Establishment of xenotransplantation model of human CN-AML with FLT3-ITD^mut/NPM1⁻ in NOD/SCID mice

Abstract

Patients with FLT3-ITD^mut/NPM1⁻ cytogenetically normal acute myeloid leukemia (CN-AML), as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD^mut/NPM1⁻ CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD^mut/NPM1⁻ CN-AML primary cells. The FLT3-ITD^mut/NPM1⁻ CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary generation models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully established xenotransplantation model of human FLT3-ITD^mut/NPM1⁻ CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

Keywords

acute myeloid leukemia / FLT3/ITD mutation / NPM1 mutation / xenotransplantation model / NOD/SCID mice

References

[1] VogelsteinB, KinzlerKW. Cancer genes and the pathways they control. Nat Med, 2004, 10(8): 789-799

[2] GillilandDG, GriffinJD. The roles of FLT3 in hematopoiesis and leukemia. Blood, 2002, 100(5): 1532-1542

[3] SchlenkRF, DohnerK, KrauterJ, et al.. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med, 2008, 358(18): 1909-1918

[4] GrimwadeD, WalkerH, OliverF, et al.. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood, 1998, 92(7): 2322-2333

[5] TerpstraW, PrinsA, VisserT, et al.. Conditions for engraftment of human acute myeloid leukemia (AML) in SCID mice. Leukemia, 1995, 9(9): 1573-1577

[6] LapidotT, FajermanY, KolletO. Immune-deficient SCID and NOD/SCID mice models as functional assays for studying normal and malignant human hematopoiesis. J Mol Med (Berl), 1997, 75(9): 664-673

[7] AillesLE, GerhardB, KawagoeH, et al.. Growth characteristics of acute myelogenous leukemia progenitors that initiate malignant hematopoiesis in nonobese diabetic/severe combined immunodeficient mice. Blood, 1999, 94(5): 1761-1772

[8] RomboutsWJ, MartensAC, PloemacherRE. Identification of variables determining the engraftment potential of human acute myeloid leukemia in the immunodeficient NOD/SCID human chimera model. Leukemia, 2000, 14(5): 889-897

[9] CheungAM, ChowHC, KwongYL, et al.. FLT3/internal tandem duplication subclones in acute myeloid leukemia differ in their engraftment potential in NOD/SCID mice. Leuk Res, 2010, 34(1): 119-122

[10] RocquainJ, CarbucciaN, TrouplinV, et al.. Combined mutations of ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in myelodysplastic syndromes and acute myeloid leukemias. BMC Cancer, 2010, 10: 401

[11] GaleRE, GreenC, AllenC, et al.. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood, 2008, 111(5): 2776-2784

[12] LevisM, AllebachJ, TseKF, et al.. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood, 2002, 99(11): 3885-3891

[13] MizukiM, FenskiR, HalfterH, 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(12): 3907-3914

[14] GrundlerR, MiethingC, ThiedeC, et al.. FLT3-ITD and tyrosine kinase domain mutants induce 2 distinct phenotypes in a murine bone marrow transplantation model. Blood, 2005, 105(12): 4792-4799

[15] PearceDJ, TaussigD, ZibaraK, et al.. AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML. Blood, 2006, 107(3): 1166-1173

[16] LumkulR, GorinNC, MalehornMT, et al.. Human AML cells in NOD/SCID mice: engraftment potential and gene expression. Leukemia, 2002, 16(9): 1818-1826

[17] PilotoO, LevisM, HusoD, 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. Cancer Res, 2005, 65(4): 1514-1522

[18] LevisM, PhamR, SmithBD, et al.. In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important to achieve synergistic cytotoxic effects. Blood, 2004, 104(4): 1145-1150

[19] LewisID, McDiarmidLA, SamelsLM, et al.. Establishment of a reproducible model of chronic-phase chronic myeloid leukemia in NOD/SCID mice using blood-derived mononuclear or CD34+ cells. Blood, 1998, 91(2): 630-640