FLT3 Ligand Stimulation Research Articles

FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy

AbstractPoint mutations of D835/I836 of the FLT3 gene have been reported in adult acute myeloid leukemia (AML), but not in pediatric AML or acute lymphoblastic leukemia (ALL). FLT3-D835/I836 mutations were found in 6 (5.4%) of 112 children with ALL older than 1 year and in 8 (16.0%) of 50 infants with ALL. Missense mutations were found in 11 patients, 3-base pair deletions in 2 patients, and a deletion/insertion in 1 patient. Remarkably, FLT3-D835/I836 mutations were found in 8 (18.2%) of 44 infants with ALL with MLL rearrangements and in 4 (21.5%) of 19 patients with hyperdiploid ALL, but they were not found in any patients older than 1 year who had TEL-AML1 (n = 11), E2APBX1 (n = 4), or BCR-ABL (n = 6) fusion genes. Although infant ALL patients with mutations had poorer prognoses than did those without mutations, pediatric ALL patients with mutations who were older than 1 year had good prognoses. We also found FLT3-D835 mutations in 2 of 11 leukemic cell lines with MLL rearrangements. FLT3 was highly phosphorylated in these cell lines with FLT3-D835 mutations, leading to constitutive activation of downstream targets such as signal transducer and activator of transcription 5 (STAT5) without FLT3 ligand stimulation. These results suggested that FLT3-D835/I836 mutations are one of the second genetic events in infant ALL with MLL rearrangements or pediatric ALL with hyperdiploidy. (Blood. 2004;103:1085-1088)

Development of Predictive Biomarker and Optimal Treatment Strategy with FLT3 Inhibitors in Acute Myeloid Leukemia

【Background】Recently, FLT3 inhibitors are approved for FLT3 mutated AML patients and other FLT3 inhibitors are also in clinical development. Since the target selectivity and the inhibitory activity of FLT3 inhibitors are varied, it is required to establish a therapeutic strategy in consideration of their characteristics. Most FLT3 mutated AML cells co-express wild type (Wt)-FLT3, and we previously demonstrated that FLT3 ligand (FL) stimulation attenuated the inhibitory effect of FLT3 inhibitors through the activation of Wt-FLT3 in Wt- and ITD-FLT3 co-expressing cells; however, little is known about this inhibitory mechanism in recently developed FLT3 inhibitors. In this study, we aimed to clarify the efficacy and characteristics of four FLT3 inhibitors including the impact of FL on AML cell lines and primary patients cells, and to identify biomarkers for drug selection and prediction of their efficacy. 【Methods】We examined the growth inhibitory effects of midostaurin, quizartinib, gilteritinib and FF-10101 at various concentrations of FL in ITD-FLT3 expressing 32D cells, Wt- and ITD-FLT3 co-expressing 32D cells,and FL and Wt-FLT3 co-expressing 32D cells. The inhibitory effects of these four FLT3 inhibitors were also examined in 87 primary AML cells with or without FLT3 mutationin vitro, and the correlation between their efficacy and clinical and molecular characteristics including genetic alterations and FLT3-ITD allelic ratio (ITD-AR) were investigated. Moreover, characteristics of residual AML cells after the treatment with FLT3 inhibitors were also examined in patient-derived xenografts (PDX) -AML model. 【Results】The inhibitory effect of FLT3 inhibitors was significantly impaired by FL stimulation dose dependently in all inhibitors except for midostaurin in Wt- and ITD-FLT3 co-expressing 32D cells. In FL and Wt-FLT3 co-expressing cells, GI50 value of gilteritinib was higher than that in ITD-FLT3 solo-expressing cells, and the similar tendency was observed with quizartinib. We examined the Growth inhibitory effects of these inhibitors in 33 FLT3 mutated and 54 FLT3 un-mutated primary AML cells. In primary cells, midostaurin showed lower selectivity to FLT3 mutation compared with other inhibitors. Subsequently, the correlation between the efficacy of FLT3 inhibitors and ITD-AR was examined. RNA or DNA based ITD-AR was not related to the growth inhibitory effect of FLT3 inhibitors except for gilteritinib in FLT3-ITD mutated patient cells; the GI50 value of gilteritinib in AML cells with RNA based ITD-AR-low were significantly higher than those in RNA based ITD-AR-high (P=0.036) (Figure).Moreover, FLT3-ITD cells with mutated NPM1 tend to have higher GI50values for all of FLT3 inhibitors, irrespective of ITD-AR. In AML-PDX treated with quizartinib or gilteritinib, FLT3-ITD-AR in residual AML cells was lower than that of non-treated cells, suggesting that co-expression level of Wt-FLT3 is related to the response to FLT3 inhibitors in vivo. 【Conclusions】FL affected the efficacy of FLT3 inhibitors in Wt- and ITD-FLT3 co-expressing cells, and the inhibitory effects on ITD-FLT3 and FL-Wt-FLT3 pathway were different among FLT3 inhibitors. Furthermore, NPM1 mutation and RNA based ITD-AR might be predictive biomarkers for the efficacy of FLT3 inhibitors in FLT3-ITD positive AML. The appropriate therapeutic strategy based on the characteristics of each inhibitor is necessary. Disclosures Ishikawa: Bristol-Myers Squibb: Honoraria; Abbvie GK.: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Celgene Co., Ltd.: Honoraria. Goto:Celgene Co., Ltd.: Honoraria; Novartis Pharma Co., Ltd.: Honoraria; JCR Pharmaceuticals Co., Ltd.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Honoraria. Ozawa:Pfizer Japan Inc.: Honoraria; Novartis: Honoraria; Kyowa-Hakko Kirin: Honoraria; Astellas Pharma Inc.: Honoraria. Kiyoi:Zenyaku Kogyo Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Pfizer Japan Inc.: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Bristol-Myers Squibb: Research Funding; Perseus Proteomics Inc.: Research Funding.

Effect of Fms-like tyrosine kinase 3 (FLT3) ligand (FL) on antitumor activity of gilteritinib, a FLT3 inhibitor, in mice xenografted with FL-overexpressing cells.

Therapeutic effects of FLT3 inhibitors have been reported in acute myeloid leukemia (AML) with constitutively activating FLT3 mutations, including internal tandem duplication (ITD) and point mutation, which are found in approximately one-third of AML patients. One of the critical issues of treatment with FLT3 inhibitors in FLT3-mutated AML is drug resistance. FLT3 ligand (FL) represents a mechanism of resistance to FLT3 inhibitors, including quizartinib, midostaurin, and sorafenib, in AML cells harboring both wild-type and mutant FLT3 (FLT3wt/FLT3mut). Here, we investigated the effect of FL on the efficacy of gilteritinib, a FLT3 inhibitor, in AML-derived cells in vitro and in mice. In contrast to other FLT3 inhibitors, FL stimulation had little effect on growth inhibition or apoptosis induction by gilteritinib. The antitumor activity of gilteritinib was also comparable between xenograft mouse models injected with FL-expressing and mock MOLM-13 cells. In the FLT3 signaling analyses, gilteritinib inhibited FLT3wt and FLT3-ITD to a similar degree in HEK293 and Ba/F3 cells, and similarly suppressed FLT3 downstream signaling molecules (including ERK1/2 and STAT5) in both the presence and absence of FL in MOLM-13 cells. Co-crystal structure analysis showed that gilteritinib bound to the ATP-binding pocket of FLT3. These results suggest that gilteritinib has therapeutic potential in FLT3-mutated AML patients with FL overexpression.

CLONAL ETIOLOGY OF EMERGENCY DENDRITIC CELL DEVELOPMENT

Dendritic cells (DCs) are critical mediators of both innate and adaptive immune responses. Increasing DC numbers by administration of Flt3 ligand (FL) provides therapeutic benefit for infection and cancer immunotherapy. However, how FL signals regulate emergency DC development at the clonal level is largely unknown. To interrogate the early cellular and molecular events within hematopoietic stem and progenitor cells (HSPCs) after FL stimulation, we develop a single cell multi-omics profiling approach. This allows simultaneous measurement of surface marker phenotype, division history and the transcriptome of single HSPCs treated with PBS or FL after transfer into non-irradiated recipients. We observe hyperproliferation of most HSPCs after FL treatment. In particular, we identify a unique group of hyper-proliferative cells that co-expresses gene signatures of both ‘multipotent progenitors’ and ‘DC progenitors’, indicative of the earliest molecular and cellular events during emergency DC development. To understand how FL affects HSPC fate towards other lineages at the clonal level, we tag individual HSPCs with unique DNA barcodes and compare their development, with or without FL stimulation in vivo. We observe no evidence of lineage diversion of HSPCs away from alternative fates and instead determine that pre-existing multi-/oligo-potent retain their potency and selectively expand a DC-producing sub-branch, leading to preferential production of DCs. Collectively, our results suggest that lineage programs within single HSPCs are established and stable, and that selective clonal expansion, not fate ‘plasticity’ of HSPCs via extrinsic regulation, explains FL-mediated emergency DC development. These findings provide new insight into the control and regulation of fate during hematopoiesis.

TGFβ1 synergizes with FLT3 ligand to induce chemoresistant quiescence in acute lymphoblastic leukemia with MLL gene rearrangements.

Fms-like tyrosine kinase 3 (FLT3) is highly expressed in mixed-lineage leukemia (MLL) gene-rearranged acute lymphoblastic leukemia (MLL + ALL) with a dismal prognosis. We previously reported that FLT3 ligand (FL) stimulation induced cell cycle arrest in MLL + ALL cells leading to resistance against anti-leukemic agents. Given that FL stimulation enhanced transforming growth factor (TGF)β1 mRNA levels in MLL + ALL cells, we extensively examined the effect of TGFβ1 on the cell cycle progression and chemosensitivity in MLL + ALL cells, and found that TGFβ1 stimulation induced MLL + ALL cells into cell cycle arrest resistant to arabinosyl cytosine; its effect was markedly enhanced in synergy with FL. Thus, it is likely that TGFβ1 and FL, both abundantly produced by bone marrow stromal cells, function in a coordinated manner to render MLL + ALL cells chemoresistant, which should lead to the development of minimal residual disease (MRD) resulting in relapse. The use of inhibitors against FLT3 and TGFβ1 may become a useful strategy for eradicating MRD in MLL + ALL.

TGFβ1 Induces MLL-Rearranged Acute Lymphoblastic Leukemia into Chemoresistant Quiescence in a Coordinated Manner with FLT3 Ligand

Background Acute lymphoblastic leukemias with rearrangements of mixed-lineage leukemia gene (MLL+ALL) frequently develop in infants and have a dismal prognosis. The refractory nature of this leukemia is known to be associated with persistence of high levels of minimal residual disease (MRD). We previously reported that FLT3 ligand (FL) stimulation of MLL+ALL cells, which express FLT3 at high levels, induced quiescence resistant to anti-leukemic agents, to the process of which up-regulation of p27, one of cyclin-dependent kinase inhibitors (CDKIs), was closely attributable. Because a vast amount of FL is secreted from bone marrow stromal cells, it was postulated that this FLT3/FL interaction should contribute, at least in part, to the high levels of MRD in MLL+ALL (Furuichi et. al. Cancer Res 2007). Of interest, in B-cell precursor ALLs other than MLL+ALL, FL stimulated their proliferation. Therefore, we performed the microarray analysis using the MLL+ALL cell line and the ETV6-RUNX1-positive cell line for comparing changes in the gene expression profiles at 24h after FL stimulation, and picked up the transforming growth factor β1 (TGFβ1) as a gene whose mRNA level was conversely regulated between two cell lines. TGFβ1 is a growth inhibitory cytokine for some cancer cells and known to have a potential to induce hematopoietic stem cell quiescence. In the present study, we examined the effects of TGFβ1 and/or FL on MLL+ALL cells particularly in terms of induction of quiescence and chemosensitivity.Materials and Methods The MLL+ALL cell line KOCL58 (MLL-AF4+) was used throughout the study to examine TGFβ1 mRNA, cell cycle progression, induction of apoptosis, and changes in chemosensitivity in the presence of TGFβ1 and/or FL.Results 1. We first examined changes in TGFβ1 mRNA after FL (20ng/ml) stimulation by real time RT-PCR in KOCL58 cells, and confirmed that TGFβ1 mRNA expression level was significantly increased (1.5 fold at 24h). Of note, when the cells were stimulated with FL and TGFβ1 (10ng/ml), TGFβ1 mRNA expression level was further increased (3.5 fold) between 7 and 24h. 2. We next evaluated the effect of TGFβ1 on proliferation. When KOCL58 cells were incubated with a low concentration of TGFβ1 (0.33ng/ml) in the presence or absence of FL (20ng/m), flow cytometric analysis using the BrdU/PI double staining showed that the G0/G1 population (35.2%; before stimulation) was increased in the presence of TGFβ1 (48.4%) or FL (53.0%), and further in the presence of TGFβ1+FL (75.1%). We also examined expression levels of cell cycle regulating molecules, and found that p27 was significantly increased in the presence of FL and further in the presence of TGFβ1+FL. This p27 up-regulation was due to post-transcriptional mechanism, because p27 mRNA remained unchanged but the half-life of p27 protein was elongated in the presence of FL and/or TGFβ1. 3. To elucidate whether TGFβ1 affects the sensitivity of MLL+ALL cells to a cell cycle dependent anti-leukemic agent AraC, we pre-incubated KOCL58 cells in the presence of TGFβ1 and/or FL for 72h and then exposed to AraC (500nM) for 24h. Flow cytometric analysis using FITC-conjugated Annexin V demonstrated that the Annexin V-negative viable population (%) was decreased by AraC exposure (25.6±1.4%), but it was significantly restored by pre-incubation with FL (39.4±1.7%, P<0.0005) and further by pre-incubation with TGFβ1+FL (49.3±5.9%, P<0.003), although not significantly different by pre-incubation with TGFβ1 alone. Flow cytometric analysis using anti-active caspase 3 antibody also demonstrated that the positive population was increased to 50.8% by AraC exposure, but it was decreased by pre-incubation with either TGFβ1 (27.5%) or FL (24.8%), and further by pre-incubation with TGFβ1+FL (10.2%) (Figure). 4.We performed chmosensitivity experiments to AraC (800nM) using KOCL58 cells adhering to bone marrow stromal cells, which secrete FL and TGFβ1, in the presence of FLT3 inhibitor (CEP701, 5nM) and/or TGFβ-receptor 1 inhibitor (HTS, 5mM), and found that KOCL58 cells adhering to stromal cells showed a marked resistance to AraC (60% increase in Annexin V-negative population), but this restoring effect was partially (~20%) alleviated by each of inhibitors.[Discussion] TGFβ1 should be involved in the development of MRD in MLL+ALL in synergy with FL, and a combined use of inhibitors against FLT3 and TGFβ1 might be effective for eradicating MRD in MLL+ALL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mechanism of FLT3 Ligand Dependent Resistance to FLT3 Inhibitors

Background:FLT3mutation, which is found in about 30% of acute myeloid leukemia (AML) patients, is involved in the signaling pathway of autonomous proliferation and differentiation block in leukemia cells. Since FLT3 mutation is associated with a poor prognosis in AML patients, mutated FLT3 serves as an important molecular target in the treatment of leukemia. To date, several FLT3 inhibitors are undergoing investigation, but their clinical efficacies were lower than expected. The inhibitory effects of FLT3 inhibitors were mainly evaluated using the sole mutant FLT3-expressing cells in pre-clinical studies, while clinicallymost AML cells harboring FLT3 mutation co-express wild-type (Wt) FLT3, suggesting that FLT3 ligand (FL)-dependent Wt-FLT3 signal might cause the lower clinical efficacies of FLT3 inhibitors. Recently, it was reported that administration of FLT3 inhibitors induced increased concentration of FL in plasma. Thus, here we analyzed how FL-dependent signal affects the inhibitory effect of FLT3 inhibitors and proliferation on Wt- and ITD-FLT3-coexpressing cells.Methods: 5 kinds of FLT3-expressing 32D cells were established: Wt-FLT3, FLT3-ITD, extra cellular domain-lacking FLT3-ITD (cyFLT3-ITD), Wt- and FLT3-ITD co-expressing and Wt- and cyFLT3-ITD co-expressing 32D cells. The growth inhibitory effects of 6 FLT3 inhibitors (AC220, CEP701, FI-700, KW2449, PKC412 and Sorafenib) with and without FL stimulation were evaluated by MTT assay. Furthermore, cell cycle analysis was performed to evaluate cell proliferation, and inhibitory effects on FLT3 kinase and its downstream molecules were also evaluated by western-blot. In vivo, cells were inoculated into C3H/Hej mice intravenously to follow survival rate and NOD/SCID mice subcutaneously to compare tumor volume between sole ITD-FLT3-expressing 32D cells and Wt and ITD-FLT3-coexpressing 32D cells.Results: The FL-stimulation reduced growth inhibitory effects by FLT3 inhibitors on Wt- and ITD-FLT3-co-expressing 32D cells, while those reducing effects were little on the sole extracellular domain lacked ITD-FLT3 (cyITD-FLT3)-expressing 32D cells. Of note, FL-stimulation induced cell cycle arrest dose-dependently, resulting reduced proliferation in Wt- and ITD-FLT3-co-expressing cells. In vivo, all syngeneic C3H mice inoculated with sole cyITD-FLT3-expressing 32D cells and sole ITD-FLT3-expressing 32D cells developed leukemia within 16 days and 72 days respectively, but mice inoculated with Wt- and ITD-FLT3-co-expressing cells survived more than 100 days (P<0.0001). Furthermore, the growth of tumors driven by sole ITD- and cyITD- FLT3-expressing 32D cells was significantly faster than tumors driven by Wt- and ITD-FLT3-co-expressing cells in NOD/SCID subcutaneous model. Western blot shows AKT and ERK are activated in Wt- and ITD-FLT3-co-expressing cells by FL stimulation. STAT3 is highly phosphorylated in Wt- and ITD-FLT3-co-expressing cells and can be further activated by FL stimulation, while the phosphorylation is weak in sole ITD expressing cells. Immunopricipitation demonstrated that FLT3 ligand activated only Wt-FLT3 but not ITD-FLT3 in co-expressing cells. Furthermore, p21 (CIP1/WAF1) can be up-regulated by FL and induce cell cycle arrest in Wt- and ITD-FLT3-co-expressing cells. FL impeded the inhibitory effect of FLT3 inhibitors by persistent activation of ERK and AKT through Wt-FLT3. Also, down-regulation of p21 and Mcl-1induced by FLT3 inhibitor can be suppressed by FL.Conclusions: These results suggested that FLT3 Ligand dependent resistance to FLT3 inhibitors were associated with reduced proliferation ability caused by up-regulation of P21 and persistent ERK and AKT activation through Wt-FLT3 signal. DisclosuresKiyoi:Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding. Naoe:Otsuka Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis Pharma: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding.

Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Hes1 Is Responsible For Notch Signaling-Mediated Suppression Of Acute Myeloid Leukemia Development Via Suppression Of FLT3 Expression

BackgroundThe transcription factor Hairy enhancer of split1 (Hes1) is well characterized as a downstream target of Notch signaling. Hes1 is a basic helix-loop-helix-type protein, and represses target gene expression. Notch signaling has been proposed to play both pro- and anti-tumorigenic roles; it promotes development of T-cell acute lymphoblastic leukemia (T-ALL), while serves as a tumor suppressor for acute myeloid leukemia (AML). Hes1 has been proven as an essential mediator of Notch signaling in T-ALL development. In contrast, we reported, in the last annual meeting, that Hes1 functions as a tumor suppressor against AML development, using a mouse model of AML induced by the MLL-AF9 fusion protein. We further explored the mechanism of Hes1-mediated suppression of AML development. MethodsCommon myeloid progenitors (CMPs) purified from RBP-Jf/f mouse bone marrow (BM) were serially transduced with MLL-AF9 and Cre recombinase (iCre) using retroviral vectors, and transplanted into lethally irradiated syngenic mice. CMPs from Hes1-/- mouse fetal liver were also retrovirally transduced with MLL-AF9 and transplanted after multiple rounds of replating, and then, expression levels of downstream targets were evaluated by cDNA array. Next Hes1 was retrovirally re-expressed in MLL-AF9/Hes1-/- cells and these cells were transplanted. MLL-AF9-transduced cells were treated with a hamster anti-mouse Notch agonistic antibody (Notch Ab). ResultsMice transplanted with MLL-AF9/RBP-J-/- cells developed leukemia at shorter latencies than those with MLL-AF9/RBPJ+/+ cells. MLL-AF9-transduced Hes1-/- cells formed the higher number of colonies at third replating compared with MLL-AF9-transduced Hes1+/+ cells. When infused into irradiated syngenic mice, MLL-AF9/Hes1-/- cells developed leukemia at shorter latencies than MLL-AF9/ Hes1+/+ cells (MLL-AF9/Hes1-/-, 7-10 weeks, n=18 vs MLL-AF9/Hes1+/+, 10-14 weeks, n=18; p<0.001). When Hes1 was retrovirally re-expressed in MLL-AF9/Hes1-/- cells, these cells developed leukemia in recipient mice at longer latencies than mock-transduced MLL-AF9/Hes1-/- cells (Hes1/MLL-AF9, 12 weeks, n=8 vs mock/MLL-AF9, 5-7 weeks, n=7 p<0.001). When treated with an anti-Notch2 Ab, MLL-AF9/Hes1+/+ cells underwent apoptosis, whereas MLL-AF9/Hes1-/- cells did not. These results indicate that Hes1 is a definitive downstream mediator for Notch signaling-mediated suppression of AML. Among the genes with different expression levels between MLL-AF9/Hes1-/- and MLL-AF9/Hes1+/+ leukemia cells, FMS-like tyrosine kinase 3 (FLT3) was expressed at significantly higher levels in MLL-AF9/Hes1-/- leukemia cells as well as RBP-J-null Background. It was also demonstrated that FLT3 and ERK were phosphorylated with FLT3 ligand stimulation in the MLL-AF9-immortalized cells specifically with the Hes1-/- Background. An FLT3 inhibitor efficiently abrogated the proliferation of MLL-AF9/Hes1-/- leukemia cells. We accessed the independent database containing mRNA expression profiles and found that the expression level of Flt3 mRNA was negatively correlated with those of Hes1 in AML samples. ConclusionCanonical Notch signaling serves as a tumor suppressor in MLL-AF9-induced AML through upregulation of Hes1. Hes1 is an essential Notch signaling mediator for AML suppression. At least a part of Hes1 function might be explained by repression of FLT3. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay

Protein-tyrosine phosphatases (PTPs) are important regulators of signal transduction processes. Essential for the functional characterization of PTPs is the identification of their physiological substrates, and an important step towards this goal is the demonstration of a physical interaction. The association of PTPs with their cellular substrates is, however, often transient and difficult to detect with unmodified proteins at endogenous levels. Density-enhanced phosphatase-1 (DEP-1/PTPRJ) is a regulator of hematopoietic cell functions, and a candidate tumor suppressor. However, association of DEP-1 with any of its proposed substrates at endogenous levels has not yet been shown. We have previously obtained functional and biochemical evidence for a direct interaction of DEP-1 with the hematopoietic receptor-tyrosine kinase Fms-like tyrosine kinase-3 (FLT3). In the current study we have used the method of in situ proximity ligation assay (in situ PLA) to validate this interaction at endogenous levels, and to further characterize it. In situ PLA readily detected association of endogenous DEP-1 and FLT3 in the human acute monocytic leukemia cell line THP-1, which was enhanced by FLT3 ligand (FL) stimulation in a time-dependent manner. Association peaked between 10 and 20 min of stimulation and returned to basal levels at 30 min. This time course was similar to the time course of FLT3 autophosphorylation. FLT3 kinase inhibition and DEP-1 oxidation abrogated association. Consistent with a functional role of DEP-1-FLT3 interaction, stable knockdown of DEP-1 in THP-1 cells enhanced FL-induced ERK1/2 activation. These findings support that FLT3 is a bona fide substrate of DEP-1 and that interaction occurs mainly via an enzyme-substrate complex formation triggered by FLT3 ligand stimulation.

P123 Type I IFN augments FLT3 ligand-dependent plasmacytoid dendritic cell development from common lymphoid progenitor

Dendritic cells (DC), belonging to the innate immune system, are specialized for antigen processing and presentation and orchestrate the adaptive immune response to infection. Among different DC populations, plasmacytoid DCs (pDCs) are highly potent type I IFN (IFN-I) producing cells during infection relative to cDCs. Aberrant secretion of IFN-I by pDCs in the absence of infection has been implicated in autoimmune diseases, including systemic lupus erythematosus (SLE) and psoriasis. However, signals dictating pDC generation and activity remain incompletely understood. DC development was done using coculture of common lymphoid progenitors (CLPs) or common dendritic cell progenitors (CDPs) with AC6 stromal cell line in the presence of Flt3 ligand (FL). We were able to derive pDC and cDC in vitro. Here, we report a synergistic role for FL and IFN-I in pDC development from CLPs. Both cDCs and pDC were generated from CLP in response to FL, but pDC generation required higher signal strength of Flt3 receptor and depended on concurrent IFN-I signaling. While CLPs predominantly generated cDCs at low dose of FL, pDCs preferentially developed at higher cytokine doses. Absence of IFN-I receptor 1 (IFNAR1) or mutation of its effector transcription factors STAT1 or STAT2 reduced pDC development in vivo and impaired FL-dependent pDC formation from CLPs in vitro and in vivo. Moreover, exogenous IFN-I enhanced FL-induced pDC genesis, and neutralizing antibody to IFNAR1 abrogated this process. Interestingly, IFN-I was induced in CLPs in response to FL stimulation, facilitating up-regulation of Flt3 expression on CLPs and their differentiation into pDCs. Collectively, these data define a cooperative effect of FL and IFN-I in pDC differentiation from CLPs through an autocrine positive feedback loop that could provide a therapeutic target for treatment of autoimmune disease.

Protein-tyrosine Phosphatase DEP-1 Controls Receptor Tyrosine Kinase FLT3 Signaling

Fms-like tyrosine kinase 3 (FLT3) plays an important role in hematopoietic differentiation, and constitutively active FLT3 mutant proteins contribute to the development of acute myeloid leukemia. Little is known about the protein-tyrosine phosphatases (PTP) affecting the signaling activity of FLT3. To identify such PTP, myeloid cells expressing wild type FLT3 were infected with a panel of lentiviral pseudotypes carrying shRNA expression cassettes targeting different PTP. Out of 20 PTP tested, expressed in hematopoietic cells, or presumed to be involved in oncogenesis or tumor suppression, DEP-1 (PTPRJ) was identified as a PTP negatively regulating FLT3 phosphorylation and signaling. Stable 32D myeloid cell lines with strongly reduced DEP-1 levels showed site-selective hyperphosphorylation of FLT3. In particular, the sites pTyr-589, pTyr-591, and pTyr-842 involved in the FLT3 ligand (FL)-mediated activation of FLT3 were hyperphosphorylated the most. Similarly, acute depletion of DEP-1 in the human AML cell line THP-1 caused elevated FLT3 phosphorylation. Direct interaction of DEP-1 and FLT3 was demonstrated by "substrate trapping" experiments showing association of DEP-1 D1205A or C1239S mutant proteins with FLT3 by co-immunoprecipitation. Moreover, activated FLT3 could be dephosphorylated by recombinant DEP-1 in vitro. Enhanced FLT3 phosphorylation in DEP-1-depleted cells was accompanied by enhanced FLT3-dependent activation of ERK and cell proliferation. Stable overexpression of DEP-1 in 32D cells and transient overexpression with FLT3 in HEK293 cells resulted in reduction of FL-mediated FLT3 signaling activity. Furthermore, FL-stimulated colony formation of 32D cells expressing FLT3 in methylcellulose was induced in response to shRNA-mediated DEP-1 knockdown. This transforming effect of DEP-1 knockdown was consistent with a moderately increased activation of STAT5 upon FL stimulation but did not translate into myeloproliferative disease formation in the 32D-C3H/HeJ mouse model. The data indicate that DEP-1 is negatively regulating FLT3 signaling activity and that its loss may contribute to but is not sufficient for leukemogenic cell transformation.

Targeting the Protein Phosphatase, Shp2, Reduces FLT3-ITD-Induced Hyperproliferation of Murine Hematopoietic Progenitors.

Abstract 827Internal tandem duplications in the FMS-like receptor tyrosine kinase receptor (FLT3-ITDs) are present in approximately 25% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. FLT3-ITDs induce FLT3 ligand (FL)-independent hyperproliferative cellular growth and promiscuous activation of STAT5; however, the molecular mechanism underlying aberrant activation of these signaling molecules is largely unknown. Upon FLT3 ligand (FL) stimulation, tyrosine 599 (Y599) of WT-FLT3 recruits the protein tyrosine phosphatase, Shp2 and induces cell proliferation and Erk activation. In several FLT3-ITDs, including N51-FLT3 and N73-FLT3, Y599 is duplicated. These findings led us to hypothesize that increased recruitment of Shp2 to N51-FLT3 or N73-FLT3, via Y599, contributes to N51-FLT3- and N73-FLT3-induced cellular hyperproliferation and promiscuous STAT5 activation. Using Baf3 cells stably expressing WT-FLT3, N51-FLT3, or N73-FLT3, co-immunoprecipitation assays demonstrated that Shp2 associates with WT-FLT3 in a FL-dependent manner, while Shp2 is constitutively associated with N51-FLT3 and N73-FLT3 independent of FL-stimulation. To investigate the functional role of Shp2 in FLT3-ITD-induced leukemogenesis, Baf3 cells expressing WT-FLT3, N51-FLT3, or N73-FLT3 were transfected with a mammalian expression vector encoding a U6 polymerase III–directed Shp2-specific short-hairpin RNA (shRNA) or a scrambled shRNA and selected in puromycin. Immunoblot analysis revealed significant reduction of Shp2 expression by the Shp2-specific shRNA and no change in Shp2 expression by the scrambled shRNA in all cell lines. Upon knock-down of Shp2 in Baf3/WT-FLT3 cells, proliferation was minimally reduced based on thymidine incorporation assays; however, knock-down of Shp2 in Baf3/N51-FLT3 and Baf3/N73-FLT3 cells significantly reduced proliferation, both at baseline and in response to FL stimulation. To further test the role of Shp2 in FLT3-ITD-induced transformation, Shp2flox/flox mice were crossed with Mx1Cre transgenic mice to produce Shp2flox/flox Mx1Cre+ mice. Shp2flox/floxMx1Cre+ and Shp2flox/floxMx1Cre- pairs were treated with 3 intraperitoneal injections of polyI/polyC to induce recombination of the Shp2 allele and generate a null allele in the Mx1Cre+ animals. PCR and immunoblot analysis revealed ablation of the flox allele and reduction of Shp2 protein, respectively, in Shp2flox/floxMx1Cre+ bone marrow as soon as 2 weeks post injection. Low density mononuclear cells (LDMNCs) isolated from polyI/polyC-treated Shp2flox/floxMx1Cre- and Shp2flox/floxMx1Cre+ mice were transduced with either pMSCV-WT-FLT3 or pMSCV-N51-FLT3. Transduced cells were sorted for EGFP (enhanced green fluorescent protein) expression and subjected to thymidine incorporation assays. Shp2flox/floxMx1Cre+ cells transduced with N51-FLT3 demonstrated significantly reduced proliferation at baseline and in response to FL stimulation compared to Shp2flox/floxMx1Cre- cells expressing N51-FLT3. To pharmacologically examine the role of Shp2 in FLT3-ITD-induced-hyperproliferation and constitutive activation of Stat5, we utilized a Shp2 inhibitor, II-B08, identified from a library of indole salicylic acid derivatives. II-B08 exhibits an IC50 of 5.5 μM for Shp2 and demonstrates excellent cellular activity. Bone marrow LDMNCs retrovirally transduced with WT-FLT3 or N51-FLT3 were incubated in the absence of or in the presence of increasing concentrations of II-B08 and plated into thymidine incorporation assays. A minimal and non-significant reduction in proliferation was observed in WT-FLT3 expressing cells in the presence of II-B08; however N51-FLT3 expressing cells demonstrated a highly significant reduction in cellular proliferation in a concentration-dependent fashion. In addition to these functional analyses, biochemical studies revealed a reduction in Stat5 activation in N51-FLT3 expressing cells treated with II-B08 compared N51-FLT3 expressing cells treated with DMSO in two independent experiments. Collectively, these data suggest that constitutive recruitment of Shp2 to N51-FLT3 and N73-FLT3 contributes to the FLT3-ITD-induced hyperproliferative phenotype and imply that inhibition of Shp2 function may provide a novel therapeutic approach to FLT3-ITD-bearing leukemias. Disclosures:No relevant conflicts of interest to declare.

The Role of Protein Tyrosine Phosphatase, Shp2, in FLT3-ITD-Induced Leukemogenesis.

Internal tandem duplications of the FMS-like receptor tyrosine kinase (FLT3-ITDs), an in-fame insertion of several amino acids within the juxtamembrane domain, are present in 25% of acute myeloid leukemia (AML) patients and confer a poor prognosis. FLT3-ITDs induce FLT3 ligand (FL)-independent hyperactivation of Erk and promiscuous activation of STAT5; however, the molecular mechanisms underlying aberrant activation of these signaling molecules is largely unknown. Tyrosine 599 (Y599) of WT FLT3 recruits the protein tyrosine phosphatase, Shp2, upon stimulation with FL, resulting Erk activation. In several FLT3-ITDs, including N51-FLT3 and N73-FLT3, Y599 is duplicated. These findings led us to hypothesize that increased recruitment of Shp2 to N51-FLT3 or N73- FLT3, via Y599, results in enhanced Shp2 activation and contributes to N51-FLT3- and N73-FLT3-induced cellular hyperproliferation, Erk hyperactivation, and promiscuous STAT5 activation. Using Baf3 cells stably expressing WT FLT3, N51-FLT3, or N73- FLT3, co-immunoprecipitation assays demonstrated that Shp2 is phosphorylated and associates with WT FLT3 in a FL-dependent manner. However, in contrast, Shp2 is constitutively hyperphosphorylated and associated with FLT3-N51 and FLT3-N73 independent of FL stimulation. To investigate the functional role of Shp2 in Flt3-ITD-induced leukemogenesis, Baf3 cells expressing WT FLT3, N51-FLT3, or N73-FLT3 were transfected with a mammalian expression vector encoding a U6 polymerase III– directed Shp2-specific short-hairpin RNA (shRNA) or a scrambled shRNA and selected in puromycin. Western blot analysis revealed significant reduction of Shp2 expression by the Shp2-specific shRNA and no change in Shp2 expression by the scrambled shRNA in all cell lines. Upon knock-down of Shp2 in Baf3/WT-FLT3 cells, proliferation was minimally reduced based on thymidine incorporation assays; however, knock-down of Shp2 in Baf3/N51-FLT3 and Baf3/N73-FLT3 cells significantly reduced proliferation, both at baseline and in response to FL stimulation. Collectively, these data suggest that constitutive recruitment of Shp2 to N51-FLT3 and N73-FLT3 contributes to the FLT3- ITD-induced hyperproliferative phenotype and imply that inhibition of Shp2 function may provide a novel therapeutic approach to FLT3-ITD-bearing leukemias.

Fms-like Tyrosine Kinase 3 Ligand Stimulation Induces MLL-Rearranged Leukemia Cells into Quiescence Resistant to Antileukemic Agents

Fms-like tyrosine kinase 3 (FLT3) is highly expressed in acute lymphoblastic leukemia with the mixed-lineage leukemia (MLL) gene rearrangement refractory to chemotherapy. We examined the biological effect of FLT3-ligand (FL) on 18 B-precursor leukemic cell lines with variable karyotypic abnormalities, and found that nine of nine MLL-rearranged cell lines with wild-type FLT3, in contrast to other leukemic cell lines, are significantly inhibited in their proliferation in a dose-dependent manner by FL. This inhibition was due to induction of the G0-G1 arrest. A marked up-regulation of p27 by suppression of its protein degradation and an abrogation of constitutive signal transducers and activators of transcription 5 phosphorylation were revealed in arrested leukemia cells after FL stimulation. Importantly, FL treatment rendered not only cell lines but also primary leukemia cells with MLL rearrangement resistant to chemotherapeutic agents. MLL-rearranged leukemia cells adhering to the bone marrow stromal cell line, which expresses FL as the membrane-bound form, were induced to quiescent state resistant to chemotherapeutic agents, but their chemosensitivity was significantly restored in the presence of neutralizing anti-FL antibody. The FL/FLT3 interaction between leukemia cells and bone marrow stromal cells expressing FL at high levels should contribute, at least in part, to persistent minimal-residual disease of MLL-rearranged leukemia in bone marrow.

FLT3 Ligand Is Required for Full Activation of FLT3 Mutants.

FLT3 is a member of the class III receptor tyrosine kinase family and is primarily expressed on hematopoietic stem/progenitor cells. It is expressed in most cases of acute myeloid leukemia (AML) and acute B-lineage leukemia. FLT3 ligand (FL) binds to wild-type FLT3 (wtFLT3) receptors and is required for the activation of wtFLT3 signaling. Somatic mutations of FLT3 involving internal tandem duplication (ITD) of the juxtamembrane domain or point mutations in the tyrosine kinase domain (TKD) have been identified in approximately 17–34% and 7–9% of acute myeloid leukemia (AML) patients, respectively. The ITD and TKD mutations appear to activate the tyrosine kinase domain of FLT3 in a FL independent manner. However, the data upon which these conclusions were based was obtained in human and murine cell lines that express FL. Thus, it is not clear whether FL also plays a role in the activation of the mutant forms of FLT3. In order to determine whether or not FLT3 mutants are completely or somewhat dependent on FL for their activation, a FL deficient murine embryo fibroblast cell line (MEF) was derived from FL deficient (FL−/−) mice by SV40 large T antigen transformation. This cell line eliminates the possibility of autocrine stimulation of transfected FLT3 receptor by FL. Expression of FLT3/ITD and FLT3/TKD mutations in FL−/−MEF cells resulted in some constitutive phosphorylation of FLT3 receptor. However, a more than 4 fold increase of FLT3 activation was induced by addition of exogenous FL. Retroviral introduction of FL in FL−/−MEF cells expressing FLT3 mutants led to more than 3 fold increases of FLT3 phosphorylation by FL. Further activation of FLT3 mutant receptors by FL was also observed in BaF3 and TF1 cells expressing FLT3/ITD or TKD mutants and in MV411 cells, a cell line expressing FLT3/ITD alone without expression of wtFLT3. Phosphorylation of downstream targets of FLT3 signaling including STAT5, AKT, or MAPK was also enhanced after FL stimulation. Biological function studies revealed that exogenous FL promoted the colony forming abilities of FLT3/ITD and FLT3/TKD mutant transduced bone marrow cells derived from FL−/− mice. However, FL did not increase the colony numbers formed from control vector transduced FL−/− bone marrow cells. In colony replating assay, FLT3 mutant transduced FL−/− bone marrow cells maintained recloning abilities in the presence of additional FL after replating in the second, third, and fouth generation. In contrast, replating abilities of FLT3 mutant expressing bone marrow cells cultured in the absence of FL and vector control transduced bone marrow cells cultured with and without FL were gradually lost after replating from the second to the fourth generation. Addition of FL also led to enhanced proliferation and/or decreased apoptosis of BaF3 and TF1 cells expressing FLT3/ITD or FLT3/TKD, and of MV411 cells under conditions of serum starvation. These data strongly suggest that full activation of FLT3 mutants is dependent on FL and thus it is likely that FL plays a role in the leukemogenesis mediated by FLT3 mutants. Therefore, FL targeted therapy might be useful in the treatment of FLT3 mutant AML.

The FLT3-D324N Variant Is a Functionally Silent Polymorphism in the FLT3 Gene and May Be Associated with a Higher Risk for AML.

Mutations within the FLT3-gene are of growing importance for classification, risk assessment and therapeutic targeting in acute myeloid leukemia (AML). An increasing number of activating mutations have been reported during the last few years. A D324N variant located in the extracellular region of the FLT3 protein has been described recently in 4/94 (4.3%) of AML patients (Ley TJ et al., PNAS 2003). We have analyzed 705 de novo AML for D324N using a LightCycler based screening assay and found a gac to aac change in codon 324 in 43 cases (6.1%). This is approximately the same frequency that has been described for tyrosine kinase domain mutations in FLT3. However, in contrast to other FLT3 mutations the D324N was associated with a low leucocyte count (6.700/μl) and had no association to any AML subtype nor a prognostic impact regarding overall survival and event free survival (235 D324N- cases vs. 13 D324+ cases with normal karyotype analyzed). To analyze whether this FLT3 variant might be a polymorphism we analyzed peripheral blood of 329 healthy donors with a similar ethnic background. In this population we could also detect the D324N variant, but only in 4 cases (1.2%). This difference between AML and healthy donors was statistically significant (p=.0001). Three of the cases were heterozygous and one was homozygous for the D324N variant. Of one of the heterozygous cases a buccal smear was evaluated and the same heterozygous pattern could be detected in this material. In addition, of three D324N positive AML at diagnosis a sample from any time point in CCR was available that was negative for the leukemia clone with a sensitivity of 10−4 to 10−6 as assessed by quantitative PML-RARA- (1 case) or CBFB-MYH11- (1 case) specific PCR or by immunophenotyping (1 case). In these remission samples again a 50% ratio of the normal and the D324N variant was detectable. To functionally characterize the FLT3-D324N in vitro, FLT3-WT, FLT3-D324N, and FLT3-ITD cDNA were retrovirally transduced into IL-3 dependent Ba/F3 cells. Stably expressing cell lines were grown for 72h in the absence of IL-3 with varying doses of human FLT3 ligand (FL) and the number of viable cells was assessed by trypan blue exclusion. In contrast to FLT3-ITD expressing cells, FLT3-D324N transduced cells were not able to grow in the absence of IL-3. The growth of FLT3-WT and FLT3-D324N, but not vector expressing cell lines could be stimulated by exogenous FL in a dose-dependent manner. No significant difference could be demonstrated between FLT3-WT and FLT3-D324N cells. In apoptosis assays using annexin-V-PE and 7-AAD staining FL stimulation protected both D324N mutant and FLT3-WT expressing Ba/F3 cells from apoptotic cell death to a similar degree. These results strongly support the hypothesis that the D324N variant in the FLT3 gene represents a functionally silent polymorphism. The fivefold higher frequency in patients with AML compared to healthy donors raises the question whether this FLT3 variant is associated with a higher risk for AML.

Proteosomal Degradation of Flt3 Is Stimulated by Leukemia-Associated Flt3 Mutations.

The hematopoietic growth factor receptor Flt3 plays an important role in both myelopoiesis and B-cell production. Mutations of Flt3 found in approximately 30% of acute myeloid leukemias cause constitutive activation of the receptor tyrosine kinase. Cells expressing these Flt3 mutants show enhanced survival and proliferation, and, when evaluated in a mouse model, leukemia-associated Flt3 mutations resulted in fatal myeloproliferative disease (Kelly et al. Blood 2002). Despite the important roles of Flt3 in both hematopoiesis and leukemogenesis, the mechanisms that regulate Flt3 remain incompletely understood. In particular, little is known about regulation of receptor quantity. To address this issue, we examined Flt3 protein turnover in human leukemic cell lines expressing wild-type or mutant Flt3. To determine protein half-life, cells were treated with cycloheximide to block new protein synthesis, and, at specific intervals thereafter, cells were lysed and remaining Flt3 protein determined by Western blotting and densitometry. Dilution studies were performed to confirm that Western blot signals showed linear variation with amounts of Flt3 protein in the relevant concentration range. In the absence of Flt3 ligand stimulation, wild-type Flt3 had a half-life of approximately 2 hours. Cell stimulation by Flt3 ligand, however, markedly accelerated Flt3 degradation: its half-life was reduced to approximately 25 minutes. Repeat immunoblotting with an anti-Actin antibody verified equivalent sample loading, and protein assays of the cell lysates revealed no overall decrease in cellular protein within the time periods studied. We next evaluated the degradation of a constitutively-active Flt3 mutant. The MV4;11 human leukemic cell line expresses a tandem duplication mutant of Flt3, the most common type of leukemia-associated Flt3 mutation. At intervals after blocking new protein synthesis, MV4;11 cells were lysed and Flt3 protein assessed by Western blot and densitometry. We found that the half-life of mutant Flt3 was significantly shorter than that of the unstimulated wild-type receptor but very similar to that seen after ligand-stimulation. This suggested that Flt3 kinase activity, or the consequent activation of intracellular signaling proteins, might regulate receptor degradation. To identify mechanisms responsible for activation-induced Flt3 degradation, we examined the effects of protein degradation inhibitors. We found that MG132 blocked degradation of the mutant receptor, implicating the proteosome pathway in activated Flt3 degradation. The Flt3 receptor has been reported to phosphorylate Cbl, a signaling protein and ubiquitin ligase. Cbl can promote proteosomal degradation of the signaling proteins it binds. However, we found no evidence of a direct association between Flt3 and Cbl by co-immunoprecipitation. Flt3 also activates Src-family tyrosine kinases, which have been shown to regulate the internalization and degradation of related receptors such as c-Kit. However, PP1, an inhibitor selective for Src-family tyrosine kinases, had no effect on Flt3 degradation. The very rapid degradation of mutant Flt3 suggests that the high levels of Flt3 mRNA expression reported for MLL leukemias might not produce equivalent increases in mutant Flt3 protein. It is possible that leukemogenic effects of Flt3 activating mutation require co-operating abnormalities of protein degradation. Our results further raise the possibility that therapeutic use of proteosome inhibitors might exacerbate Flt3-related leukemias.

The Src Related Tyrosine Kinase Lyn Mediates Proliferative Signals from Leukemia-Related Flt3 Mutants.

Fms-like tyrosine kinase-3 (Flt3) is a hematopoietic growth factor receptor that regulates the survival and proliferation of myeloid and B-cell precursors. Mutations of Flt3 resulting in constitutive activation are the most common known molecular abnormalities in acute myeloid leukemia. Cells expressing activated Flt3 mutants show enhanced survival and proliferation, and leukemia-associated Flt3 mutations produced a fatal myeloproliferative disorder, in a mouse model (Kelly et al. Blood 2002). Nevertheless, the signaling pathways mediating Flt3 effects on proliferation are incompletely understood. Src kinases, such as Lyn, have also been linked to myeloproliferative disease, but the participation of Src-related kinases in Flt3 signaling has not been established. We therefore determined whether Src-family kinases were activated by Flt3 in human leukemic cell lines that express either wild-type or mutant Flt3. Flt3-ligand stimulation of THP1 cells, which express wild-type Flt3, significantly increased the phosphorylation of Lyn, the principal Src-family kinase found in these cells. In MV4;11 cells, which express a constitutively-active tandem duplication mutant of Flt3, Lyn phosphorylation is constitutively elevated. In both cases, a Lyn phosphorylation state specific antibody confirmed phosphorylation at tyrosine 397 of human Lyn; phosphorylation at this site is indicative of Lyn kinase activation. To determine whether the increase in Lyn phosphorylation seen in MV4;11 cells could be attributed specifically to effects of mutant Flt3, we examined Lyn phosphorylation in the murine leukemic Baf3 cells transduced with either wild-type or mutant human Flt3. Lyn phosphorylation and activation in Baf3 cells expressing the Flt3 mutant was significantly increased, compared to basal Lyn phosphorylation in Baf3 cells expressing wild-type Flt3 or vector alone. This result suggested that the constitutive Lyn phosphorylation seen in MV4;11 cells could be caused by mutant Flt3. In Baf3 cells expressing wild-type Flt3, Lyn phosphorylation was stimulated by Flt3-ligand treatment as expected. The Baf3 cell line is growth factor dependent, requiring IL-3 for survival and proliferation. However, as previously reported, expression of activated Flt3 mutants render the cells growth factor independent. We found that the Src-family specific inhibitors PP1 and PP2, but not the inactive analog PP3, inhibited the growth factor-independent proliferation of Baf3 cells expressing mutant Flt3. Moreover, anti-sense oligonucleotides that specifically reduced Lyn expression also inhibited proliferation of cells expressing Flt3 mutants. Previous studies identified Stat5 and Erk as mediators of the proliferation signal from mutant Flt3. We therefore examined the effects of Src kinase inhibitors on the activation of these signaling proteins by mutant Flt3. No change in Stat5 phosphorylation was detected, but inhibition of Lyn did inhibit Erk phosphorylation. These results suggest that Lyn is upstream from Erk in a Flt3 signaling pathway important for proliferation. Flt3 activation of Lyn also raises the possibility that Src kinase inhibitors could have therapeutic efficacy in Flt3-related acute leukemias.

Flt-3 and Its Ligand Are Expressed in Neural Crest–Derived Tumors and Promote Survival and Proliferation of Their Cell Lines

Flt-3 ligand (FL) is a cytokine that promotes the survival, proliferation, and differentiation of hematopoietic progenitors in synergy with other growth factors, such as stem cell factor. Previously we have demonstrated that stem cell factor and its receptor c-kit are expressed in neural crest–derived tumor cells and that a c-kit block induces their apoptosis. Here we have evaluated the expression of flt-3 and its ligand in 12 neuroectodermal tumor cell lines from neuroblastoma (NB), neuroepithelioma (NE), Ewing sarcoma (ES), and peripheral neuroectodermal tumor (PNET) and in 38 biopsies: 19 from NB and 19 from ES and PNET. RT-PCR demonstrated the expression of flt-3 and FL in all lines. Coexpression was observed in 42% of NB and in 74% of ES and PNET biopsies. Flow cytometry confirmed the presence of membrane and cytoplasmic flt-3 and membrane FL in all lines, whereas soluble FL protein was not measurable in their supernatants. Microphysiometric demonstration of acidification of the medium provided evidence of the specific response of cell lines to FL stimulation. Specific flt-3 phosphorylation after FL treatment was also demonstrated by Western blotting analysis. In cells growing in RPMI plus 1% fetal calf serum, FL revealed a significant proliferating activity, more evident in NB and NE lines (mean increase of viable cells, 73 ± 26% after 1 day). Treatment with flt-3 antisense oligonucleotides significantly inhibited cell growth. FL also displayed an antiapoptotic activity: after a 12-hour culture in the presence of 0.1% fetal calf serum, FL caused a 50% reduction of apoptotic cells. These results provide further evidence that neuroectodermal and hematopoietic cells share common regulatory pathways, and could be of interest in the clinical management of neuroectodermal tumors.