FLT3/D835Y mutation knock-in mice display less SEE COMMENTARY aggressive disease compared with FLT3/internal tandem duplication (ITD) mice Emily Baileya,b,LiLia, Amy S. Duffieldc, Hayley S. Maa, David L. Husob, and Don Smalla,d,1 Departments of aOncology, cPathology, and dPediatrics, The Johns Hopkins School of Medicine, Baltimore, MD 21231; and bDepartment of Molecular and Comparative Pathobiology, The Johns Hopkins School of Medicine, Baltimore, MD 21205 Edited by Kevin Shannon, University of California, San Francisco, CA, and accepted by the Editorial Board October 23, 2013 (received for review June 24, 2013) FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately one and SH2-containing inositol phosphatase (SHIP), suggesting its third of acute myeloid leukemia cases. The most common FLT3 involvement in the PI3K and mitogen-activated protein kinases mutations in acute myeloid leukemia are internal tandem dupli- (RAS-MAPK) pathway (4–6, 14). Wild-type FLT3 activates STAT5 cation (ITD) mutations in the juxtamembrane domain (23%) at a low level (15). FLT3/ITD mutations activate these same and point mutations in the tyrosine kinase domain (10%). The downstream targets but, in contrast, activate STAT5 at an elevated mutation substituting the aspartic acid at position 838 (equivalent level (16, 17). Evidence from cell lines has shown that FLT3/ITD to the human aspartic acid residue at position 835) with a tyrosine and FLT3/KD mutant signal transduction pathways have some (referred to as FLT3/D835Y hereafter) is the most frequent kinase differences, even though both mutations result in the constitutive domain mutation, converting aspartic acid to tyrosine. Although activation of FLT3 independent of its ligand (3–5, 9, 14, 18). FLT3/ both of these mutations constitutively activate FLT3, patients with KD mutations also induce activation of the RAS, ERK, and AKT an ITD mutation have a significantly poorer prognosis. To eluci- pathways but demonstrate relatively weak STAT5 activity (19). The date the mechanisms behind this prognostic difference, we have differences in signal transduction between FLT3/KD and FLT3/ generated a knock-in mouse model with a D838Y point mutation ITD mutations may help explain the distinctive disease pro- in FLT3 that corresponds to the FLT3/D835Y mutation described in gression and prognosis in leukemias harboring these mutations. humans. Compared with FLT3/ITD knock-in mice, the FLT3/D835Y Cytogenetic, PCR, and sequencing studies show that FLT3 knock-in mice survive significantly longer. The majority of these mutations frequently occur together with mutations/alterations mice develop myeloproliferative neoplasms with a less-aggressive of other genes, including DNA methyltransferase 3a (DNMT3a, phenotype. In addition, FLT3/D835Y mice have distinct hemato- 13.3%), nucleophosmin 1 (NPM1, 6.8%), Wilms tumor 1 (WT1, poietic development patterns. Unlike the tremendous depletion 5%), runt-related transcription factor 1 (RUNX1, 3.5%), mixed- lineage leukemia (MLL, 2.5%), CCAAT/enhancer binding pro- of the hematopoietic stem cell compartment we have observed in α FLT3/ITD mice, FLT3/D835Y mutant mice are not depleted in tein alpha (CEBP , 1.5%) and core-binding factor (CBF, 1.5%) hematopoietic stem cells. Further comparisons of these FLT3/ (20, 21). These studies also show that both the FLT3 mutations (FLT3/ITD or FLT3/KD), as well as the collaborating mutations, D835Y knock-in mice with FLT3/ITD mice should provide an ideal fi platform for dissecting the molecular mechanisms that underlie can have prognostic signi cance. Dissecting the role of each type the prognostic differences between the two different types of of FLT3 mutation in the development of leukemia in the setting FLT3 mutations. of the different cooperating mutations promises to improve our understanding, and thus therapy, of patients with AML. LT3 (FMS-like tyrosine kinase 3, CD135) is a type 3 receptor Significance Ftyrosine kinase that plays important roles in cell survival, proliferation, and differentiation during normal hematopoiesis MEDICAL SCIENCES (1–3). It is one of the most frequently mutated genes in acute FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately myeloid leukemia (AML), with about one-third of AML cases a third of acute myeloid leukemia cases, predominantly in the affected (4–6). Two predominant types of FLT3-activating muta- forms of FLT3/internal tandem duplication mutations in the tions have been described in association with AML. The first juxtamembrane domain or point mutations in the kinase do- main. Although both mutations activate FLT3, they confer involves internal tandem duplication (ITD) mutations mapping to distinctive prognosis. To elucidate the mechanisms behind this the juxtamembrane domain of FLT3, found in about 25% of adult prognostic difference, we have generated a knock-in mouse and 15% of pediatric AML cases (4–9). AML patients with FLT3/ model with a FLT3/D835Y mutation that converts aspartic acid ITD mutations have a poorer prognosis than patients without to tyrosine. Further comparisons of the FLT3/D835Y mice with these mutations. The second type is point mutations, which most the previously generated, otherwise genetically identical FLT3/ frequently occur in the activation loop of the tyrosine kinase do- ∼ – ITD knock-in mice should provide an ideal platform for dis- main (KD). FLT3/KD mutations occur in 8 10% of both adult secting the molecular mechanisms that underlie the prognostic and pediatric AML (10). AML patients who have FLT3/KD differences and for drug screening against these two different mutations have a similar prognosis to AML patients with unmu- types of FLT3 mutations. tated FLT3 (10–13). The most common KD mutation is at codon 835, converting aspartic acid to tyrosine (D835Y). Also seen are Author contributions: E.B., L.L., and D.S. designed research; E.B., L.L., A.S.D., H.S.M., D.L.H., mutations D835V, D835E, and D835H, converting aspartic acid and D.S. performed research; E.B., L.L., A.S.D., H.S.M., D.L.H., and D.S. analyzed data; and to valine, glutamic acid, and histidine at residue 835, respectively, E.B., L.L., and D.S. wrote the paper. mutations converting glycine to glutamic acid at residue 831 The authors declare no conflict of interest. (G831E) and arginine to glutamine at residue 834 (R834Q), as This article is a PNAS Direct Submission. K.S. is a guest editor invited by the Editorial well as the deletion of isoleucine at residue 836 (10–12). Board. On dimerization by FL stimulation, wild-type FLT3 activates See Commentary on page 20860. a series of downstream signaling targets, including the p85 subunit 1To whom correspondence should be addressed. E-mail: [email protected]. of phosphoinositide 3-kinase (PI3K), growth factor receptor-bound This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. protein 2 (GRB2), proto-oncogene tyrosine-protein kinase SRC, 1073/pnas.1310559110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1310559110 PNAS | December 24, 2013 | vol. 110 | no. 52 | 21113–21118 Downloaded by guest on September 30, 2021 The prevalence and prognostic implications of FLT3 muta- AB tions make them a promising therapeutic target in AML. A number of small-molecule tyrosine kinase inhibitors (TKI) against FLT3 are currently in clinical trials, with varying degrees of clinical responses, but even those patients who respond de- velop resistance to monotherapy (22). One of the mechanisms of acquired resistance to several FLT3 TKIs is the selection for FLT3/KD mutations documented in relapsed patients (23, 24). Further understanding of the mechanisms involved in FLT3 ac- C tivation, signaling, inhibition, and resistance will aid in un- derstanding the complexities of both pediatric and adult AML and help develop more tailored and effective treatments. We have previously generated and characterized a FLT3/ITD knock- Fig. 2. Mice with a FLT3/D835Y mutation develop less aggressive disease in mouse model that consistently develops predominantly compared with FLT3/ITD mutant mice. (A) Kaplan-Meier survival curve of a myeloproliferative neoplastic (MPN) phenotype (25). Here we FLT3/ITD and FLT3/D835Y mice on a C57BL/6 background. P < 0.0001. (B) report on the generation of a FLT3/D835Y knock-in mouse Disease distribution of moribund FLT3/D835Y mice (n = 24). (C) Levels of + + + + model in which an aspartic acid residue was mutated to a tyrosine Mac1 , Mac1 /Gr1 , and Gr1 as a percentage of whole BM in moribund at residue D838Y (corresponding to D835Y in the human gene, FLT3/ITD (n = 20), FLT3/D835Y (n = 21; hemangiosarcoma diagnoses ex- Fig. 1A). In both models, Flt3 is under control of the endogenous cluded), and wild-type (n = 11) controls. Flt3 promoter mimicking physiological expression. By comparing the phenotypic and signaling differences between FLT3/ITD and FLT3/D835Y mice, we hope to continue to explore the distinctive populations or with different cooperating mutations. To de- roles the two mutations play in leukemogenesis and why only the termine whether the prognostic difference reported in humans FLT3/ITD mutation results in poor prognosis. would also be observed in genetically identical mouse models that differ only in the type of FLT3 mutation, we directly com- Results pared FLT3/ITD mice with the newly generated FLT3/D835Y mice. Mice were monitored for survival and killed when mori- Mice with a FLT3/D835Y Mutation Develop MPN That Is Less bund or demonstrating obvious clinical distress. The median Aggressive Than That of FLT3/ITD Mutant Mice. AML patients with survival of the heterozygous FLT3/D835Y mice was 678 d and FLT3/KD mutations develop less-aggressive disease compared ranged from 394 to 826 d. This is prolonged survival compared with those with FLT3/ITD mutations, as evidenced by lower with the heterozygous FLT3/ITD mice that have a median sur- WBC and better prognosis (13). There are a number of potential vival of 430 d (P < 0.0001; Fig.