Mechanisms of resistance

Activating JAK-mutations confer resistance to FLT3 kinase inhibitors in FLT3-ITD positive AML in vitro and in vivo


An important limitation of FLT3 tyrosine kinase inhibitors (TKIs) in FLT3-ITD positive AML is the development of resistance. To better understand resistance to FLT3 inhibition, we examined FLT3-ITD positive cell lines which had acquired resistance to midostaurin or sorafenib. In 6 out of 23 TKI resistant cell lines we were able to detect a JAK1 V658F mutation, a mutation that led to reactivation of the CSF2RB–STAT5 pathway. Knockdown of JAK1, or treatment with a JAK inhibitor, resensitized cells to FLT3 inhibition. Out of 136 patients with FLT3-ITD mutated AML and exposed to FLT3 inhibitor, we found seven different JAK family mutations in six of the cases (4.4%), including five bona fide, activating mutations. Except for one patient, the JAK mutations occurred de novo (n = 4) or displayed increasing variant allele frequency after exposure to FLT3 TKI (n = 1). In vitro each of the five activating variants were found to induce resistance to FLT3-ITD inhibition, which was then overcome by dual FLT3/JAK inhibition. In conclusion, our data characterize a novel mechanism of resistance to FLT3-ITD inhibition and may offer a potential therapy, using dual JAK and FLT3 inhibition.

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Fig. 1: FLT3-ITD independent STAT5 and CSF2RB phosphorylation/activation confers resistance to FLT3 TKIs in vitro.
Fig. 2: Mutant JAK1 mediates resistance to FLT3 inhibitors that can be overcome by dual inhibition of FLT3 and JAK1/2.
Fig. 3: Dual FLT3 and JAK inhibition overcomes FLT3-TKI resistance mediated by JAK mutations found in FLT3-TKI treated patients.
Fig. 4: Dual FLT3 and JAK inhibition blocks downstream signaling of cells simulating the resistance mechanisms found in FLT3-TKI treated patients.


  1. 1.

    Thiede C, Steudel C, Mohr B, Schaich M, Schäkel U, Platzbecker U, et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood. 2002;99:4326–35.

    CAS  Article  Google Scholar 

  2. 2.

    Schlenk RF, Döhner K, Krauter J, Fröhling S, Corbacioglu A, Bullinger L, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med. 2008;358:1909–18.

    CAS  Article  Google Scholar 

  3. 3.

    Stone RM, DeAngelo DJ, Klimek V, Galinsky I, Estey E, Nimer SD, et al. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood. 2005;105:54–60.

    CAS  Article  Google Scholar 

  4. 4.

    Knapper S, Burnett AK, Littlewood T, Kell WJ, Agrawal S, Chopra R, et al. A phase 2 trial of the FLT3 inhibitor lestaurtinib (CEP701) as first-line treatment for older patients with acute myeloid leukemia not considered fit for intensive chemotherapy. Blood. 2006;108:3262–70.

    CAS  Article  Google Scholar 

  5. 5.

    Borthakur G, Kantarjian H, Ravandi F, Zhang W, Konopleva M, Wright JJ, et al. Phase I study of sorafenib in patients with refractory or relapsed acute leukemias. Haematologica. 2011;96:62–8.

    CAS  Article  Google Scholar 

  6. 6.

    Cortes J, Perl AE, Döhner H, Kantarjian H, Martinelli G, Kovacsovics T, et al. Quizartinib, an FLT3 inhibitor, as monotherapy in patients with relapsed or refractory acute myeloid leukaemia: an open-label, multicentre, single-arm, phase 2 trial. Lancet Oncol. 2018;19:889–903.

    CAS  Article  Google Scholar 

  7. 7.

    Perl AE, Altman JK, Cortes J, Smith C, Litzow M, Baer MR, et al. Selective inhibition of FLT3 by gilteritinib in relapsed/refractory acute myeloid leukemia: a multicenter, first-in-human, open-label, phase 1/2 study. Lancet Oncol. 2017;18:1061–75.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Fiedler W, Serve H, Döhner H, Schwittay M, Ottmann OG, O’Farrell A-M, et al. A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood. 2005;105:986–93.

    CAS  Article  Google Scholar 

  9. 9.

    Cortes JE, Kantarjian HM, Kadia TM, Borthakur G, Konopleva M, Garcia-Manero G, et al. Crenolanib besylate, a type I pan-FLT3 inhibitor, to demonstrate clinical activity in multiply relapsed FLT3-ITD and D835 AML. Clin Oncol. 2016;34:7008–7008.

    Google Scholar 

  10. 10.

    Serve H, Krug U, Wagner R, Sauerland MC, Heinecke A, Brunnberg U, et al. Sorafenib in combination with intensive chemotherapy in elderly patients with acute myeloid leukemia: results from a randomized, placebo-controlled trial. J Clin Oncol Off J Am Clin Oncol. 2013;31:3110–8.

    CAS  Article  Google Scholar 

  11. 11.

    Röllig C, Serve H, Hüttmann A, Noppeney R, Müller-Tidow C, Krug U, et al. Addition of sorafenib versus placebo to standard therapy in patients aged 60 years or younger with newly diagnosed acute myeloid leukaemia (SORAML): a multicentre, phase 2, randomised controlled trial. Lancet Oncol. 2015;16:1691–9.

    Article  CAS  PubMed  Google Scholar 

  12. 12.

    Burchert A, Bug G, Finke J, Stelljes M, Rollig C, Wäsch R, et al. Sorafenib as maintenance therapy post allogeneic stem cell transplantation for FLT3-ITD positive AML: results from the randomized, double-blind, placebo-controlled multicentre sormain trial. Blood. 2018;132:661–661.

    Article  Google Scholar 

  13. 13.

    Mathew NR, Baumgartner F, Braun L, O’Sullivan D, Thomas S, Waterhouse M, et al. Sorafenib promotes graft-versus-leukemia activity in mice and humans through IL-15 production in FLT3-ITD-mutant leukemia cells. Nat Med. 2018;24:282–91.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Stone RM, Mandrekar SJ, Sanford BL, Laumann K, Geyer S, Bloomfield CD, et al. Midostaurin plus chemotherapy for acute myeloid leukemia with a FLT3 mutation. N Engl J Med. 2017;377:454–64.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Perl AE, Martinelli G, Cortes JE, Neubauer A, Berman E, Paolini S, et al. Gilteritinib or chemotherapy for relapsed or refractory FLT3-Mutated AML. N Engl J Med. 2019;381:1728–40.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    von Bubnoff N, Engh RA, Aberg E, Sänger J, Peschel C, Duyster J. FMS-like tyrosine kinase 3-internal tandem duplication tyrosine kinase inhibitors display a nonoverlapping profile of resistance mutations in vitro. Cancer Res. 2009;69:3032–41.

    Article  CAS  Google Scholar 

  17. 17.

    Heidel F, Solem FK, Breitenbuecher F, Lipka DB, Kasper S, Thiede MH, et al. Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain. Blood. 2006;107:293–300.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    von Bubnoff N, Rummelt C, Menzel H, Sigl M, Peschel C, Duyster J. Identification of a secondary FLT3/A848P mutation in a patient with FLT3-ITD-positive blast phase CMML and response to sunitinib and sorafenib. Leukemia. 2010;24:1523–5.

    Article  CAS  Google Scholar 

  19. 19.

    Smith CC, Paguirigan A, Jeschke GR, Lin KC, Massi E, Tarver T, et al. Heterogeneous resistance to quizartinib in acute myeloid leukemia revealed by single-cell analysis. Blood. 2017;130:48–58.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Zhang H, Savage S, Schultz AR, Bottomly D, White L, Segerdell E, et al. Clinical resistance to crenolanib in acute myeloid leukemia due to diverse molecular mechanisms. Nat Commun. 2019;10:244.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Smith CC, Wang Q, Chin C-S, Salerno S, Damon LE, Levis MJ, et al. Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia. Nature. 2012;485:260–3.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    McMahon CM, Ferng T, Canaani J, Wang ES, Morrissette JJD, Eastburn DJ, et al. Clonal selection with RAS pathway activation mediates secondary clinical resistance to selective FLT3 inhibition in acute myeloid leukemia. Cancer Disco. 2019;9:1050–63.

    CAS  Article  Google Scholar 

  23. 23.

    Mizuki M, Fenski R, Halfter H, Matsumura I, Schmidt R, Müller C, et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood. 2000;96:3907–14.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Duyster J, Baskaran R, Wang JY. Src homology 2 domain as a specificity determinant in the c-Abl-mediated tyrosine phosphorylation of the RNA polymerase II carboxyl-terminal repeated domain. Proc Natl Acad Sci USA. 1995;92:1555–9.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Shimoda K, Feng J, Murakami H, Nagata S, Watling D, Rogers NC, et al. Jak1 plays an essential role for receptor phosphorylation and stat activation in response to granulocyte colony-stimulating factor. Blood. 1997;90:597–604.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    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. N Engl J Med. 2005;352:1779–90.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Xiang Z, Zhao Y, Mitaksov V, Fremont DH, Kasai Y, Molitoris A, et al. Identification of somatic JAK1 mutations in patients with acute myeloid leukemia. Blood. 2008;111:4809–12.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Jeong EG, Kim MS, Nam HK, Min CK, Lee S, Chung YJ, et al. Somatic mutations of JAK1 and JAK3 in acute leukemias and solid cancers. Clin Cancer Res J Am Assoc Cancer Res. 2008;14:3716–21.

    CAS  Article  Google Scholar 

  29. 29.

    Mullighan CG, Zhang J, Harvey RC, Collins-Underwood JR, Schulman BA, Phillips LA, et al. JAK mutations in high-risk childhood acute lymphoblastic leukemia. Proc Natl Acad Sci USA. 2009;106:9414–8.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Harvey RC, Mullighan CG, Chen I-M, Wharton W, Mikhail FM, Carroll AJ, et al. Rearrangement of CRLF2 is associated with mutation of JAK kinases, alteration of IKZF1, Hispanic/Latino ethnicity, and a poor outcome in pediatric B-progenitor acute lymphoblastic leukemia. Blood. 2010;115:5312–21.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Quintás-Cardama A, Vaddi K, Liu P, Manshouri T, Li J, Scherle PA, et al. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood. 2010;115:3109–17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Fujii H. Receptor expression is essential for proliferation induced by dimerized Jak kinases. Biochem Biophys Res Commun. 2008 ;370:557–60.

    CAS  Article  Google Scholar 

  33. 33.

    Degryse S, Bock CE, de, Cox L, Demeyer S, Gielen O, Mentens N, et al. JAK3 mutants transform hematopoietic cells through JAK1 activation, causing T-cell acute lymphoblastic leukemia in a mouse model. Blood. 2014;124:3092–100.

    CAS  Article  Google Scholar 

  34. 34.

    Hart S, Goh KC, Novotny-Diermayr V, Hu CY, Hentze H, Tan YC, et al. SB1518, a novel macrocyclic pyrimidine-based JAK2 inhibitor for the treatment of myeloid and lymphoid malignancies. Leukemia. 2011;25:1751–9.

    CAS  Article  Google Scholar 

  35. 35.

    Andraos R, Qian Z, Bonenfant D, Rubert J, Vangrevelinghe E, Scheufler C, et al. Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent. Cancer Discov. 2012;2:512–23.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Haan C, Rolvering C, Raulf F, Kapp M, Drückes P, Thoma G, et al. Jak1 has a dominant role over Jak3 in signal transduction through γc-containing cytokine receptors. Chem Biol. 2011;18:314–23.

    CAS  Article  Google Scholar 

  37. 37.

    Singer JW, Al-Fayoumi S, Ma H, Komrokji RS, Mesa R, Verstovsek S. Comprehensive kinase profile of pacritinib, a nonmyelosuppressive Janus kinase 2 inhibitor. J Exp Pharm. 2016;8:11–9.

    CAS  Article  Google Scholar 

  38. 38.

    Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D, et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature. 2012;481:157–63.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Metzeler KH, Herold T, Rothenberg-Thurley M, Amler S, Sauerland MC, Görlich D, et al. Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia. Blood. 2016;128:686–98.

    CAS  Article  Google Scholar 

  40. 40.

    Bellanger D, Jacquemin V, Chopin M, Pierron G, Bernard OA, Ghysdael J, et al. Recurrent JAK1 and JAK3 somatic mutations in T-cell prolymphocytic leukemia. Leukemia. 2014;28:417–9.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Walters DK, Mercher T, Gu T-L, O’Hare T, Tyner JW, Loriaux M, et al. Activating alleles of JAK3 in acute megakaryoblastic leukemia. Cancer Cell. 2006;10:65–75.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Bouchekioua A, Scourzic L, de Wever O, Zhang Y, Cervera P, Aline-Fardin A, et al. JAK3 deregulation by activating mutations confers invasive growth advantage in extranodal nasal-type natural killer cell lymphoma. Leukemia. 2014;28:338–48.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Flex E, Petrangeli V, Stella L, Chiaretti S, Hornakova T, Knoops L, et al. Somatically acquired JAK1 mutations in adult acute lymphoblastic leukemia. J Exp Med. 2008;205:751–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Malinge S, Ragu C, Della-Valle V, Pisani D, Constantinescu SN, Perez C, et al. Activating mutations in human acute megakaryoblastic leukemia. Blood. 2008;112:4220–6.

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Bains T, Heinrich MC, Loriaux MM, Beadling C, Nelson D, Warrick A, et al. Newly described activating JAK3 mutations in T-cell acute lymphoblastic leukemia. Leukemia. 2012;26:2144–6.

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Kiyoi H, Yamaji S, Kojima S, Naoe T. JAK3 mutations occur in acute megakaryoblastic leukemia both in Down syndrome children and non-Down syndrome adults. Leukemia. 2007;21:574–6.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Hanna DMT, Fellowes A, Vedururu R, Mechinaud F, Hansford JR. A unique case of refractory primary mediastinal B-cell lymphoma with JAK3 mutation and the role for targeted therapy. Haematologica. 2014;99:e156–8.

    Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Genovese G, Kähler AK, Handsaker RE, Lindberg J, Rose SA, Bakhoum SF, et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N Engl J Med. 2014;371:2477–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Jaiswal S, Fontanillas P, Flannick J, Manning A, Grauman PV, Mar BG, et al. Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med. 2014;371:2488–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Levis M, Brown P, Smith BD, Stine A, Pham R, Stone R, et al. Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors. Blood. 2006;108:3477–83.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Knapper S, Mills KI, Gilkes AF, Austin SJ, Walsh V, Burnett AK. The effects of lestaurtinib (CEP701) and PKC412 on primary AML blasts: the induction of cytotoxicity varies with dependence on FLT3 signaling in both FLT3-mutated and wild-type cases. Blood. 2006;108:3494–503.

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Mony U, Jawad M, Seedhouse C, Russell N, Pallis M. Resistance to FLT3 inhibition in an in vitro model of primary AML cells with a stem cell phenotype in a defined microenvironment. Leukemia. 2008;22:1395–401.

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Sung PJ, Sugita M, Koblish H, Perl AE, Carroll M. Hematopoietic cytokines mediate resistance to targeted therapy in FLT3-ITD acute myeloid leukemia. Blood Adv. 2019;3:1061–72.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Patel AB, Pomicter AD, Yan D, Eiring AM, Antelope O, Schumacher JA, et al. Dasatinib overcomes stroma-based resistance to the FLT3 inhibitor quizartinib using multiple mechanisms. Leukemia. 2020;34:2981–91.

  55. 55.

    Weisberg E, Liu Q, Nelson E, Kung AL, Christie AL, Bronson R, et al. Using combination therapy to override stromal-mediated chemoresistance in mutant FLT3-positive AML: synergism between FLT3 inhibitors, dasatinib/multi-targeted inhibitors and JAK inhibitors. Leukemia. 2012;26:2233–44.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Cook AM, Li L, Ho Y, Lin A, Li L, Stein A, et al. Role of altered growth factor receptor-mediated JAK2 signaling in growth and maintenance of human acute myeloid leukemia stem cells. Blood. 2014;123:2826–37.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Ikezoe T, Kojima S, Furihata M, Yang J, Nishioka C, Takeuchi A, et al. Expression of p-JAK2 predicts clinical outcome and is a potential molecular target of acute myelogenous leukemia. Int J Cancer. 2011;129:2512–21.

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Zhou J, Bi C, Janakakumara JV, Liu S-C, Chng W-J, Tay K-G, et al. Enhanced activation of STAT pathways and overexpression of survivin confer resistance to FLT3 inhibitors and could be therapeutic targets in AML. Blood. 2009;113:4052–62.

    CAS  Article  PubMed  Google Scholar 

  59. 59.

    Ikezoe T, Yang J, Nishioka C, Kojima S, Takeuchi A, Phillip Koeffler H, et al. Inhibition of signal transducer and activator of transcription 5 by the inhibitor of janus kinases stimulates dormant human leukemia CD34+ /CD38- cells and sensitizes them to antileukemia agents. Int J Cancer. 2011;128:2317–25.

    CAS  Article  PubMed  Google Scholar 

  60. 60.

    Tyner JW, Deininger MW, Loriaux MM, Chang BH, Gotlib JR, Willis SG, et al. RNAi screen for rapid therapeutic target identification in leukemia patients. Proc Natl Acad Sci. 2009;106:8695–700.

    CAS  Article  PubMed  Google Scholar 

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This work was supported by a grant to NvB from the Deutsche Forschungsgemeinschaft No. BU 2508/4–1, by a grant to JD and NvB from the Bundesministerium für Bildung und Forschung (NGFNplus) and by a grant to JD and NvB from the José Carreras Stiftung No. 106682. Proofreading was performed by Dr. Marie Follo

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NvB, JD, and CR designed experiments and wrote the manuscript. CR, SPG, AC, and CE performed experiments. MM, KD, FH, TF, and TH provided patient samples and patient data. TF provided constructs.

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Correspondence to Nikolas von Bubnoff.

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NvB received honoraria from Amgen, Astra Zeneca, BMS and Novartis, and research funding from Novartis. JD received honoraria from Novartis. The authors have no additional financial interests.

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Rummelt, C., Gorantla, S.P., Meggendorfer, M. et al. Activating JAK-mutations confer resistance to FLT3 kinase inhibitors in FLT3-ITD positive AML in vitro and in vivo. Leukemia (2020).

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