Differences in growth promotion, drug response and intracellular protein trafficking of FLT3 mutants

Document Type : Original Article


1 Department of Medical Biochemistry, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Discipline of Medical Biochemistry, School of Biomedical Sciences and Pharmacy, University of Newcastle, NSW 2308, Australia

2 School of Medical Sciences/Pharmacology, University of New South Wales, Sydney, NSW 2052, Australia School of Environmental and Life Sciences, University of Newcastle, NSW 2308, Australia

3 Discipline of Medical Biochemistry, School of Biomedical Sciences and Pharmacy, University of Newcastle, NSW 2308, Australia


Objective(s): Mutant forms FMS-like tyrosine kinase-3 (FLT3), are reported in 25% of childhood acute lymphoid leukemia (ALL) and 30% of acute myeloid leukemia (AML) patients. In this study, drug response, growth promoting, and protein trafficking of FLT3 wild-type was compared with two active mutants (Internal Tandem Duplication (ITD)) and D835Y.
Materials and Methods:FLT3 was expressed on factor-dependent cells (FDC-P1) using retroviral transduction. The inhibitory effects of CEP701, imatinib, dasatinib, PKC412 and sunitinib were studied on cell proliferation and FLT3 tyrosine phosphorylation. Total expression and proportion of intracellular and surface FLT3 was also determined.
Results: FDC-P1 cells became factor-independent after expression of human FLT3 mutants (ITD and D835Y). FDC-P1 cells expressing FLT3-ITD grow 3 to 4 times faster than those expressing FLT3-D835Y. FD-FLT3-ITD cells were three times more resistant to sunitinib than the FD-FLT3-WT cells. The Geo means for surface FLT3 expression in FD-FLT3-ITD and –D835Y were 65 and 70% less than the FD-FLT3-WT cells. About 40% of expressed FLT3 was detected as intracellular in FD-FLT3-D835Y cell compared to 4 and 4.5% in FD-FLT3-WT and –ITD cells.
Conclusion: Retention of D835Y FLT3 mutant protein may cause altered signaling, endoplasmic reticulum stress and activation of apoptotic signaling pathways leading to lower proliferation rate in FD-FLT3-D835Y than the FLT3-WT and ITD mutant., these may also also contribute,  along with the preferential affinity, to the increased sensitivity of D835Y of CEP701 and PKC412. Studying these genetic variations can help determining the prognosis and designing a therapeutic plan for the patients with FLT3 mutations.


1. Gotze KS, Ramirez M, Tabor K, Small D, Matthews W, Civin CI. Flt3 high and Flt3 low CD34+ progenitor cells isolated from human bone marrow are functionally distinct. Blood 1998; 91:1947-1958.
2. Christensen JL, Weissman IL. Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proc Natl Acad Sci U S A 2001; 98:14541-1456.
3. Holmes ML, Carotta S, Corcoran LM, Nutt SL. Repression of Flt3 by Pax5 is crucial for B-cell lineage commitment. Genes Dev 2006; 20:933-938.
4. Drexler HG, Quentmeier H. FLT3: receptor and ligand. Growth Factors 2004; 22:71-73.
5. Carow CE, Levenstein M, Kaufmann SH, Chen J, Amin S, Rockwell P, et al. Expression of the hematopoietic growth factor receptor FLT3 (STK-1/Flk2) in human leukemias. Blood 1996; 87:1089-1096.
6. Meshinchi S, Appelbaum FR. Structural and functional alterations of FLT3 in acute myeloid leukemia. Clin Cancer Res 2009; 15:4263-4269.
7. Hubbard SR. Juxtamembrane autoinhibition in receptor tyrosine kinases. Nat Rev Mol Cell Biol 2004; 5:464-471.
8. Spencer DH, Abel HJ, Lockwood CM, Payton JE, Szankasi P, Kelley TW, et al. Detection of FLT3 internal tandem duplication in targeted, short-read-length, next-Generation sequencing data. J Mol Diagn 2012;15:81-93.
9. Meshinchi S, Stirewalt DL, Alonzo TA, Boggon TJ, Gerbing RB, Rocnik JL, et al. Structural and numerical variation of FLT3/ITD in pediatric AML. Blood 2008; 111:4930-4933.
10. Armstrong SA, Mabon ME, Silverman LB, Li A, Gribben JG, Fox EA, et al. FLT3 mutations in childhood acute lymphoblastic leukemia. Blood 2004; 103:3544-3546.
11. Abu-Duhier FM, Goodeve AC, Wilson GA, Care RS, Peake IR, Reilly JT. Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukemia. Brit J Haemat 2001; 113:983-988.
12. Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, Miyawaki S, et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 2001; 97:2434-2439.
13. Griffin JD. Point mutations in the FLT3 gene in AML. Blood 2001; 97:2193A-.
14. Levis M, Small D. FLT3: ITDoes matter in leukemia. Leukemia 2003; 17:1738-1752.
15. Daver N, Strati P, Jabbour E, Kadia T, Luthra R, Wang S, et al. FLT3 mutations in myelodysplastic syndrome and chronic myelomonocytic leukemia. Am J Hematol 2013; 88:56-59.
16. Shih L, Huang C, Wang P, Wu J, Lin T, Dunn P, et al. Acquisition of FLT3 or N-ras mutations is frequently associated with progression of myelodysplastic syndrome to acute myeloid leukemia. Leukemia 2004; 18:466-475.
17. Tiesmeier J, Müller-Tidow C, Westermann A, Czwalinna A, Hoffmann M, Krauter J, et al. Evolution of FLT3-ITD and D835 activating point mutations in relapsing acute myeloid leukemia and response to salvage therapy. Leuk Res 2004; 28:1069-1074.
18. Whartenby KA, Calabresi PA, McCadden E, Nguyen B, Kardian D, Wang T, et al. Inhibition of FLT3 signaling targets DCs to ameliorate autoimmune disease. Proc Natl Acad Sci U S A 2005; 102:16741-16746.
19. Andersson SE, Svensson MN, Erlandsson MC, Dehlin M, Andersson KM, Bokarewa MI. Activation of Fms-Like tyrosine kinase 3 signaling enhances survivin expression in a mouse model of rheumatoid arthritis. PloS One 2012; 7:e47668.
20. Chilton PM, Rezzoug F, Fugier-Vivier I, Weeter LA, Xu H, Huang Y, et al. Flt3-ligand treatment prevents diabetes in NOD mice. Diabetes 2004; 53:1995-2002.
21. Schmidt-Arras D, Schwable J, Bohmer FD, Serve H. Flt3 receptor tyrosine kinase as a drug target in leukemia. Curr Pharm Des 2004; 10:1867-1883.
22. Taylor JR, Brownlow N, Domin J, Dibb NJ. FMS receptor for M-CSF (CSF-1) is sensitive to the kinase inhibitor imatinib and mutation of Asp-802 to Val confers resistance. Oncogene 2006; 25:147–151.
23. Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer 2003; 3:650-665.
24. Small D. FLT3 mutations: biology and treatment. Hematol Am Soc Hematol Educ Program 2006; 178-184.
25. Teller S, Kramer D, Bohmer SA, Tse KF, Small D, Mahboobi S, et al. Bis(1H-2-indolyl)-1-methanones as inhibitors of the hematopoietic tyrosine kinase Flt3. Leukemia 2002; 16:1528-1534.
26. Tabone-Eglinger S, Subra F, El Sayadi H, Alberti L, Tabone E, Michot JP, et al. KIT mutations induce intracellular retention and activation of an immature form of the KIT protein in gastrointestinal stromal tumors. Clin Cancer Res 2008; 14:2285-2294.
27. 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; 25:1751-1759.
28. Frost MJ, Ferrao PT, Hughes TP, Ashman LK. Juxtamembrane mutant V560GKit is more sensitive to Imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant. Mol Cancer Ther 2002; 1:1115-1124.
29. Roberts KG, Odell AF, Byrnes EM, Baleato RM, Griffith R, Lyons AB, et al. Resistance to c-KIT kinase inhibitors conferred by V654A mutation. Mol Cancer Ther 2007; 6:1159-1166.
30. Nishimura N, Nishioka Y, Shinohara T, Sone S. Enhanced efficiency by centrifugal manipulation of adenovirus-mediated interleukin 12 gene transduction into human monocyte-derived dendritic cells. Hum Gene Ther 2001; 12:333-346.
31. Dexter TM, Garland J, Scott D, Scolnick E, Metcalf D. Growth of factor-dependent hemopoietic precursor cell lines. J Exp Med 1980; 152:1036-1047.
32. Cleveland JL, Dean M, Rosenberg N, Wang JY, Rapp UR. Tyrosine kinase oncogenes abrogate interleukin-3 dependence of murine myeloid cells through signaling pathways involving c-myc: conditional regulation of c-myc transcription by temperature-sensitive v-abl. Mol Cell Biol 1989; 9:5685-5695.
33. Mashkani B, Griffith R, Ashman LK. Colony
stimulating factor-1 receptor as a target for small molecule inhibitors. Bioorg Med Chem 2010; 18:1789-1797.
34. Foster R, Griffith R, Ferrao P, Ashman L. Molecular basis of the constitutive activity and STI571 resistance of Asp816Val mutant KIT receptor tyrosine kinase. J Mol Graph Model 2004; 23:139-152.
35. Sanjay A, Horne WC, Baron R. The Cbl family: ubiquitin ligases regulating signaling by tyrosine kinases. Sci STKE 2001; 2001:pe40.
36. Schmidt-Arras DE, Bohmer A, Markova B, Choudhary C, Serve H, Bohmer FD. Tyrosine phosphorylation regulates maturation of receptor tyrosine kinases. Mol Cell Biol 2005; 25:3690-3703.
37. Sitia R, Braakman I. Quality control in the endoplasmic reticulum protein factory. Nature 2003; 426:891-894.
38. Cho J, Chen L, Sangji N, Okabe T, Yonesaka K, Francis JM, et al. Cetuximab Response of Lung Cancer-Derived EGF Receptor Mutants Is Associated with Asymmetric Dimerization. Cancer Res 2013; 73:6770-6779.
39. Gajiwala KS, Wu JC, Christensen J, Deshmukh GD, Diehl W, DiNitto JP, et al. KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients. Proc Natl Acad Sci USA 2009; 106:1542-1547.
40. Williams AB, Nguyen B, Li L, Brown P, Levis M, Leahy D, et al. Mutations of FLT3/ITD confer resistance to multiple tyrosine kinase inhibitors. Leukemia 2013; 27:48-55.
41. Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, Miyawaki S, et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 2001; 97:2434-2439.
42. Malhotra JD, Kaufman RJ. Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? Antioxid Redox Signal 2007; 9:2277-2293.
43. Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 2012; 13:89-102.
44. Schroder M, Kaufman RJ. ER stress and the unfolded protein response. Mut Res 2005; 569:29-63