Advances in Clinical and Experimental Medicine
2015, vol. 24, nr 2, March-April, p. 195–202
Publication type: original article
Pro-Apoptotic Activity of Ruxolitinib Alone and in Combination with Hydroxyurea, Busulphan, and PI3K/mTOR Inhibitors in JAK2-Positive Human Cell Lines
1 Laboratory of Clinical and Transplant Immunology and Genetics, Copernicus Memorial Hospital, Łódź, Poland
2 Department of Experimental Hematology, Medical University of Lodz, Poland
3 Department of Hematology, Medical University of Lodz, Poland
Background. The JAK2V617F mutation plays a crucial role in the pathogenesis of myeloproliferative neoplasms (MPNs). Inhibition of JAK2 activity by ruxolitinib (RX) results in growth inhibition and apoptosis of cells carrying the JAK2V617F mutation however the exact mechanisms regulating apoptosis have not been fully elucidated.
Objectives. This study assessed the potential cytotoxicity of RX against JAK2-positive human cell lines (SET-2 and HEL), either alone or in combination with hydroxyurea, busulphan, rapamycin or LY294002.
Material and Methods. Cell viability, the apoptosis rate (annexin-V staining), drop of mitochondrial transmembrane potential (Δψm) and caspase activation, were measured using flow cytometry. Additionally, the expression of several apoptosis-regulating proteins was evaluated.
Results. RX showed cytotoxicity against both SET-2 and HEL cell lines. The main mechanism of this action was apoptosis, with significant drop of Δψm, caspase-3 and -9 activation, and moderate activation of caspase-8 (only for SET-2 cells). Corresponding to enhanced apoptosis, the expression levels of some apoptosis-regulating proteins were changed, the most pronounced in both cell lines being up-regulation of Bax and down-regulation of Bcl-2 proteins. Additionally, up-regulation of Bak and Bad (SET-2) and down-regulation of Mcl-1 (HEL) were observed. Of the studied compounds, a combination of RX + LY294002 induced the greatest cytotoxicity in both SET-2 and HEL cell lines, and rapamycin the least.
Conclusion. . This study shows that the combination of RX and a PI3K kinase inhibitor provokes a significant pro-apoptotic effect in JAK2V617F mutated cells, which may justify the beginning of clinical trials based on the combination of these drugs.
ruxolitinib, hydroxyurea, busulphan, rapamycin, LY294002.
- James C, Ugo V, Le Couédic JP, Staerk J, Delhommeau F, Lacout C, Garçon L, Raslova H, Berger R, Bennaceur-Griscelli A, Villeval JL, Constantinescu SN, Casadevall N, Vainchenker W: A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005, 434, 1144–1148.
- Treliński J, Robak T: JAK inhibitors: pharmacology and clinical activity in chronic myeloprolipherative neoplasms. Curr Med Chem 2013, 20, 1147–1161.
- Harrison C, Vannucchi AM: Ruxolitinib: a potent and selective Janus kinase 1 and 2 inhibitor in patients with myelofibrosis. An update for clinicians. Ther Adv Hematol 2012, 3, 341–354.
- Verstovsek S: Ruxolitinib: an oral Janus kinase 1 and Janus kinase 2 inhibitor in the management of myelofibrosis. Postgrad Med 2013, 125, 128–135.
- Mascarenhas J, Mughal TI, Verstovsek S: Biology and clinical management of myeloproliferative neoplasms and development of the JAK inhibitor ruxolitinib. Curr Med Chem 2012, 19, 4399–4413.
- Quintás-Cardama A, Vaddi K, Liu P, Manshouri T, Li J, Scherle PA, Caulder E, Wen X, Li Y, Waeltz P, Rupar M, Burn T, Lo Y, Kelley J, Covington M, Shepard S, Rodgers JD, Haley P, Kantarjian H, Fridman JS, Verstovsek S: Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood 2010, 115, 3109–3117.
- Will B, Siddiqi T, Jordà MA, Shimamura T, Luptakova K, Staber PB, Costa DB, Steidl U, Tenen DG, Kobayashi S: Apoptosis induced by JAK2 inhibition is mediated by Bim and enhanced by the BH3 mimetic ABT-737 in JAK2 mutant human erythroid cells. Blood 2010, 115, 2901–2909.
- Rubert J, Qian Z, Andraos R, Guthy DA, Radimerski T: Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival. BMC Cancer 2011, 11, 24.
- Baffert F, Régnier CH, De Pover A, Pissot-Soldermann C, Tavares GA, Blasco F, Brueggen J, Chène P, Drueckes P, Erdmann D, Furet P, Gerspacher M, Lang M, Ledieu D, Nolan L, Ruetz S, Trappe J, Vangrevelinghe E, Wartmann M, Wyder L, Hofmann F, Radimerski T: Potent and selective inhibition of polycythemia by the quinoxaline JAK2 inhibitor NVP-BSK805. Mol Cancer Ther 2010, 9, 1945–1955.
- Bumm TG, Elsea C, Corbin AS, Loriaux M, Sherbenou D: Characterization of murine JAK2V617F-positive myeloproliferative disease. Cancer Res 2006, 66, 11156–11165.
- Bartalucci N, Guglielmelli P, Vannucchi AM: Rationale for targeting the PI3K/Akt/mTOR pathway in myeloproliferative neoplasms. Clin Lymphoma Myeloma Leuk 2013, 13, Suppl 2, 307–309.
- Bogani C, Bartalucci N, Martinelli S, Tozzi L, Guglielmelli P, Bosi A, Vannucchi AM: Associazione Italiana per la Ricerca sul Cancro AGIMM Gruppo Italiano Malattie Mieloproliferative. mTOR inhibitors alone and in combination with JAK2 inhibitors effectively inhibit cells of myeloproliferative neoplasms. PLoS One 2013, 8, e54826.
- Bartalucci N, Tozzi L, Bogani C, Martinelli S, Rotunno G, Villeval JL, Vannucchi AM: Co-targeting the PI3K/ /mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms. J Cell Mol Med 2013, 17, 1385–1396.
- Guglielmelli P, Barosi G, Rambaldi A, Marchioli R, Masciulli A, Tozzi L, Biamonte F, Bartalucci N, Gattoni E, Lupo ML, Finazzi G, Pancrazzi A, Antonioli E, Susini MC, Pieri L, Malevolti E, Usala E, Occhini U, Grossi A, Caglio S, Paratore S, Bosi A, Barbui T, Vannucchi AM: AIRC-Gruppo Italiano Malattie Mieloproliferative (AGIMM) investigators. Safety and efficacy of everolimus, a mTOR inhibitor, as single agent in a phase 1/2 study in patients with myelofibrosis. Blood 2011, 118, 2069–2076.
- Janus A, Linke A, Cebula B, Robak T, Smolewski P: Rapamycin, the mTOR kinase inhibitor, sensitizes acute myeloid leukemia cells, HL-60 cells, to the cytotoxic effect of arabinozide cytarabine. Anti-Cancer Drugs 2009, 20, 693–701.
- Vannucchi AM, Lasho TL, Guglielmelli P, Biamonte F, Pardanani A, Pereira A, Finke C, Score J, Gangat N, Mannarelli C, Ketterling RP, Rotunno G, Knudson RA, Susini MC, Laborde RR, Spolverini A, Pancrazzi A, Pieri L, Manfredini R, Tagliafico E, Zini R, Jones A, Zoi K, Reiter A, Duncombe A, Pietra D, Rumi E, Cervantes F, Barosi G, Cazzola M, Cross NC, Tefferi A: Mutations and prognosis in primary myelofibrosis. Leukemia 2013, 27, 1861–1869.
- Odenike O: Beyond JAK inhibitor therapy in myelofibrosis. Hematology Am Soc Hematol Educ Program 2013, 2013, 545–552.
- Vicari L, Martinetti D, Buccheri S, Colarossi C, Aiello E, Stagno F, Villari L, Cavalli M, Di Raimondo F, Gulisano M, De Maria R, Vigneri P: Increased phospho-mTOR expression in megakaryocytic cells derived from CD34+ progenitors of essential thrombocythaemia and myelofibrosis patients. Br J Haematol 2012, 159, 237–240.
- Vannucchi AM, Bogani C, Bartalucci N: Inhibition of PI3K/Akt and/or mTOR Inhibit the Growth of Cells of Myeloproliferative Neoplasms and Synergize with JAK2 Inhibitor and Interferon. ASH International Conference. Blood 2011, 118, 1638–1639.
- Gozgit JM, Bebernitz G, Patil P, Ye M, Parmentier J, Wu J, Su N, Wang T, Ioannidis S, Davies A, Huszar D, Zinda M: Effects of the JAK2 inhibitor, AZ960, on Pim/BAD/BCL-xL survival signaling in the human JAK2 V617F cell line SET-2. J Biol Chem 2008, 283, 32334–32343.
- Baker SJ, Rane SG, Reddy EP: Hematopoietic cytokine receptor signaling. Oncogene 2007, 26, 6724–6737.
- Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF: Fetal anemia and apoptosis of red cell progenitors in Stat5a−/−5b−/− mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 1999, 98, 181–191.
- Adams JM, Cory S: The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 2007, 26, 1324– –1337.
- Ley R, Ewings KE, Hadfield K, Cook SJ: Regulatory phosphorylation of Bim: sorting out the ERK from the JNK. Cell Death Differ 2005, 12, 1008–1014.
- Treliński J, Chojnowski K, Cebula-Obrzut B, Smolewski P: Impaired apoptosis of megakaryocytes and bone marrow mononuclear cells in essential thrombocythemia: correlation with JAK2V617F mutational status and cytoreductive therapy. Med Oncol 2012, 29, 2388–2395.
- Opferman JT, Iwasaki H, Ong CC, Suh H, Mizuno S, Akashi K, Korsmeyer SJ: Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science 2005, 307, 1101–1104.
- Kim R: Unknotting the roles of Bcl-2 and Bcl-xL in cell death. Biochem Biophys Res Commun 2005, 29, 333, 336–343.
- Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ: Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 1995, 27, 80, 285–291.
- Green DR, Kroemer G. Pharmacological manipulation of cell death: clinical applications in sight? J Clin Invest 2005, 115, 2610–2617.