefGoogle Scholar
  12. 12.
    Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212CrossRefGoogle Scholar
  13. 13.
    Thiele J, Kvasnicka HM, Orazi A, Gianelli U, Barbui T, Barosi G, Tefferi A (2017) Primary myelofibrosis. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Arber DA, Hasserjian RP, Beau MML, Orazi A, Siebert R (eds) WHO classification of tumors of haematopoietic and lymphoid tissues. IARC, Lyon, pp 44–50Google Scholar
  14. 14.
    Passamonti F, Cervantes F, Vannucchi AM, Morra E, Rumi E, Pereira A, Guglielmelli P, Pungolino E, Caramella M, Maffioli M et al (2010) A dynamic prognostic model to predict survival in primary myelofibrosis: a study by the IWG-MRT (International Working Group for Myeloproliferative Neoplasms Research and Treatment). Blood 115:1703–1708CrossRefGoogle Scholar
  15. 15.
    Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760CrossRefGoogle Scholar
  16. 16.
    MA DP, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del Angel G, Rivas MA, Hanna M et al (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43:491–498CrossRefGoogle Scholar
  17. 17.
    Ye K, Schulz MH, Long Q, Apweiler R, Ning Z (2009) Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25:2865–2871CrossRefGoogle Scholar
  18. 18.
    Larson DE, Harris CC, Chen K, Koboldt DC, Abbott TE, Dooling DJ, Ley TJ, Mardis ER, Wilson RK, Ding L (2012) SomaticSniper: identification of somatic point mutations in whole genome sequencing data. Bioinformatics 28:311–317CrossRefGoogle Scholar
  19. 19.
    Cibulskis K, Lawrence MS, Carter SL, Sivachenko A, Jaffe D, Sougnez C, Gabriel S, Meyerson M, Lander ES, Getz G (2013) Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat Biotechnol 31:213–219CrossRefGoogle Scholar
  20. 20.
    Wang K, Li M, Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38:e164CrossRefGoogle Scholar
  21. 21.
    Genomes Project C, Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, Korbel JO, Marchini JL, McCarthy S, McVean GA et al (2015) A global reference for human genetic variation. Nature 526:68–74CrossRefGoogle Scholar
  22. 22.
    Sherry ST, Ward MH, Kholodov M, Baker J, Phan L, Smigielski EM, Sirotkin K (2001) dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 29:308–311CrossRefGoogle Scholar
  23. 23.
    Li G, Miskimen KL, Wang Z, Xie XY, Brenzovich J, Ryan JJ, Tse W, Moriggl R, Bunting KD (2010) STAT5 requires the N-domain for suppression of miR15/16, induction of bcl-2, and survival signaling in myeloproliferative disease. Blood 115:1416–1424CrossRefGoogle Scholar
  24. 24.
    Sepulveda P, Encabo A, Carbonell-Uberos F, Minana MD (2007) BCL-2 expression is mainly regulated by JAK/STAT3 pathway in human CD34+ hematopoietic cells. Cell Death Differ 14:378–380CrossRefGoogle Scholar
  25. 25.
    Guo J, Roberts L, Chen Z, Merta PJ, Glaser KB, Shah OJ (2015) JAK2V617F drives Mcl-1 expression and sensitizes hematologic cell lines to dual inhibition of JAK2 and Bcl-xL. PLoS One 10:e0114363CrossRefGoogle Scholar
  26. 26.
    Miao LJ, Huang FX, Sun ZT, Zhang RX, Huang SF, Wang J (2014) Stat3 inhibits Beclin 1 expression through recruitment of HDAC3 in nonsmall cell lung cancer cells. Tumour Biol 35:7097–7103CrossRefGoogle Scholar
  27. 27.
    Laplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell 149:274–293CrossRefGoogle Scholar
  28. 28.
    Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3:542–545CrossRefGoogle Scholar
  29. 29.
    Green DR, Galluzzi L, Kroemer G (2011) Mitochondria and the autophagy-inflammation-cell death axis in organismal aging. Science 333:1109–1112CrossRefGoogle Scholar
  30. 30.
    Takahashi Y, Hori T, Cooper TK, Liao J, Desai N, Serfass JM, Young MM, Park S, Izu Y, Wang HG (2013) Bif-1 haploinsufficiency promotes chromosomal instability and accelerates Myc-driven lymphomagenesis via suppression of mitophagy. Blood 121:1622–1632CrossRefGoogle Scholar
  31. 31.
    Okamoto K (2014) Organellophagy: eliminating cellular building blocks via selective autophagy. J Cell Biol 205:435–445CrossRefGoogle Scholar
  32. 32.
    Can G, Ekiz HA, Baran Y (2011) Imatinib induces autophagy through BECLIN-1 and ATG5 genes in chronic myeloid leukemia cells. Hematology 16:95–99CrossRefGoogle Scholar
  33. 33.
    Bagca BG, Ozalp O, Kurt CC, Mutlu Z, Saydam G, Gunduz C, Avci CB (2016) Ruxolitinib induces autophagy in chronic myeloid leukemia cells. Tumour Biol 37:1573–1579CrossRefGoogle Scholar
  34. 34.
    Ianniciello A, Dumas PY, Drullion C, Guitart A, Villacreces A, Peytour Y, Chevaleyre J, Brunet de la Grange P, Vigon I, Desplat V et al (2017) Chronic myeloid leukemia progenitor cells require autophagy when leaving hypoxia-induced quiescence. Oncotarget 8:96984–96992CrossRefGoogle Scholar
  35. 35.
    Seglen PO, Gordon PB (1982) 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A 79:1889–1892CrossRefGoogle Scholar
  36. 36.
    Heckmann BL, Yang X, Zhang X, Liu J (2013) The autophagic inhibitor 3-methyladenine potently stimulates PKA-dependent lipolysis in adipocytes. Br J Pharmacol 168:163–171CrossRefGoogle Scholar
  37. 37.
    Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y (1998) Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 23:33–42CrossRefGoogle Scholar
  38. 38.
    Maclean KH, Dorsey FC, Cleveland JL, Kastan MB (2008) Targeting lysosomal degradation induces p53-dependent cell death and prevents cancer in mouse models of lymphomagenesis. J Clin Invest 118:79–88CrossRefGoogle Scholar
  39. 39.
    Maycotte P, Aryal S, Cummings CT, Thorburn J, Morgan MJ, Thorburn A (2012) Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy. Autophagy 8:200–212CrossRefGoogle Scholar
  40. 40.
    Slater AF (1993) Chloroquine: mechanism of drug action and resistance in Plasmodium falciparum. Pharmacol Ther 57:203–235CrossRefGoogle Scholar
  41. 41.
    Subramony H, Tangpukdee N, Krudsood S, Poovorawan K, Muangnoicharoen S, Wilairatana P (2016) Evaluation of efficacy of chloroquine for Plasmodium vivax infection using parasite clearance times: a 10-year study and systematic review. Ann Acad Med Singap 45:303–314Google Scholar
  42. 42.
    Goldsmith K (1946) A controlled field trial of SN 7618–5 (chloroquine) for the suppression of malaria. J Malar Inst India 6:311–316Google Scholar
  43. 43.
    Haydu GG (1953) Rheumatoid arthritis therapy; a rationale and the use of chloroquine diphosphate. Am J Med Sci 225:71–75CrossRefGoogle Scholar
  44. 44.
    Loeb F, Clark WM, Coatney GR, Coggeshall LT, Dieuaide FR, Dochez AR, Hakansson EG, Marshall EK Jr, Marvel CS, McCoy OR et al (1946) Acitivity of a new antimalarial agent, chloroquine (SN 7618). J Am Med Assoc 130:1069–1070CrossRefGoogle Scholar
  45. 45.
    Gomez-Puerto MC, Folkerts H, Wierenga AT, Schepers K, Schuringa JJ, Coffer PJ, Vellenga E (2016) Autophagy proteins ATG5 and ATG7 are essential for the maintenance of human CD34(+) hematopoietic stem-progenitor cells. Stem Cells 34:1651–1663CrossRefGoogle Scholar
  46. 46.
    Huang J, Ge M, Lu S, Shi J, Yu W, Li X, Wang M, Zhang J, Feng S, Dong S et al (2016) Impaired autophagy in adult bone marrow CD34+ cells of patients with aplastic anemia: possible pathogenic significance. PLoS One 11:e0149586CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • João Agostinho Machado-Neto
    • 1
    • 2
  • Juan Luiz Coelho-Silva
    • 1
  • Fábio Pires de Souza Santos
    • 3
    • 4
  • Priscila Santos Scheucher
    • 1
  • Paulo Vidal Campregher
    • 3
    • 4
  • Nelson Hamerschlak
    • 3
  • Eduardo Magalhães Rego
    • 1
  • Fabiola Traina
    • 1
    Email author
  1. 1.Department of Medical Images, Hematology and Clinical OncologyUniversity of São Paulo at Ribeirão Preto Medical SchoolRibeirão PretoBrazil
  2. 2.Department of PharmacologyInstitute of Biomedical Sciences of the University of São PauloSão PauloBrazil
  3. 3.Centro de Oncologia e Hematologia Familia Dayan-DaycovalHospital Israelita Albert Einstein São PauloSão PauloBrazil
  4. 4.Instituto de Ensino e PesquisaHospital Israelita Albert Einstein São PauloSão PauloBrazil

Personalised recommendations