FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model

Abstract

Activating FMS-like tyrosine kinase 3 (FLT3) mutations have been identified in 30% of patients with acute myelogenous leukemia (AML), and recently in a smaller subset of patients with acute lymphoblastic leukemia (ALL). To explore the in vivo consequences of an activating FLT3 internal tandem duplication mutation (FLT3-ITD), we created a transgenic mouse model in which FLT3-ITD was expressed under the control of the vav hematopoietic promoter. Five independent lines of vav-FLT3-ITD transgenic mice developed a myeloproliferative disease with high penetrance and a disease latency of 6–12 months. The phenotype was characterized by splenomegaly, megakaryocytic hyperplasia, and marked thrombocythemia, but without leukocytosis, polycythemia, or marrow fibrosis, displaying features reminiscent of the human disease essential thrombocythemia (ET). Clonal immature B- or T-lymphoid disease was observed in two additional founder mice, respectively, that could be secondarily transplanted to recipient mice that rapidly developed lymphoid disease. Treatment of these mice with the FLT3 tyrosine kinase inhibitor, PKC412, resulted in suppression of disease and a statistically significant prolongation of survival. These results demonstrate that FLT3-ITD is capable of inducing myeloproliferative as well as lymphoid disease, and indicate that small-molecule tyrosine kinase inhibitors may be an effective treatment for lymphoid malignancies in humans that are associated with activating mutations in FLT3.

Introduction

FMS-like tyrosine kinase 3 (FLT3) also known as fetal liver kinase-2 (FLK-2) is a member of the class III receptor tyrosine kinase (RTK) family and shares strong sequence and structural similarities to other class III tyrosine kinase members, including FLT1, FMS, PDGFβR, and KIT (Rosnet and Birnbaum, 1993). FLT3 is expressed in the placenta, gonads, and brain (Maroc et al., 1993; deLapeyriere et al., 1995), as well as a variety of human and murine cell lines of both myeloid and B-lymphoid lineage (Brasel et al., 1995; Turner et al., 1996). In normal bone marrow, expression is restricted to immature hematopoietic progenitors, including CD34+ cells with high levels of expression of CD117 (c-KIT) (Rasko et al., 1995; Rosnet et al., 1996). Interestingly, although targeted disruption of FLT3 results in healthy adult mice with normal mature hematopoietic populations, these mice demonstrate deficiencies in primitive B-lymphoid progenitors, and bone marrow transplantation (BMT) studies reveal a reduced ability of stem cells lacking FLT3 to reconstitute both T cells and myeloid cells (Mackarehtschian et al., 1995). Together, these data implicate an important role for FLT3 in the development of multipotent hematopoietic stem cells and lymphoid cells.

FLT3 has been shown to be expressed at high levels in a number of hematopoietic malignancies, including 70% or greater cases of acute myelogenous leukemia (AML) of all French–American–British (FAB) subtypes, and a smaller fraction of T-cell ALLs, and chronic myelogenous leukemia (CML) in lymphoid blast crisis (Drexler, 1996; Rosnet et al., 1996). Significantly, high levels of FLT3 expression have also been detected in a large percentage of cases of B-precursor cell acute lymphoblastic leukemia (ALL), as well as in a subset of ALLs carrying a chromosomal translocation involving the mixed-lineage leukemia (MLL) gene (Drexler, 1996; Rosnet et al., 1996; Armstrong et al., 2002). FLT3 is also expressed at high levels in both leukemia and lymphoma cell lines (DaSilva et al., 1994; Meierhoff et al., 1995) including pre-B, myeloid, and monocytic cell lines. Moreover, FLT3 ligand expression can also be detected in a majority of cell lines, and studies suggest that autocrine stimulation may play a role in the proliferation of leukemic blasts (Meierhoff et al., 1995).

The presence of internal tandem duplications (ITDs) in the juxtamembrane (JM) domain of FLT3 in patients with AML, first reported by Nakao et al. (1996) suggested that these mutations might play an important role in the pathogenesis of AML. Subsequent studies have confirmed this initial observation, and to date the overall frequency of FLT3-ITD mutations in adult AML in the literature is reported to be 24% (Gilliland and Griffin, 2002). FLT3-ITDs have been detected in 15% secondary AML (Horiike et al., 1997), and significantly may be associated with disease progression or relapse of AML (Horiike et al., 1997; Ishii et al., 1999). An additional cohort of AML patients contains mutations in the so-called activation loop of FLT3, and, in particular, substitution mutations at aspartic acid residue 835. D835 mutations have been reported in 7% of AML patients, as well as a smaller percentage (3%) of myelodysplastic syndrome (MDS) patients (Abu-Duhier et al., 2001; Griffin, 2001; Yamamoto et al., 2001), with D835Y being the most commonly reported substitution. More recently, activation loop mutations in FLT3 have been described in cases of ALLs that harbor rearrangements of the MLL gene on chromosome 11q23 (Armstrong et al., 2003), implicating FLT3 in the pathogenesis of both lymphoid as well as myeloid leukemias. Overall, approximately 31% of AML patients have acquired mutations in FLT3 composed of either FLT3-ITD mutations (24%) or FLT3 activation-loop mutations (7%). Thus, FLT3 is the single most commonly mutated gene in AML, surpassing the incidence of any known chromosomal translocations or point mutations.

Both FLT3-ITD mutations in the JM domain and activation-loop mutations result in constitutive activation of the FLT3 kinase. ITD mutations result in ligand-independent dimerization and tyrosine autophosphorylation (Kiyoi et al., 1998), as well as activation of the STAT5, RAS/MAPK, and PI3 pathways (Hayakawa et al., 2000; Mizuki et al., 2000). FLT3-ITDs confer factor-independent growth to the murine hematopoietic cell lines 32D and Ba/F3 that are dependent on IL-3 for growth (Hayakawa et al., 2000; Mizuki et al., 2000). In contrast, wild-type (WT) FLT3, even in the presence of FLT3 ligand, does not confer factor-independent growth to these cells. Injection of 32D or Ba/F3 cells stably transfected with constitutively activated FLT3 into syngeneic recipient mice results in the development of a leukemia phenotype (Mizuki et al., 2000; Tse et al., 2000). We have previously shown that retroviral transduction of FLT3-ITD mutations into primary murine bone marrow cells results in a myeloproliferative phenotype in a bone marrow transplant assay (Kelly et al., 2002). These studies demonstrated that, although FLT3-ITDs have been associated primarily with AML in humans, FLT3-ITD expression alone is not sufficient to induce AML in primary murine hematopoietic cells. Furthermore, point mutations that inactivate the FLT3 kinase in the context of the FLT3-ITD abrogate the disease, indicating an absolute requirement of the FLT3 kinase activity for development of myeloproliferative disease in this model.

To further examine the effects and oncogenic potential of activated FLT3 in vivo, we have created a transgenic mouse model in which expression of FLT3-ITD was directed to the hematopoietic compartment by the vav promoter. In this model, the majority of transgenic mice developed a myeloproliferative syndrome (MPS). However, unlike the myeloproliferative disease (MPD) phenotype induced by retroviral transduction of FLT3-ITD in the BMT assay (Kelly et al., 2002), the MPS in these transgenic mice did not display marked leukocytosis and neutrophilia, but rather were characterized by a striking megakaryocytic hyperplasia and significant thrombocytosis. In addition, we also observed immature B- or T-lymphoid disease that developed in two animals and assessed the in vivo efficacy of PKC412 (Weisberg et al., 2002), a known FLT3 tyrosine kinase inhibitor, against FLT3-induced lymphoid disease in secondary transplant experiments. Overall, these studies indicate that activating FLT3 mutations are capable of inducing a variety of hematopoietic disorders in vivo, and demonstrate that small-molecule inhibitors may be efficacious in the treatment of FLT3-ITD-induced lymphoid disease.

Results

Construction of vav-FLT3-ITD transgenic mice

We sought to examine the in vivo consequences of expressing an activated FLT3 RTK by constructing a transgenic mouse model whereby expression of human FLT3 harboring an ITD mutation was directed toward the hematopoietic system using the vav promoter which has been shown to drive transgene expression broadly throughout the entire hematopoietic compartment (Figure 1) (Ogilvy et al., 1999a). A total of nine founder mice were identified as having integrated the vav-FLT3-ITD construct. Germline transmission allowed the propagation of eight transgenic lines. One founder (mouse 6028) failed to successfully breed before becoming moribund due to diffuse lymphadenopathy, and was killed for analysis at 76 days of age (see below).

Figure 1
figure1

vav-FLT3-ITD transgenic construct. Human FLT3 cDNA harboring a W51 ITD mutation (Kelly et al., 2002) was cloned into the HS321/45 vav hematopoietic vector between the SfiI and NotI sites. The vav FLT3-ITD transgenic vector contains the vav promoter and 7.8 kb of additional upstream vav sequence 5′ of, and 4.4 kb of, the sequence from the vav first intron 3′ of the FLT3-ITD cDNA insert

vav-FLT3-ITD induces a MPS

We noted splenomegaly upon palpation in five independent transgenic founder lines between 6 and 10 months of age. Clinically, however, these mice were not moribund and did not exhibit lymphadenopathy, weight loss, ruffled coats, labored breathing, or other signs of distress. Splenomegaly was confirmed upon necropsy and measurement of spleen weights (Figure 2a and Table 1). Splenomegaly was variable, ranging from mild (1.9-fold increase in spleen weight) to marked (8.3-fold increase in size). Histopathologic examination of the spleens from four independent founders (6019, 6024, 6035, 2908) demonstrated an abnormal splenic architecture with marked expansion of red pulp, with a striking proliferation of the megakaryocytic population (Figure 2c–f). Megakaryocytes were clustered, abnormally large in size, and cytologically atypical (Figure 2e, f). The increase in megakaryocytes was also corroborated by flow-cytometric analysis of splenic single-cell suspensions with staining for glycoprotein IIb integrin (CD41), a platelet receptor for fibrinogen expressed on megakaryocytes and platelets during adult hematopoiesis, which demonstrated 8-fold greater CD41-positive population of cells in the spleens of these transgenic animals than splenic cells derived from a WT FVB control animal (Figure 3a). Of note, prominent eosinophilia was not observed in the spleen or marrow sections.

Figure 2
figure2

Gross and histopathology of vav-FLT3-ITD mice with MPS phenotype. (a) Transgenic animal (6035) displaying marked splenomegaly (arrow). (b) Spleen from WT FVB animal ( × 100). (c, d) Spleen from two different, representative transgenic animals with MPS phenotype ( × 100 and × 600) (e, f). (g) Peripheral blood smear demonstrating thrombocythemia and the presence of giant platelets (arrow). (h) Bone marrow section displaying marked hypercellularity and myeloid hyperplasia consisting predominantly of mature granulocytic elements. (i, j) Extramedullary hematopoiesis in the liver. (k) Human FLT3-ITD expression in spleen cells from vav FLT3-ITD transgenic (Tg) animals versus nontransgenic WT (non-Tg WT) control. Protein lysates of Ba/F3 cells transformed with human FLT3-ITD were used as a positive control (+ control). Bands from a nonspecific protein were utilized as protein loading control

Table 1 Characteristics of vav FLT3-ITD transgenic animals
Figure 3
figure3

Flow-cytometric analysis of vav-FLT3-ITD mice with MPS phenotype. (a) Expanded CD41-positive population in spleens from transgenic vav-FLT3-ITD animals (6019, 6024) versus WT FVB control. (b) Increased Gr-1+/Mac-1+ cell populations in bone marrows from transgenic mice (6019, 6024) as compared to bone marrow cells from a control nontransgenic WT animal (non-Tg)

Wright–Giemsa stains of peripheral blood smears from these different founders demonstrated thrombocytosis and the presence of giant platelets (Figure 2g). Platelet counts derived from an automated hematology analyzer showed a median platelet count of 3 000 000/ml (Table 1). Bone marrow sections stained with hematoxylin–eosin demonstrated hypercellularity with a marked predominance of maturing myeloid lineage cells within the marrow compartment of all transgenic mice (Figure 2h). This was substantiated by flow-cytometric analysis of marrow single-cell suspensions, as evident by the increased percentage of Mac-1+/Gr-1+ cells in transgenic mice compared to bone marrow cells from control nontransgenic mice (Figure 3b). Interestingly, the marrow from these mice did not display a significant degree of reticulin fibrosis, as demonstrated by silver stain that has been previously noted in marrow sections from FLT3-ITD mice in BMT assays (data not shown). While marked splenic megakaryocytic proliferation was found consistently in four different founder lines, only one mouse (6019) displayed a prominent increase in megakaryocytes in the bone marrow in addition to the myeloid hyperplasia (data not shown). Analysis of the liver demonstrated extramedullary hematopoiesis in a sinusoidal and perivascular distribution pattern comprised of maturing myeloid elements, with admixed erythroid and megakaryocytic elements similar to that seen in the spleen (Figure 2i, j). Myeloproliferative vav-FLT3-ITD syndrome was not transplantable into sublethally irradiated secondary FVB recipient mice (n=12) when 1 × 106 spleens cells from three independent MPD founder lines (6019, 6024, and 6035) were used as donors with a follow-up of more than 80 days. Expression of human FLT-ITD protein was confirmed in the vav-FLT3-ITD transgenic animals by Western blot analysis of protein lysates derived from single-cell splenic suspensions, whereas no human FLT3 protein was detected from WT control animals (Figure 2k).

The fifth independent founder (6033) with mild splenomegaly (1.9-fold increase in spleen weight) also displayed increased numbers of megakaryocytes in the red pulp, however, not to the extent as seen in the four other founder animals (data not shown). Moreover, while the platelet count from this animal was increased over normal WT FVB platelet numbers (1654 k/mm3), the extent of thrombocythemia was not as marked as observed with the other transgenic founders.

Measurements of serum levels of thrombopoietin (TPO) from vav FLT3-ITD transgenic animals were comparable to TPO levels from WT control animals, indicating that the megakaryocytic hyperplasia that we observed in these animals was a cell-autonomous effect (TPO levels (pg/ml): WT nontransgenic FVB, n=3; mean 436.1, median 377.6 (range 356.0–574.7); vav-FLT3-ITD, n=3; mean 485.8, median 395.4 (range 291.3–770.7)).

We examined a number of offspring from each of these founder animals that exhibited a myeloproliferative (MPS) phenotype. While the degree of thrombocytosis and splenomegaly was variable among the offspring of these founder animals, these mice were found to exhibit a statistically significant increased platelet count and degree of splenomegaly, but not in leukocyte counts when compared with the nontransgenic control animals (Table 2).

Table 2 Characteristics of vav FLT3-ITD offspring animals from MPS founders

FLT3-ITD induces an immature B-lymphoid lymphoma

As noted above, one founder animal (6028) became moribund and immobile due to hindlimb paralysis at 2.5 months of age and was unable to successfully breed before succumbing to disease. Analysis of this mouse revealed diffuse (including cervical, axillary, mesenteric, inguinal) lymphadenopathy at necropsy as well as splenomegaly (340 mg). Complete blood counts did not show leukocytosis (WBC 8.8 × 109 cells/l; Table 1); however, the presence of numerous large atypical cells was noted upon examination of peripheral blood smears (Figure 4c). Histopathologic examination of enlarged lymph node masses showed effacement of normal nodal architecture by a monotonous population of intermediate-to-large atypical lymphoid cells with round to oval nuclei, variably prominent nucleoli, and scant cytoplasm (Figure 4a, b). Frequent tingible-body macrophages and a brisk mitotic rate were also noted with extension of the tumor infiltrate into perinodal adipose tissue (Figure 4a, b). The neoplastic lymphoid cells were noted to completely replace normal hematopoietic elements within the bone marrow of the vertebral column, with prominent extension into the paraspinal skeletal muscle, peripheral nerves, and ganglion structures accounting for the animal's hindlimb paralysis (Figure 4d). Splenic architecture was effaced by expansion of the white pulp by the large atypical lymphoid cells. Moreover, tumor cell infiltrate was also focally present in lung parenchyma, gastrointestinal-associated lymphoid tissue, and liver (data not shown). Flow-cytometric analysis of tumor cells was performed to characterize the lymphoid infiltrate, and demonstrated a dominant population of CD19+, B220+ (moderate), CD3−, CD43+, and surface IgM−, consistent with an immature B-cell lymphoma (Figure 4e). Clonality of tumors was assessed by Southern blot that demonstrated oligoclonal rearrangements in B-cell lymphoid cells with probes for the immunoglobulin heavy chain (IgH), but not the T-cell receptor (βTCR) loci, consistent with the histopathologic and flow-cytometry data (Figure 4l).

Figure 4
figure4

FLT3-ITD induces an immature B-cell (ae) and T-cell lymphoma (fj). (a, b) Effacement of lymph node architecture by B-cell lymphoblastic lymphoma ( × 100 and × 1000). (c) Peripheral blood smear with atypical lymphocytes ( × 1000; Wright–Giemsa). (d) Infiltration of tumor cells in the paraspinal skeletal muscle, peripheral nerve, ganglion, and bone marrow. (e) Flow-cytometric analysis of B-cell lymph node tumor. (f, g) T-cell lymphoma mediastinal mass ( × 100, × 1000). (h) Tumor infiltration in lung and (i) spleen, inset ( × 1000). (j) Flow-cytometric analysis of T-cell lymphoma mediastinal mass. (k) Western blot analysis of human FLT3-ITD protein expression from protein lysates derived from splenic single-cell suspensions of vav-FLT3-ITD transgenic animals with an immature B-cell and T-cell lymphoma. Nontransgenic WT control (non-Tg WT) and Ba/F3 cells transformed with human FLT3-ITD as a positive control (+ control). Bands from a nonspecific protein were utilized as protein loading control. (l) Southern blot analysis of genomic DNA derived from mediastinal mass of mouse 6045 (T cell), lymph node of mouse 6028 (B cell), or WT FVB tail DNA as control. Arrowheads denote germline bands of the IgH or βTCR loci

FLT3-ITD induces an immature T-lymphoid lymphoma

One vav-FLT3-ITD transgenic founder (6045) was found to exhibit labored breathing and a scruffy coat at 8.0 months of age. Necropsy of this animal revealed a massive fleshy mediastinal mass. Histopathologic examination of the mass showed a monotonous population of intermediate-to-large lymphoid cells, again demonstrating the histologic features of lymphoblastic lymphoma (Figure 4f, g). Prominent infiltration of tumor cells was noted in the lung and spleen, and focal involvement in bone marrow (Figure 4h, i and data not shown). Flow-cytometric analysis of the mediastinal mass demonstrated a predominant population of CD4+, CD8+, B220−, and CD19− cells consistent with an immature T-cell lymphoma (Figure 4j). Southern blot analysis confirmed clonal rearrangement of the βTCR locus, but not of the IgH (Figure 4l). We confirmed human FLT3 protein expression in both the vav FLT3-ITD immature B- and T-cell lymphoid disease by performing Western blot analysis on single-cell suspensions of spleen from the founder mice (Figure 4k).

To date, we have not observed any clinical evidence of T-cell lymphoblastic lymphoma in offspring derived from founder 6045, nor through complete necroscopic examination of several offspring from this line. While this may indicate that the degree of penetrance for T-cell disease is low, the age of disease onset exhibited in the founder mouse may be reflective of the long latency required for the disease to manifest itself.

FLT3-ITD-induced B- and T-cell lymphoid disease is transplantable and responds to the FLT3 inhibitor, PKC412

To confirm the malignant phenotype of lymphoma cells, 1 × 105 cells in single-cell suspensions derived from either B- or T-cell FLT3-ITD-induced lymphomas from organ cells harvested from lymph node or mediastinal tumor masses were transplanted by intravenous injection into sublethally irradiated syngeneic mice. Transplanted mice receiving B-cell lymphoma cells rapidly became moribund approximately 2 weeks after transplantation. Specifically, the majority of animals exhibited scruffy coats, developed hindlimb paralysis, lymphadenopathy, and either leukocytosis or leukopenia. Similarly, secondary recipient animals receiving FLT3-ITD-induced T-cell lymphoma cells also became moribund 21–25 days after transplantation. These animals also developed hindlimb paralysis, splenomegaly, lymphadenopathy, as well as leukocytosis on a consistent basis. Histopathologic and flow-cytometric analysis was consistent with either B- or T-cell lymphoblastic lymphoma, demonstrating that the transplanted tumors retained the same morphology and immunophenotype as their primary B- or T-cell tumor, respectively.

To assess the efficacy of the FLT3 inhibitor, PKC412, against FLT3-ITD-induced B- or T-cell lymphoid disease in this secondary transplant model, we administered 1 × 105 of either B- or T-cell lymphoid cells into secondary recipients (n=19, B-cell disease; n=17, T-cell disease) and monitored them for the onset of disease by the aforementioned parameters. Mice in both the B- or T-cell/PKC412 drug trials were gavaged with either PKC412 (100 mg/kg/day) or vehicle control daily. These doses were based on our experience with PKC412 from numerous prior murine transplant drug trial studies in which IC50 levels of PKC412 would be well below those where nonspecific toxicity effects might be observed (Weisberg et al., 2002; Cools et al., 2003; Chen et al., 2004). Mice in the B-cell drug trial received treatment beginning 8 days after transplantation for 30 consecutive days, and mice in the T-cell drug trial received treatment beginning 14 days after transplantation for 30 consecutive days. For both the B- and T-cell drug trials, there was a statistically significant prolongation of survival in mice treated with PKC412 as compared with placebo controls (Figure 5a, b), as well as a significant reduction in spleen sizes and leukocytosis (with the T-cell disease only) (Table 3).

Figure 5
figure5

FLT3-ITD-induced lymphoid disease is transplantable and responds to PKC412. Kaplan–Meier curves and histopathology of organs from representative placebo- and PKC412-treated animals secondarily transplanted with FLT3-ITD induced immature (a) B-cell or (b) T-cell lymphoma cells. Histologic sections of PKC412-treated animals in both trials were taken from animals at the trial end point (41 days after initial injection of diseased cells) and sections of placebo-treated mice were taken from representative animals 5 and 13 days after initiation of oral gavage treatments for the B-cell and T-cell trials, respectively

Table 3 PKC412 drug trial

Histopathologic analysis of multiple organs from representative animals in both trials further supported the interpretation of a dramatic reduction in FLT3-ITD-induced disease in the drug-treated mice compared to placebo controls. Tumor infiltration of the spleen, liver, kidney, lung, and bone marrow was clearly evident in placebo-treated animals, whereas tumor burden was dramatically decreased in PKC412-treated mice (Figure 5a, b).

Discussion

Activating mutations in FLT3 are present in approximately 30–35% of cases of AML, and have been identified in lymphoid malignancies in a smaller proportion of cases. We chose to express FLT3 under the control of the vav promoter in a transgenic mouse model, because vav is a well-characterized panhematopoietic promoter that is widely expressed at high levels in virtually all nucleated cells of adult murine hematopoietic tissues, including both the myeloid and lymphoid compartments (Ogilvy et al., 1998, 1999a, 1999b). Consistent with the pattern of expression of vav, we obtained founder mice with myeloproliferative as well as B- and T-lymphoid tumors.

A myeloproliferative phenotype was observed in five independent founders characterized by thrombocytosis, myeloid hyperplasia in the bone marrow, extramedullary hematopoiesis in the spleen associated with prominent megakaryopoiesis, and minimal fibrosis. The mice thus have several phenotypic similarities to human essential thrombocythemia (ET), suggesting that mutational analysis for activating mutations in FLT3 in ET may be warranted. The development of a phenotype characterized by thrombocytosis in vav-FLT3 transgenic mice is not likely to be an integration site effect, because it was observed in five independent founders. It is most likely that the observed phenotype is due to preferential or enhanced activity of the vav promoter in megakaryocytic precursors, but it may also be due to selective effects of activated FLT3 on megakaryocytic progenitors.

Unlike the FLT3-ITD bone marrow transplant (BMT) mouse model (Kelly et al., 2002), the transgenic vav FLT3 model does not exhibit a fatal myeloproliferative disorder characterized by leukocytosis with neutrophilia, but appears to be a well-tolerated syndrome manifested by splenomegaly and thrombocytosis, features suggestive of ET, a fairly well-tolerated MPS. These phenotypic differences may be reflective of a number of factors, including the strength of the viral LTR versus the vav promoter, effects of retroviral integration, as well as modifier influences of the mouse strains used in these two different model systems (Balb/c versus FVB). It is interesting to note that, to our knowledge, activating FLT3 mutations have not been identified in patients with myeloproliferative disease. As might be suggested by our transgenic FLT3 model, it is plausible that there may be a population of patients who harbor activating FLT3 mutations that may never come to clinical attention.

Activating mutations of FLT3 have been reported in a spectrum of myeloid malignancies, and rarely reported in cases of megakaryocytic leukemia (Kiyoi et al., 1999; Gilliland and Griffin, 2002). These mice may therefore also be useful in developing murine models of acute megakaryocytic leukemias.

The range of hematopoietic malignancies in which activating FLT3 mutations have been identified in humans now span lymphoid as well as myeloid neoplasms. Two lines of vav-FLT3 transgenic mice also developed lymphoid malignancies. One line developed a B-cell lymphoma, while another developed a T-lymphoma. Although rare, activating mutations in FLT3 have been reported in both B- and T-leukemia/lymphoma in humans (Ferrando et al., 2002; Armstrong et al., 2003). These transgenic mice thus afford an opportunity to study these tumors in a murine model. In both lines, the lymphoid tumors that developed were clonally derived, indicating that second mutations are necessary for development of tumors. The tumors were readily transplantable into secondary recipient mice, affording an opportunity to test small-molecule inhibitors of FLT3 as therapeutic agents. We observed that there was a statistically significant prolongation of survival in animals treated with the FLT3-selective inhibitor PKC412 (Weisberg et al., 2002). PKC412 is currently in Phase II trials for treatment of relapsed AML associated with activating mutations of FLT3. These data indicate that it may also be appropriate to test the efficacy of PKC412, or other small-molecule inhibitors of FLT3, in treatment of lymphoid malignancies associated with activating mutations in FLT3.

Materials and methods

Construction of transgenic mice

Human FLT3 cDNA harboring the W51 ITD mutation isolated from Kelly et al. (2002) was subcloned into HS321/45 vav hematopoietic vector (Ogilvy et al., 1999a) between the SfiI and NotI sites. Prokaryotic sequences were removed by digesting with SacII and gel purification using Qiaex II beads (Qiagen). Purified constructs were microinjected into single-cell fertilized FVB embryos that were subsequently transferred into the oviduct of a pseudopregnant FVB mouse in the Transgenic Core Facility at Brigham and Women's Hospital. Founder mice (nine) were identified by both PCR and Southern blot analysis of DNA isolated from tail clippings. Crossing with normal FVB mice generated progeny of the founder lines.

Assessment of transgenic mice

Animals were monitored several times per week for the development of disease by general inspection and palpation of the spleen and cervical, femoral, and axillary lymph nodes. Mice with clinically evident disease were killed and analysed. Peripheral blood was collected from the retro-orbital cavity using a heparinized glass capillary. A blood smear was prepared and stained with Wright and Giemsa (Sigma). Manual white blood counts were performed and an aliquot of blood was analysed using the ADVIA 120 Hematology system (Bayer) for total and differential white blood cell (WBC) and platelet counts. Mice were anesthetized using methoxyfluorane (Medical Developments, Australia) and then killed by cervical dislocation. The spleen, liver, heart, lungs, intestines, hind limb bones, kidneys, as well as abnormally enlarged lymph nodes or tumor masses were examined and collected. Spleen weights and any other pathologic findings at necropsy were recorded. Cells from bone marrow were isolated by flushing femurs and tibias with phosphate-buffered saline (PBS). Single-cell suspensions were prepared by passing spleen and lymph node tumor tissue through nylon mesh (Falcon, Lincoln Park, NJ, USA) dampened with PBS and were stored in 10% dimethylsulfoxide/90% fetal calf serum at −80°C following lysis of RBCs performed using red cell lysis solution (Puregene).

Histopathology

Murine tissues were fixed for at least 24 h in 10% neutral buffered formalin (Sigma), dehydrated in alcohol, cleared in xylene, and infiltrated with paraffin on an automated processor (Leica, Bannockburn, IL, USA). Tissue sections (4 μm thick) were placed on charged slides, deparaffinized in xylene, rehydrated through graded alcohol solutions, and stained with hematoxylin and eosin (H&E).

Western blot analysis

Single-cell suspensions of mouse organs prepared at necropsy were collected by centrifugation, washed in PBS and lysed with 1 × lysis buffer (Cell Signaling) containing 1 mM PMSF and complete protease inhibitor cocktail tablets (Roche). Loading buffer (1 × ) containing 2% SDS and 40 μ M DTT was added to equivalent micrograms of protein cell lysate which was subsequently separated using 7.5% SDS–PAGE and transferred to membranes. Western analysis was performed using a 1 : 200 dilution of anti-FLT3 SC-479 (Santa Cruz, CA, USA) as the primary antibody, followed by horseradish peroxidase-conjugated secondary antibody (Amersham Life Science) and detection performed using the Western Lightning system (Perkin-Elmer).

Flow-cytometric immunophenotyping

Single-cell suspensions of bone marrow, spleen, and lymph nodes were prepared. Red blood cells were lysed in ammonium chloride solution (150 mmol/l NH4Cl, 10 mmol/l KHCO3, 0.1 mmol/l EDTA, pH 7.4) for 5 min at room temperature. The cells were washed in PBS with 0.1% NaN3 and 0.1% bovine serum albumin (BSA; staining buffer). To block nonspecific Fc receptor-mediated binding, the cells were preincubated with supernatant from the 2.4G2 hybridoma line (anti-CD16/CD32; Cell Line American Type Culture Collection, Rockville, MD, USA) for 20 min on ice. Aliquots of 0.5–1.0 × 106 cells were then stained for 20 min on ice with monoclonal antibodies specific for B220 (CD45R), CD19, IgM, CD3, CD43, CD41, Gr-1, Mac-1, CD4, or CD8 conjugated with fluorescein isothiocyanate (FITC), phycoerthyrin (PE), allophycocyanin or biotin. Binding of biotinylated primary antibodies was detected using PE-conjugated streptavidin (Immunotech, Westbrook, ME, USA). Cells were washed once in staining buffer, followed by four-color flow-cytometric analysis with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). A minimum of 10 000 events was acquired and viable cells gated for analysis on the basis of forward- and side-scatter signals.

Mouse TPO measurements

Murine TPO levels were ascertained from both nontransgenic WT FVB mice and transgenic vav-FLT3-ITD animals, using the Quantikine M Mouse TPO Immunoassay Kit (R&D Systems, Minneapolis, MN, USA) and following the manufacturer's protocol. Plasma was collected from animals by a rapid centrifugation of EDTA-treated blood samples that were used immediately to determine TPO levels or stored at −20°C until further use. Standard curves were generated and samples run in triplicate.

Immunoglobulin gene rearrangement analysis

Genomic DNA was prepared from single-cell suspensions of tumor cells and control DNA was isolated from a WT littermate using a PUREGENE DNA isolation kit according to the manufacturer's protocol (Gentra Systems, Minneapolis, MN, USA). A 20-μg quantity of genomic DNA was digested with either EcoRI or BglII, subjected to electrophoresis on a 0.8% agarose gel and transferred to nylon membranes (Hybond N+; Amersham, Arlington Heights, IL, USA). A 1.9 kb BamHI–EcoRI IgH fragment and a 1.5 kb BamHI–KpnI βTCR fragment were used as probes (a gift of Dr Ruben Carrasco), and random-labeled using 32PαdCTP (Boehringer Manheim, Indianapolis, IN, USA). Hybridization was performed at 65°C for 16 h and membranes were washed with 2 × SSC, 0.1% SDS for 20 min at room temperature, and exposed to photographic film (BioMax; Eastman Kodak, Rochester, NY, USA) at −80°C overnight.

Tumor cell transplantation

Single-cell suspensions from lymph node tumors were prepared as above. Cells were counted and diluted to a concentration of 2 × 105/ml. Recipient mice were subjected to a single dose of 450 rad and 0.5 ml of the tumor cell suspension was administered by tail vein injection.

Mouse drug trials

Secondary transplants of 1 × 105 cells of either B- or T-lymphoid disease were administered to sublethally irradiated FVB recipient mice as described above. 6% w/w PKC412 in Gelucire 44/14 (Gattefosse, France) was stored at 4oC as a waxy-solid formation. Prior to administration, the Gelucire/PKC412 waxy solid mixture was warmed in a 44oC water bath until liquid. The liquid mixture was then diluted with sterilized deionized water to produce a final PKC412 concentration of 12.0 mg/ml. The animals were weighed prior to treatment and administered 100 mg/kg/day of drug. Dosing was performed every 24 h by gavage of a maximum volume of 150 μl per animal using 22-gauge gavage needles (Hornbecks). Placebo animals received the same volume of a Gelucire 44/14 and water solution prepared in the same way as described above for the PKC412/Gelucire mixture. In each trial, mice were dosed with either drug or placebo. In comparing the survival time of the mice, all times were measured from the day of the secondary transplant. Survival curves were produced using the Kaplan–Meier estimates, and the significance of any difference in survival was assessed with the log-rank test. Spleen weights and WBC count differences between PKC412- and placebo-treated animals were compared with the Mann–Whitney U-test. All calculations were performed with the SPSS statistical package (SPSS, Chicago, IL, USA).

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Acknowledgements

We thank members of the Gilliland laboratory for valuable advice and discussions. This work was supported in part by NIH grants CA66996 and DK50654, and by a Leukemia and Lymphoma Society SCOR grant. BHL is a recipient of a Physician-Scientist Fellowship from the Leukemia Research Foundation and was previously supported by NIH departmental training grant 5T32HL 07627-16, and DGG is an Investigator in the Howard Hughes Medical Institute.

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Correspondence to Dwight Gary Gilliland.

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Lee, B., Williams, I., Anastasiadou, E. et al. FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model. Oncogene 24, 7882–7892 (2005). https://doi.org/10.1038/sj.onc.1208933

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Keywords

  • FLT3 tyrosine kinase
  • transgenic
  • mouse
  • leukemia

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