JAK2V617F is the most common mutation in patients with BCR-ABL negative myeloproliferative neoplasms (MPNs). The eradication of JAK2V617F hematopoietic stem cells (HSCs) is critical for achieving molecular remissions and cure. We investigate the distinct effects of two therapies, ruxolitinib (JAK1/2 inhibitor) and interferon-alpha (IFN-α), on the disease-initiating HSC population. Whereas ruxolitinib inhibits Stat5 activation in erythroid progenitor populations, it fails to inhibit this same pathway in HSCs. In contrast, IFN-α has direct effects on HSCs. Furthermore, STAT1 phosphorylation and pathway activation is greater after IFN-α stimulation in Jak2V617F murine HSCs with increased induction of reactive oxygen species, DNA damage and reduction in quiescence after chronic IFN-α treatment. Interestingly, ruxolitinib does not block IFN-α induced reactive oxygen species and DNA damage in Jak2V617F murine HSCs in vivo. This work provides a mechanistic rationale informing how pegylated IFN-α reduces JAK2V617F allelic burden in the clinical setting and may inform future clinical efforts to combine ruxolitinib with pegylated IFN-α in patients with MPN.
Subscribe to Journal
Get full journal access for 1 year
only $102.00 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Ishii T, Bruno E, Hoffman R, Xu M. Involvement of various hematopoietic-cell lineages by the JAK2V617F mutation in polycythemia vera. Blood. 2006;108:3128–34.
Jamieson CH, Gotlib J, Durocher JA, Chao MP, Mariappan MR, Lay M, et al. The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation. Proc Natl Acad Sci USA. 2006;103:6224–9.
Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365:1054–61.
James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434:1144–8.
Kralovics R, Passamonti F, Buser AS, Teo S-S, Tiedt R, Passweg JR, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. New Engl J Med. 2005;352:1779–90.
Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005;7:387–97.
Mullally A, Lane SW, Ball B, Megerdichian C, Okabe R, Al-Shahrour F, et al. Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells. Cancer Cell. 2010;17:584–96.
Mullally A, Lane SW, Brumme K, Ebert BL. Myeloproliferative neoplasm animal models. hematology/oncology. Clinics. 2012;26:1065–81.
Mullally A, Poveromo L, Schneider RK, Al-Shahrour F, Lane SW, Ebert BL. Distinct roles for long-term hematopoietic stem cells and erythroid precursor cells in a murine model of Jak2V617F-mediated polycythemia vera. Blood. 2012;120:166–72.
Reinisch A, Thomas D, Corces MR, Zhang X, Gratzinger D, Hong WJ, et al. A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells. Nat Med. 2016;22:812–21.
Deininger M, Radich J, Burn TC, Huber R, Paranagama D, Verstovsek S. The effect of long-term ruxolitinib treatment on JAK2p.V617F allele burden in patients with myelofibrosis. Blood. 2015;126:1551–4.
Verstovsek S, Mesa RA, Gotlib J, Levy RS, Gupta V, DiPersio JF, et al. A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. New Engl J Med. 2012;366:799–807. 2012/03/01.
Harrison C, Kiladjian J-J, Al-Ali HK, Gisslinger H, Waltzman R, Stalbovskaya V, et al. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. New Engl J Med. 2012;366:787–98.
Vannucchi AM, Kiladjian JJ, Griesshammer M, Masszi T, Durrant S, Passamonti F, et al. Ruxolitinib versus standard therapy for the treatment of polycythemia vera. New Engl J Med. 2015;372:426–35.
Vannucchi AM, Verstovsek S, Guglielmelli P, Griesshammer M, Burn TC, Naim A, et al. Ruxolitinib reduces JAK2 p.V617F allele burden in patients with polycythemia vera enrolled in the RESPONSE study. Ann Hematol. 2017;96:1113–20.
Harrison CN, Vannucchi AM, Kiladjian JJ, Al-Ali HK, Gisslinger H, Knoops L, et al. Long-term findings from COMFORT-II, a phase 3 study of ruxolitinib vs best available therapy for myelofibrosis. Leukemia. 2016;30:1701–7.
Kiladjian JJ, Cassinat B, Chevret S, Turlure P, Cambier N, Roussel M. Pegylated interferon-alfa-2a induces complete hematologic and molecular responses with low toxicity in polycythemia vera. Blood. 2008;112:3065–72.
Quintas-Cardama A, Kantarjian H, Manshouri T, Luthra R, Estrov Z, Pierce S. Pegylated interferon alfa-2a yields high rates of hematologic and molecular response in patients with advanced essential thrombocythemia and polycythemia vera. J Clin Oncol. 2009;27:5418–24.
Them NC, Bagienski K, Berg T, Gisslinger B, Schalling M, Chen D, et al. Molecular responses and chromosomal aberrations in patients with polycythemia vera treated with peg-proline-interferon alpha-2b. Am J Hematol. 2015;90:288–94.
Stauffer Larsen T, Iversen KF, Hansen E, Mathiasen AB, Marcher C, Frederiksen M, et al. Long term molecular responses in a cohort of Danish patients with essential thrombocythemia, polycythemia vera and myelofibrosis treated with recombinant interferon alpha. Leuk Res. 2013;37:1041–5.
Essers MA, Offner S, Blanco-Bose WE, Waibler Z, Kalinke U, Duchosal MA, et al. IFNalpha activates dormant haematopoietic stem cells in vivo. Nature. 2009;458:904–8.
Sato T, Onai N, Yoshihara H, Arai F, Suda T, Ohteki T. Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent exhaustion. Nat Med. 2009;15:696–700.
Kleppe M, Spitzer MH, Li S, Dong L, Papalexi E, Hill C, et al. JAK1 as a convergent regulator of hematopoietic stem cell function and stress hematopoiesis. Blood. 2016;128:722.
Walter D, Lier A, Geiselhart A, Thalheimer FB, Huntscha S, Sobotta MC, et al. Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells. Nature. 2015;520:549–52.
Mullally A, Bruedigam C, Poveromo L, Heidel FH, Purdon A, Vu T, et al. Depletion of Jak2V617F myeloproliferative neoplasm-propagating stem cells by interferon-alpha in a murine model of polycythemia vera. Blood. 2013;121:3692–702.
Chen E, Ahn JS, Massie CE, Clynes D, Godfrey AL, Li J, et al. JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response. Proc Natl Acad Sci. 2014;111:15190–5.
Mikkelsen SU, Kjaer L, Bjorn ME, Knudsen TA, Sorensen AL, Andersen CBL, et al. Safety and efficacy of combination therapy of interferon-alpha2 and ruxolitinib in polycythemia vera and myelofibrosis. Cancer Med. 2018;7:3571–81.
Mikkelsen SU, Kjær L, Skov V, Bjørn ME, Andersen CL, Bjerrum OW, et al. Safety and efficacy of combination therapy of interferon-alpha2 + JAK1-2 inhibitor in the philadelphia-negative chronic myeloproliferative neoplasms. Preliminary results from the Danish combi-trial—an open label, single arm, non-randomized multicenter phase ii study. Blood. 2015;126:824.
Swann JB, Hayakawa Y, Zerafa N, Sheehan KCF, Scott B, Schreiber RD, et al. Type I IFN contributes to NK cell homeostasis, activation, and antitumor function. J Immunol. 2007;178:7540–9. 2007.
Bhagwat N, Koppikar P, Keller M, Marubayashi S, Shank K, Rampal R, et al. Improved targeting of JAK2 leads to increased therapeutic efficacy in myeloproliferative neoplasms. Blood. 2014;123:2075–83.
Vu T, Austin R, Paine Kuhn C, Bruedigam C, Song A, Guignes S, et al. Jak2V617F driven myeloproliferative neoplasm occurs independently of interleukin-3 receptor beta common signaling. Haematologica 2016;101:e77–80.
Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004;3. https://doi.org/10.2202/1544-6115.1027.
Shi JG, Chen X, McGee RF, Landman RR, Emm T, Lo Y, et al. The pharmacokinetics, pharmacodynamics, and safety of orally dosed INCB018424 phosphate in healthy volunteers. J Clin Pharmacol. 2011;51:1644–54.
Van Putten LM, Croon F. The life span of red cells in the rat and the mouse as determined by labeling with DFP32 in vivo. Blood. 1958;13:789.
Koulnis M, Pop R, Porpiglia E, Shearstone JR, Hidalgo D, Socolovsky M Identification and analysis of mouse erythroid progenitors using the CD71/TER119 flow-cytometric assay. J Vis Exp. 2017. https://doi.org/10.3791/55305.
Kato Y, Iwama A, Tadokoro Y, Shimoda K, Minoguchi M, Akira S, et al. Selective activation of STAT5 unveils its role in stem cell self-renewal in normal and leukemic hematopoiesis. J Exp Med. 2005;202:169–79.
Yan D, Hutchison RE, Mohi G. Critical requirement for Stat5 in a mouse model of polycythemia vera. Blood. 2012;119:3539–49.
Rampal R, Al-Shahrour F, Abdel-Wahab O, Patel JP, Brunel JP, Mermel CH, et al. Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis. Blood. 2014;123:e123–133.
Tothova Z, Kollipara R, Huntly BJ, Lee BH, Castrillon DH, Cullen DE, et al. FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. Cell. 2007;128:325–39.
Marty C, Lacout C, Droin N, Le Couedic JP, Ribrag V, Solary E, et al. A role for reactive oxygen species in JAK2 V617F myeloproliferative neoplasm progression. Leukemia. 2013;27:2187–95.
Plo I, Nakatake M, Malivert L, de Villartay JP, Giraudier S, Villeval JL, et al. JAK2 stimulates homologous recombination and genetic instability: potential implication in the heterogeneity of myeloproliferative disorders. Blood. 2008;112:1402–12.
Chen E, Ahn JS, Sykes DB, Breyfogle LJ, Godfrey AL, Nangalia J, et al. RECQL5 suppresses oncogenic JAK2-induced replication stress and genomic instability. Cell Rep. 2015;13:2345–52.
Flach J, Bakker ST, Mohrin M, Conroy PC, Pietras EM, Reynaud D, et al. Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells. Nature. 2014;512:198.
Barzilai A, Yamamoto K. DNA damage responses to oxidative stress. DNA Repair. 2004;3:1109–15.
Tasdogan A, Kumar S, Allies G, Bausinger J, Beckel F, Hofemeister H, et al. DNA damage-induced HSPC malfunction depends on ros accumulation downstream of ifn-1 signaling and bid mobilization. Cell Stem Cell. 2017;20:415
Miyamoto K, Araki KY, Naka K, Arai F, Takubo K, Yamazaki S, et al. Foxo3a is essential for maintenance of the hematopoietic stem cell pool. Cell Stem Cell. 2007;1:101–12.
Eliasson P, Jönsson J-I. The hematopoietic stem cell niche: Low in oxygen but a nice place to be. J Cell Physiol. 2010;222:17–22.
Ito K, Hirao A, Arai F, Matsuoka S, Takubo K, Hamaguchi I, et al. Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells. Nature. 2004;431:997–1002.
Czech J, Cordua S, Weinbergerova B, Baumeister J, Crepcia A, Han L, et al. JAK2V617F but not CALR mutations confer increased molecular responses to interferon-α via JAK1/STAT1 activation. Leukemia. 2019;33:995–1010.
Ianotto JC, Chauveau A, Boyer-Perrard F, Gyan E, Laribi K, Cony-Makhoul P, et al. Benefits and pitfalls of pegylated interferon-alpha2a therapy in patients with myeloproliferative neoplasm-associated myelofibrosis: a French Intergroup of Myeloproliferative neoplasms (FIM) study. Haematologica. 2018;103:438–46.
Boulais PE, Frenette PS. Making sense of hematopoietic stem cell niches. Blood. 2015;125:2621–9.
Ostojic A, Vrhovac R, Verstovsek S. Ruxolitinib: a new JAK1/2 inhibitor that offers promising options for treatment of myelofibrosis. Future Oncol. 2011;7:1035–43.
HIGHLIGHTS OF PRESCRIBING INFORMATION: JAKAFI™ (ruxolitinib) tablets, for oral use. 2011 [cited 2018 10/01/2018]. https://www.accessdata.fda.gov/drugsatfda_docs/label/2011/202192lbl.pdf.
Corbin AS, Agarwal A, Loriaux M, Cortes J, Deininger MW, Druker BJ. Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. J Clin Invest. 2011;121:396–409.
Chu S, McDonald T, Lin A, Chakraborty S, Huang Q, Snyder DS, et al. Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment. Blood. 2011;118:5565–72.
Mullally A, Bruedigam C, Poveromo L, Heidel FH, Purdon A, Vu T, et al. Depletion of Jak2V617F myeloproliferative neoplasm-propagating stem cells by interferon-α in a murine model of polycythemia vera. Blood. 2013;121:3692–702.
Li J, Spensberger D, Ahn JS, Anand S, Beer PA, Ghevaert C, et al. JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia. Blood. 2010;116:1528–38.
Pietras EM, Lakshminarasimhan R, Techner JM, Fong S, Flach J, Binnewies M, et al. Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons. J Exp Med. 2014;211:245–62.
Nieborowska-Skorska M, Maifrede S, Dasgupta Y, Sullivan K, Flis S, Le BV, et al. Ruxolitinib-induced defects in DNA repair cause sensitivity to PARP inhibitors in myeloproliferative neoplasms. Blood. 2017;130:2848–59.
We are grateful for the assistance of the QIMR Berghofer animal house, flow cytometry facility and business development office. We gratefully acknowledge the support of the MPN Research Foundation, MPN Alliance of Australia, CSL Centenary Fellowship, NHMRC, Gordon and Jessie Gilmour Trust, Cure Cancer Australia Foundation (SWL) and Leukaemia Foundation of Australia (SWL and RA). Murine Ropeginterferon-α (mP1101) was a gift from PharmaEssentia.
Conflict of interest
SWL has participated in advisory boards for Novartis. Pegylated murine IFN-α (mP1101) was a gift from Pharmaessentia. FHH has served as a consultant for and has received research funding from Novartis Inc. SJL, NTC, and CWL are employees of PharmaEssentia Co. The other authors declare that they have no conflict of interest.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Austin, R.J., Straube, J., Bruedigam, C. et al. Distinct effects of ruxolitinib and interferon-alpha on murine JAK2V617F myeloproliferative neoplasm hematopoietic stem cell populations. Leukemia (2019). https://doi.org/10.1038/s41375-019-0638-y