FLT3-ITD mutations occur in 20–30% of AML patients and are associated with aggressive disease. Patients with relapsed FLT3-mutated disease respond well to 2nd generation FLT3 TKIs but inevitably relapse within a short timeframe. In this setting, until overt relapse occurs, the bone marrow microenvironment facilitates leukemia cell survival despite continued on-target inhibition. We demonstrate that human bone marrow derived conditioned medium (CM) protects FLT3-ITD+ AML cells from the 2nd generation FLT3 TKI quizartinib and activates STAT3 and STAT5 in leukemia cells. Extrinsic activation of STAT5 by CM is the primary mediator of leukemia cell resistance to FLT3 inhibition. Combination treatment with quizartinib and dasatinib abolishes STAT5 activation and significantly reduces the IC50 of quizartinib in FLT3-ITD+ AML cells cultured in CM. We demonstrate that CM protects FLT3-ITD+ AML cells from the inhibitory effects of quizartinib on glycolysis and that this is partially reversed by treating cells with the combination of quizartinib and dasatinib. Using a doxycycline-inducible STAT5 knockdown in the FLT3-ITD+ MOLM-13 cell line, we show that dasatinib-mediated suppression of leukemia cell glycolytic activity is STAT5-independent and provide a preclinical rationale for combination treatment with quizartinib and dasatinib in FLT3-ITD+ AML.
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.
Small D, Levenstein M, Kim E, Carow C, Amin S, Rockwell P, et al. STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells. Proc Natl Acad Sci USA. 1994;91:459–63.
Beaudin AE, Boyer SW, Forsberg EC. Flk2/Flt3 promotes both myeloid and lymphoid development by expanding non-self-renewing multipotent hematopoietic progenitor cells. Exp Hematol. 2014;42:218–29.e4.
Dehmel U, Quentmeier H, Drexler HG. Effects of FLT3 ligand on human leukemia cells. II. Agonistic and antagonistic effects of other cytokines. Leukemia. 1996;10:271–8.
Hannum C, Culpepper J, Campbell D, Mcclanahan T, Zurawski S, Bazan JF, et al. Ligand for Flt3 Flk2 receptor tyrosine kinase regulates growth of hematopoietic stem-cells and is encoded by variant Rnas. Nature. 1994;368:643–8.
Drexler HG. Expression of FLT3 receptor and response to FLT3 ligand by leukemic cells. Leukemia. 1996;10:588–99.
Smith CC, Wang Q, Chin CS, 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.
Kiyoi H, Naoe T, Nakano Y, Yokota S, Minami S, Miyawaki S, et al. Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia. Blood. 1999;93:3074–80.
Kiyoi H, Ohno R, Ueda R, Saito H, Naoe T. Mechanism of constitutive activation of FLT3 with internal tandem duplication in the juxtamembrane domain. Oncogene. 2002;21:2555–63.
Choudhary C, Brandts C, Schwable J, Tickenbrock L, Sargin B, Ueker A, et al. Activation mechanisms of STAT5 by oncogenic Flt3-ITD. Blood. 2007;110:370–4.
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.
Fischer T, Stone RM, Deangelo DJ, Galinsky I, Estey E, Lanza C, et al. Phase IIB trial of oral midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high-risk myelodysplastic syndrome with either wild-type or mutated FLT3. J Clin Oncol. 2010;28:4339–45.
Short NJ, Kantarjian H, Ravandi F, Daver N. Emerging treatment paradigms with FLT3 inhibitors in acute myeloid leukemia. Ther Adv Hematol. 2019;10:2040620719827310.
Cortes JE, Khaled S, Martinelli G, Perl AE, Ganguly S, Russell N, et al. Quizartinib versus salvage chemotherapy in relapsed or refractory FLT3-ITD acute myeloid leukaemia (QuANTUM-R): a multicentre, randomised, controlled, open-label, phase 3 trial. Lancet Oncol. 2019;20:984–97.
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.
Alvarado Y, Kantarjian HM, Luthra R, Ravandi F, Borthakur G, Garcia-Manero G, et al. Treatment with FLT3 inhibitor in patients with FLT3-mutated acute myeloid leukemia is associated with development of secondary FLT3-tyrosine kinase domain mutations. Cancer. 2014;120:2142–9.
Pratz KW, Sato T, Murphy KM, Stine A, Rajkhowa T, Levis M. FLT3-mutant allelic burden and clinical status are predictive of response to FLT3 inhibitors in AML. Blood. 2010;115:1425–32.
Antar AI, Otrock ZK, Jabbour E, Mohty M, Bazarbachi A. FLT3 inhibitors in acute myeloid leukemia: ten frequently asked questions. Leukemia. 2020;34:682–96.
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.
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.
Berger A, Hoelbl-Kovacic A, Bourgeais J, Hoefling L, Warsch W, Grundschober E, et al. PAK-dependent STAT5 serine phosphorylation is required for BCR-ABL-induced leukemogenesis. Leukemia. 2014;28:629–41.
Eiring AM, Kraft IL, Page BD, O’Hare T, Gunning PT, Deininger MW. STAT3 as a mediator of BCR-ABL1-independent resistance in chronic myeloid leukemia. Leuk Suppl. 2014;3(Suppl 1):S5–6.
Traer E, MacKenzie R, Snead J, Agarwal A, Eiring AM, O’Hare T, et al. Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors. Leukemia. 2012;26:1140–3.
Quentmeier H, Reinhardt J, Zaborski M, Drexler HG. FLT3 mutations in acute myeloid leukemia cell lines. Leukemia. 2003;17:120–4.
Wu H, Hu C, Wang A, Weisberg EL, Wang W, Chen C, et al. Ibrutinib selectively targets FLT3-ITD in mutant FLT3-positive AML. Leukemia. 2016;30:754–7.
Traer E, Martinez J, Javidi-Sharifi N, Agarwal A, Dunlap J, English I, et al. FGF2 from marrow microenvironment promotes resistance to FLT3 Inhibitors in acute myeloid leukemia. Cancer Res. 2016;76:6471–82.
Weisberg E, Liu Q, Zhang X, Nelson E, Sattler M, Liu F, et al. Selective Akt inhibitors synergize with tyrosine kinase inhibitors and effectively override stroma-associated cytoprotection of mutant FLT3-positive AML cells. PLoS ONE. 2013;8:e56473.
Weisberg EL, Puissant A, Stone R, Sattler M, Buhrlage SJ, Yang J, et al. Characterization of midostaurin as a dual inhibitor of FLT3 and SYK and potentiation of FLT3 inhibition against FLT3-ITD-driven leukemia harboring activated SYK kinase. Oncotarget. 2017;8:52026–44.
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.
Fang Y, Zhong L, Lin M, Zhou X, Jing H, Ying M, et al. MEK/ERK dependent activation of STAT1 mediates dasatinib-induced differentiation of acute myeloid leukemia. PLoS ONE. 2013;8:e66915.
Gallipoli P, Giotopoulos G, Tzelepis K, Costa ASH, Vohra S, Medina-Perez P, et al. Glutaminolysis is a metabolic dependency in FLT3(ITD) acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition. Blood. 2018;131:1639–53.
Fletcher L, Joshi S, Traer E. Profile of quizartinib for the treatment of adult patients with relapsed/refractory FLT3-ITD-positive acute myeloid leukemia: evidence to date. Cancer Manag Res. 2020;2020:151–63.
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.
Dumas PY, Naudin C, Martin-Lanneree S, Izac B, Casetti L, Mansier O, et al. Hematopoietic niche drives FLT3-ITD acute myeloid leukemia resistance to quizartinib via STAT5-and hypoxia-dependent upregulation of AXL. Haematologica. 2019;104:2017–27.
Bewry NN, Nair RR, Emmons MF, Boulware D, Pinilla-Ibarz J, Hazlehurst LA. Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance. Mol Cancer Ther. 2008;7:3169–75.
Eiring AM, Page BDG, Kraft IL, Mason CC, Vellore NA, Resetca D, et al. Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia. Leukemia. 2015;29:586–97.
Kuepper MK, Butow M, Herrmann O, Ziemons J, Chatain N, Maurer A, et al. Stem cell persistence in CML is mediated by extrinsically activated JAK1-STAT3 signaling. Leukemia. 2019;33:1964–77.
Pricola KL, Kuhn NZ, Haleem-Smith H, Song Y, Tuan RS. Interleukin-6 maintains bone marrow-derived mesenchymal stem cell stemness by an ERK1/2-dependent mechanism. J Cell Biochem. 2009;108:577–88.
Manshouri T, Estrov Z, Quintas-Cardama A, Burger J, Zhang Y, Livun A, et al. Bone marrow stroma-secreted cytokines protect JAK2(V617F)-mutated cells from the effects of a JAK2 inhibitor. Cancer Res. 2011;71:3831–40.
Gordon PM, Dias S, Williams DA. Cytokines secreted by bone marrow stromal cells protect c-KIT mutant AML cells from c-KIT inhibitor-induced apoptosis. Leukemia. 2014;28:2257–60.
Park IK, Mundy-Bosse B, Whitman SP, Zhang X, Warner SL, Bearss DJ, et al. Receptor tyrosine kinase Axl is required for resistance of leukemic cells to FLT3-targeted therapy in acute myeloid leukemia. Leukemia. 2015;29:2382–9.
Furukawa M, Ohkawara H, Ogawa K, Ikeda K, Ueda K, Shichishima-Nakamura A, et al. Autocrine and paracrine interactions between multiple myeloma cells and bone marrow stromal cells by growth arrest-specific gene 6 cross-talk with interleukin-6. J Biol Chem. 2017;292:4280–92.
Ghosh AK, Secreto C, Boysen J, Sassoon T, Shanafelt TD, Mukhopadhyay D, et al. The novel receptor tyrosine kinase Axl is constitutively active in B-cell chronic lymphocytic leukemia and acts as a docking site of nonreceptor kinases: implications for therapy. Blood. 2011;117:1928–37.
Meyer AS, Miller MA, Gertler FB, Lauffenburger DA. The receptor AXL diversifies EGFR signaling and limits the response to EGFR-targeted inhibitors in triple-negative breast cancer cells. Sci Signal. 2013;6:ra66.
Vouri M, Croucher DR, Kennedy SP, An Q, Pilkington GJ, Hafizi S. Axl-EGFR receptor tyrosine kinase hetero-interaction provides EGFR with access to pro-invasive signalling in cancer cells. Oncogenesis. 2016;5:e266.
Goruppi S, Ruaro E, Varnum B, Schneider C. Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts. Mol Cell Biol. 1997;17:4442–53.
Gay CM, Balaji K, Byers LA. Giving AXL the axe: targeting AXL in human malignancy. Br J Cancer. 2017;116:415–23.
Davis MI, Hunt JP, Herrgard S, Ciceri P, Wodicka LM, Pallares G, et al. Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol. 2011;29:1046–51.
Kitagawa D, Yokota K, Gouda M, Narumi Y, Ohmoto H, Nishiwaki E, et al. Activity-based kinase profiling of approved tyrosine kinase inhibitors. Genes Cells. 2013;18:110–22.
Rankin EB, Fuh KC, Castellini L, Viswanathan K, Finger EC, Diep AN, et al. Direct regulation of GAS6/AXL signaling by HIF promotes renal metastasis through SRC and MET. Proc Natl Acad Sci USA. 2014;111:13373–8.
Jin Y, Cai Q, Shenoy AK, Lim S, Zhang Y, Charles S, et al. Src drives the Warburg effect and therapy resistance by inactivating pyruvate dehydrogenase through tyrosine-289 phosphorylation. Oncotarget. 2016;7:25113–24.
Zhang J, Wang S, Jiang B, Huang L, Ji Z, Li X, et al. c-Src phosphorylation and activation of hexokinase promotes tumorigenesis and metastasis. Nat Commun. 2017;8:13732.
DeNicola GM, Cantley LC. Cancer’s fuel choice: New Flavors for a picky eater. Mol Cell. 2015;60:514–23.
Jin L, Chun J, Pan C, Alesi GN, Li D, Magliocca KR, et al. Phosphorylation-mediated activation of LDHA promotes cancer cell invasion and tumour metastasis. Oncogene. 2017;36:3797–806.
Banerjee M, Cui X, Li Z, Yu H, Cai L, Jia X, et al. Na/K-ATPase Y260 phosphorylation-mediated Src regulation in control of aerobic glycolysis and tumor growth. Sci Rep. 2018;8:12322.
Cheng LC, Chen YL, Cheng AN, Lee AY, Cho CY, Huang JS, et al. AXL phosphorylates and up-regulates TNS2 and its implications in IRS-1-associated metabolism in cancer cells. J Biomed Sci. 2018;25:80.
Simsek T, Kocabas F, Zheng J, Deberardinis RJ, Mahmoud AI, Olson EN, et al. The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche. Cell Stem Cell. 2010;7:380–90.
Moschoi R, Imbert V, Nebout M, Chiche J, Mary D, Prebet T, et al. Protective mitochondrial transfer from bone marrow stromal cells to acute myeloid leukemic cells during chemotherapy. Blood. 2016;128:253–64.
Jain N, Keating M, Thompson P, Ferrajoli A, Burger J, Borthakur G, et al. Ibrutinib and venetoclax for First-line treatment of CLL. N Engl J Med. 2019;380:2095–103.
Luskin M, Murakami MA, Stevenson KE, Wadleigh M, McMasters M, Winter P, et al. A phase I Study of Asciminib (ABL001) in combination with dasatinib and prednisone for untreated BCR-ABL1-positive ALL in older adults. Blood. 2019;134(Suppl_1):3879.
DeAngelo DJ, Mauro MJ, Kim D-W, Cortes J, Réa D, Hughes TP, et al. Combination of asciminib + nilotinib or asciminib + dasatinib in previously treated chronic myeloid leukemia (CML) patients: phase 1 study results. Clin Lymphoma Myeloma Leuk. 2019;19:S290–1.
This work was supported by the National Cancer Institute (NCI) at the National Institutes of health (NIH) through grant R21CA20593601 (MWD), the V Foundation for Cancer Research Translational Research Grant T2017–008 (MWD), NIH grant R01CA178397 (MWD and TOH), and the American Society of Hematology Research Training Award for Fellows (ABP). DY is supported by the International Award from the Lady Tata Memorial Trust. AME is supported by NIH grant 1K22CA216008. We thank the Metabolic Phenotyping Core Facility at the University of Utah for assistance with Seahorse experiments. This work was funded in part by the University of Utah Flow Cytometry Core Facility and the NCI through award 5P30CA042014–24 awarded to the Huntsman Cancer Institute and the National Center for Research Resources of the NIH award 1S10RR026802–01.
Conflict of interest
MWD reports research funding from and is a paid advisory board member and/or consultant for the following companies: Pfizer Inc, TRM Blueprint, Fusion Pharma, Takeda, Ascentage Pharma, Humana, Adelphi, Medscape, Novartis, Incyte and Sangamo. Inc.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Patel, A.B., Pomicter, A.D., Yan, D. et al. Dasatinib overcomes stroma-based resistance to the FLT3 inhibitor quizartinib using multiple mechanisms. Leukemia (2020). https://doi.org/10.1038/s41375-020-0858-1