Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways.

Continuous treatment of malignancies with tyrosine kinase inhibitors (TKIs) may select for resistant clones (ie, imatinib mesylate). To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in acute myelogenous leukemia (AML), we developed resistant human cell lines through prolonged coculture with FLT3 TKIs. FLT3 TKI-resistant cell lines and primary samples still exhibit inhibition of FLT3 phosphorylation on FLT3 TKI treatment. However, FLT3 TKI-resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these signaling pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI-resistant cell lines revealed activating N-Ras mutations in 2 cell lines that were not present in the parental FLT3 TKI-sensitive cell line. Taken together, these data indicate that FLT3 TKI-resistant cells most frequently become FLT3 independent because of activation of parallel signaling pathways that provide compensatory survival/proliferation signals when FLT3 is inhibited. Anti-FLT3 mAb treatment was still cytotoxic to FLT3 TKI-resistant clones. An approach combining FLT3 TKIs with anti-FLT3 antibodies and/or inhibitors of important pathways downstream of FLT3 may reduce the chances of developing resistance.

[1]  H. Drexler,et al.  Expression of FLT3 receptor and response to FLT3 ligand by leukemic cells. , 1996, Leukemia.

[2]  H. Kaneko,et al.  Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. , 1996, Leukemia.

[3]  W. Leonard,et al.  Essential Role of Signal Transducer and Activator of Transcription (Stat)5a but Not Stat5b for Flt3-Dependent Signaling , 2000, The Journal of experimental medicine.

[4]  D. Gilliland,et al.  Variable sensitivity of FLT3 activation loop mutations to the small molecule tyrosine kinase inhibitor MLN518. , 2004, Blood.

[5]  J. Allebach,et al.  Inhibition of the transforming activity of FLT3 internal tandem duplication mutants from AML patients by a tyrosine kinase inhibitor , 2002, Leukemia.

[6]  H. Broxmeyer,et al.  p85 subunit of PI3 kinase does not bind to human Flt3 receptor, but associates with SHP2, SHIP, and a tyrosine-phosphorylated 100-kDa protein in Flt3 ligand-stimulated hematopoietic cells. , 1999, Biochemical and biophysical research communications.

[7]  M. Heinrich,et al.  SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. , 2003, Blood.

[8]  H. Saito,et al.  Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product , 1998, Leukemia.

[9]  J. Radich,et al.  The role of FLT3 in haematopoietic malignancies , 2003, Nature Reviews Cancer.

[10]  G. Ehninger,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 , 2022 .

[11]  D. Hicklin,et al.  IMC-EB10, an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples. , 2006, Cancer research.

[12]  D. Hicklin,et al.  Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody. , 2004, Blood.

[13]  D. Hicklin,et al.  FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells. , 2004, Blood.

[14]  D. Small,et al.  Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor , 2001, Leukemia.

[15]  R. Stone,et al.  Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain. , 2006, Blood.

[16]  D. Birnbaum,et al.  FLT3 signaling in hematopoietic cells involves CBL, SHC and an unknown P115 as prominent tyrosine-phosphorylated substrates , 1998, Leukemia.

[17]  B. Smith,et al.  A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. , 2002, Blood.

[18]  C. Miething,et al.  Sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor. , 2003, Blood.

[19]  H. Broxmeyer,et al.  Flt3 ligand induces tyrosine phosphorylation of gab1 and gab2 and their association with shp-2, grb2, and PI3 kinase. , 2000, Biochemical and biophysical research communications.

[20]  D. Birnbaum,et al.  Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. , 1996, Leukemia.

[21]  D. Birnbaum,et al.  Expression and signal transduction of the FLT3 tyrosine kinase receptor. , 1996, Acta haematologica.

[22]  B. Smith,et al.  A FLT3 tyrosine kinase inhibitor is selectively cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem duplication mutations. , 2001, Blood.

[23]  山本 幸也,et al.  Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies , 2002 .

[24]  J. Reilly,et al.  Identification of novel FLT‐3 Asp835 mutations in adult acute myeloid leukaemia , 2001, British journal of haematology.

[25]  E. Estey,et al.  Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors. , 2006, Blood.

[26]  R. Arceci,et al.  Pediatric AML primary samples with FLT3/ITD mutations are preferentially killed by FLT3 inhibition. , 2004, Blood.

[27]  D. Birnbaum,et al.  Expression of the FMS/KIT-like gene FLT3 in human acute leukemias of the myeloid and lymphoid lineages. , 1992, Blood.

[28]  Richard Clark,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. , 2004, Blood.

[29]  T. Naoe,et al.  Mechanism of constitutive activation of FLT3 with internal tandem duplication in the juxtamembrane domain , 2002, Oncogene.

[30]  K. Horibe,et al.  Prognostic value of internal tandem duplication of the FLT3 gene in childhood acute myelogenous leukemia. , 1999, Medical and pediatric oncology.

[31]  M. Borowitz,et al.  Expression of the hematopoietic growth factor receptor FLT3 (STK-1/Flk2) in human leukemias. , 1996, Blood.

[32]  M. Levis,et al.  FLT3: ITDoes matter in leukemia , 2003, Leukemia.

[33]  Wolfgang Hiddemann,et al.  FLT3-ITD-TKD dual mutants associated with AML confer resistance to FLT3 PTK inhibitors and cytotoxic agents by overexpression of Bcl-x(L). , 2005, Blood.

[34]  D. Linch,et al.  Prognostic Implications of the Presence of FLT3 Mutations in Patients with Acute Myeloid Leukemia , 2003, Leukemia & lymphoma.

[35]  G. Mcmahon,et al.  SU5416 and SU5614 inhibit kinase activity of wild-type and mutant FLT3 receptor tyrosine kinase. , 2002, Blood.

[36]  D. Hicklin,et al.  Inhibitory anti-FLT3 antibodies are capable of mediating antibody-dependent cell-mediated cytotoxicity and reducing engraftment of acute myelogenous leukemia blasts in nonobese diabetic/severe combined immunodeficient mice. , 2005, Cancer research.

[37]  Doriano Fabbro,et al.  Prediction of Resistance to Small Molecule FLT3 Inhibitors , 2004, Cancer Research.

[38]  Doriano Fabbro,et al.  Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. , 2002, Cancer cell.

[39]  H. Kantarjian,et al.  Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia. , 2004, Blood.

[40]  H. Griesser,et al.  Leukemic predisposition of mice transplanted with gene-modified hematopoietic precursors expressing flt3 ligand. , 1998, Blood.

[41]  M. Kutlubaev,et al.  [The influence of some psychotropic drugs on the processes of free radical oxidation in model systems]. , 2005, Zhurnal Nevrologii i Psikhiatrii imeni S.S. Korsakova.

[42]  P. N. Rao,et al.  Clinical Resistance to STI-571 Cancer Therapy Caused by BCR-ABL Gene Mutation or Amplification , 2001, Science.

[43]  D. Small,et al.  FLT3 inhibitors: a paradigm for the development of targeted therapeutics for paediatric cancer. , 2004, European journal of cancer.

[44]  W. Hiddemann,et al.  Mutations in the tyrosine kinase domain of FLT3 define a new molecular mechanism of acquired drug resistance to PTK inhibitors in FLT3-ITD-transformed hematopoietic cells. , 2004, Blood.

[45]  Randy Allred,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. , 2005, Blood.

[46]  J. Griffin,et al.  The roles of FLT3 in hematopoiesis and leukemia. , 2002, Blood.

[47]  H. Broxmeyer,et al.  Flt3 signaling involves tyrosyl‐phosphorylation of SHP‐2 and SHIP and their association with Grb2 and Shc in Baf3/Flt3 cells , 1999, Journal of leukocyte biology.

[48]  D. Small,et al.  Constitutive activation of FLT3 stimulates multiple intracellular signal transducers and results in transformation , 2000, Leukemia.

[49]  M. Taniwaki,et al.  Internal tandem duplication of the FLT3 gene and clinical evaluation in childhood acute myeloid leukemia , 1999, Leukemia.

[50]  E. Estey,et al.  Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412 , 2004 .