FLT3 Receptor Research Articles

Role of VEGF-Flt-1 signaling in malignant behaviors of oral cancer

Vascular endothelial growth factor (VEGF) plays a critical role in tumor angiogenesis. VEGF and its receptor FLT-1 signaling may contribute to cell migration of macrophages, osteoclasts and some tumor cells. However, the direct role of VEGF-Flt-1 signaling in malignant behaviors of oral squamous cell carcinoma (OSCC) is not well understood. To clarify the effects of VEGF-Flt-1 signaling in OSCC bone invasion, VEGF expression in OSCC cells and number of Flt-1+ osteoclasts at bone invasion front in 55 gingival OSCC cases were evaluated by immunohistochemistry. Twenty-four of 55 cases (43.63%) strongly expressed VEGF. The VEGF highly expressing cases were statistically correlated with the increased number of Flt-1+ osteoclasts and the more aggressive radiographic pattern of bone invasion (p In conclusion, VEGF-Flt-1 signaling promotes osteoclastogenesis, proliferation and facilitating invasion and migration of OSCC through production of MMP-1 and 3. Blocking of VEGF-Flt-1 signaling may be beneficial for OSCC treatment.

Follicular fluid placental growth factor is increased in polycystic ovarian syndrome: correlation with ovarian stimulation.

BackgroundPolycystic ovarian syndrome (PCOS) is characterized by increased ovarian angiogenesis and vascularity. Accumulating evidence indicates that vascular endothelial growth factor (VEGF) is increased in PCOS and may play an important role in these vascular changes and the pathogenesis of this disease. Placental growth factor (PlGF), a VEGF family member, has not been previously characterized in PCOS women. We investigated levels and temporal expression patterns of PlGF and its soluble receptor sFlt-1 (soluble Fms-like tyrosine kinase) in serum and follicular fluid (FF) of women with PCOS during controlled ovarian stimulation.MethodsThis was a prospective cohort study of 14 PCOS women (Rotterdam criteria) and 14 matched controls undergoing controlled ovarian stimulation. Serum was collected on day 3, day of hCG and day of oocyte retrieval. FF was collected on retrieval day. PlGF, sFlt-1 and anti-mullerian hormone (AMH) protein concentrations were measured using ELISA. Since sFlt-1 binds free PlGF, preventing its signal transduction, we calculated PlGF bioavailability as PlGF/sFlt-1 ratio.ResultsSerum PlGF and sFlt-1 levels were constant throughout controlled ovarian stimulation, and no significant differences were observed in either factor in PCOS women compared with non-PCOS controls at all three measured time points. However, FF PlGF levels were increased 1.5-fold in PCOS women compared with controls (p < 0.01). Moreover, FF PlGF correlated positively with number of oocytes retrieved and the ovarian reserve marker anti-mullerian hormone (AMH) and negatively with age. In addition, FF sFlt-1 levels were decreased 1.4-fold in PCOS women compared to controls (p = 0.04). PlGF bioavailability in FF was significantly greater (2-fold) in PCOS women compared with non-PCOS controls (p < 0.01).ConclusionsThese data provide evidence that FF PlGF correlates with ovarian stimulation and that its bioavailability is increased in women with PCOS undergoing controlled ovarian stimulation. This suggests that PlGF may play a role in PCOS pathogenesis and its angiogenic dysregulation.

Expression of VEGF and Flk-1 and Flt-1 Receptors during Blood-Brain Barrier (BBB) Impairment Following Phoneutria nigriventer Spider Venom Exposure

Apart from its angiogenic and vascular permeation activity, the vascular endothelial growth factor (VEGF) has been also reported as a potent neuronal protector. Newborn rats with low VEGF levels develop neuron degeneration, while high levels induce protective mechanisms in several neuropathological conditions. Phoneutria nigriventer spider venom (PNV) disrupts the blood-brain barrier (BBB) and causes neuroinflammation in central neurons along with excitotoxic signals in rats and humans. All these changes are transient. Herein, we examined the expression of VEGF and its receptors, Flt-1 and Flk-1 in the hippocampal neurons following envenomation by PNV. Adult and neonatal rats were evaluated at time limits of 2, 5 and 24 h. Additionally, BBB integrity was assessed by measuring the expression of occludin, β-catenin and laminin and neuron viability was evaluated by NeuN expression. VEGF, Flt-1 and Flk-1 levels increased in PNV-administered rats, concurrently with respective mRNAs. Flt-1 and Flk-1 immunolabeling was nuclear in neurons of hippocampal regions, instead of the VEGF membrane-bound typical location. These changes occurred simultaneously with the transient decreases in BBB-associated proteins and NeuN positivity. Adult rats showed more prominent expressional increases of the VEGF/Flt-1/Flk-1 system and earlier recovery of BBB-related proteins than neonates. We conclude that the reactive expressional changes seen here suggest that VEGF and receptors could have a role in the excitotoxic mechanism of PNV and that such role would be less efficient in neonate rats.

Immunohistochemical expression of vascular endothelial growth factor receptors Flt1 and KDR in the endometrium during the estrus cycle in albino rats

Background Angiogenesis is an important process in endometrial development and embryonic implantation and is regulated through vascular endothelial growth factor (VEGF); its receptors Flt1 and KDR. Aim This work aimed to study the immunoexpression of VEGF receptors (VEGF-Rs) in the endometrium at different ages and reproductive phases and correlate them with the histological profiles in these phases. Materials and methods Seventy female albino rats were included in this study. They were divided into seven groups of 10 rats each: one group consisted of rats in the prepubertal period at age 4–6 weeks; five groups consisted of rats in the reproductive period at age 6–10 months, which were divided according to estrus cycle phases into proestrus, estrus, metestrus, diestrus, and pregnant groups; and the sixth group consisted of rats in the postmenopausal period at age 15–18 months. The uteri of all rats were removed and processed for staining with H&E and were subjected to immunohistochemical staining for Flt1 and KDR. For morphometric measurements, uterine wall thickness and Flt1 and KDR optical density in the endometrial surface epithelium, glandular epithelium, stromal cells, and endometrial endothelial cells were measured using image analysis. Results were statistically compared. Results The expression of VEGF-Rs was highest in the pubertal age group with marked expression of these receptors in the proestrus phase followed by the estrus phase. This supports the role of sex hormones, especially the estrogen hormone, in regulating VEGF-R expression. The Flt1 receptor was predominantly expressed in endometrial and stromal cells as well as in blastocysts, whereas the KDR receptor was predominantly expressed in endometrial endothelial cells. Comparison among all groups and then between each two groups revealed statistically significant differences in the measured morphometric parameters. Conclusion The upregulation of Flt1 and KDR could be involved in the regulation of endometrial endothelial cell proliferation and in increase in endometrial vascular permeability, especially at implantation sites.

MUC1-C oncoprotein promotes FLT3 receptor activation in acute myeloid leukemia cells

Blasts from approximately one-third of patients with acute myeloid leukemia (AML) harbor activating mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase that confer a poor prognosis. The Mucin 1-C-terminal subunit (MUC1-C) oncoprotein is aberrantly expressed in AML blasts and stem cells; however, there is no known interaction between MUC1-C and FLT3. The present studies demonstrate that MUC1-C associates with wild-type and mutant FLT3 in AML cells. Targeting MUC1-C with the cell-penetrating peptide inhibitor GO-203 disrupts MUC1-C/FLT3 complexes and downregulates FLT3 activation. GO-203 treatment of AML cells was also associated with inhibition of the FLT3 downstream effectors AKT, extracellular signal-regulated kinase, and STAT5. The results further show that AML cells with FLT3-activating mutations and resistant to the FLT3 inhibitor midostaurin/PKC412 are sensitive to GO-203-induced growth arrest and death. Moreover, GO-203 increases sensitivity of mutant FLT3 AML cells to FLT3 inhibitor treatment. These results indicate that MUC1-C contributes to FLT3 activation in AML cells and that targeting MUC1-C inhibits the FLT3 signaling pathway. Our findings support the development of MUC1-C inhibitors alone and in combination with agents that target FLT3 for the treatment of wild-type and mutant FLT3 AML.

Crosstalk between VEGF and MTA1 signaling pathways contribute to aggressiveness of breast carcinoma

The expression of metastasis associated protein (MTA1) correlates well with tumor metastasis; however its role as a proangiogenic protein and the molecular mechanisms underlying the same are not fully understood. In this study the MTA1 protein was expressed and purified to evaluate its angiogenic potential. In both MCF-7 and MDA-MB-231 cells, endogenous MTA1 protein was localized in the nucleus; while added recombinant MTA1 protein was bound to cell membrane as per immunofluorescence data. MTA1 was detected both in conditioned media and in human serum samples. Recombinant MTA1 regulated cellular functions of HUVEC's such as, proliferation, tube formation, and migration. MTA1 was more potent than VEGF in inducing invasion of breast cancer cells. Analogous to VEGF, MTA1 could induce angiogenesis in both non-tumor and tumor context, as verified by rat cornea, shell less CAM and xenograft models respectively. However MTA-1 was more potent an inducer of angiogenesis. VEGF or Flt-1 gene promoter, luciferase gene reporter analysis revealed that MTA1 up regulates the expression of VEGF and its receptor Flt-1 genes. Kinetics of VEGF-induced expression of MTA1 and qPCR studies showed that there is an increased expression of MTA1 in tumor cells. VEGF induced phosphorylation of endogenous MTA1 on tyrosine residues; phosphorylation was mediated through VEGFR2 and p38-MAP kinase. Recombinant MTA1 activated signaling, in MCF-7 and MDA-MB-231 cells, involved ERK and JNK pathways. In conclusion, MTA1 is a potent angiogenic molecule and cross talk between VEGF and MTA1 protein regulates tumor angiogenesis and metastasis.

Differentiation Induction In Acute Myeloid Leukemia Using Site-Specific DNA-Targeting

Hoxa9 and Meis1 are overexpressed in >70% of acute myeloid leukemia (AML) and associated with poor prognosis and survival. Hoxa9 and Meis1 interact with DNA and PBX to achieve transcription of differentiation-blocking genes. We tested transcriptional repression at Hoxa9-PBX-Meis1 genomic binding sites to induce differentiation in a model of human AMLWe designed a DNA-recognition strategy based on the known structure of the Hoxa9-PBX-DNA complex by fusing the DNA binding helices of Hoxa9 and PBX to create concise homeodomain fusion proteins that target the Hoxa9-PBX DNA recognition sequence. To confer transcription-repressing properties to the proteins, we attached a transcriptional repressor (sin3 interacting) domain and ectopically expressed this protein in Hoxa9-Meis1 immortalized murine progenitors. Introduction of this transcription repressor protein significantly enabled cell differentiation versus control (51.2% Mac-1high Gr-1high cells versus 11.3% for control). Multiple gene transcripts indicative of differentiation, such as GCSFR, myeloperoxidase, neutrophil elastase, and the calcium binding protein, S100A8, were also elevated in repressor-expressing cells. Furthermore, direct transcriptional targets of Hoxa9 (e.g. SOX2, CD34, FOXP1, FLT3R, DNAJC10) were down regulated in repressor-expressing cells. Importantly, a mutant repressor lacking the DNA-interacting amino acids did not affect transcription of Hoxa9 targets, demonstrating on-target specificity. Repressor-expressing cells also exhibited lower surface expression of c-Kit and Flt3 receptors and when transplanted into mice resulted in a significant increase in disease latency with a 94 day median latency versus 62 day latency for the control group (p value = 0.002).Our results demonstrate that site-specific DNA-targeting using homeodomain fusion proteins can enable AML cell differentiation and significantly increase disease latency. Disclosures:Scadden:Fate Therapeutics: Consultancy, Equity Ownership.

Identification and Characterization Of Human NK Lineage Restricted Progenitor (NKP)

Natural Killer (NK) cells, the third lymphoid lineage after T and B cells, are large granular lymphocytes belonging to innate immune system, classically defined as CD3-CD56+CD16+ in human. NK cells can kill target cells without prior activation, based on the missing self-hypothesis, either due to the lack of MHC-class I molecule or up-regulation of NKG2D ligands, two properties commonly associated with transformed cells (D.H. Raulet, N. Guerra, 2009). Moreover, by secreting cytokines (such as TNF-α and IFN-γ), NK cells can also activate the response of the adaptive immune system. Experiments in mice have shown that NK cells are primarily responsible for the in vivo elimination of transplanted tumor cells (J. Wu, L. L. Lanier, 2003) and, in human, a direct correlation between low NK cytotoxicity and elevated cancer incidence was reported in a large cohort of Japanese aged over 40 (K. Imai et al., 2000). The important anti-tumor role for alloreactive NK cells has been shown in patients with acute myeloid leukemia where transplanted donor NK cells, expressing mismatch inhibitory receptors, provided graft versus leukemia effect in the absence of graft versus host disease (L. Ruggeri et al., 2006). These and many other findings are the basis of a large number of ongoing studies and clinical trials for NK cell immunotherapy against cancer (M. Terme et al., 2008). However, in contrast to B and T cell development where both cellular and regulatory pathways are well studied, the development of NK cells from hematopoietic stem cells (HSCs) is not well understood and the identity of restricted NK cell progenitor (NKP) is unknown (S. Doulatov et al., 2012).To identify human NKP, we applied the expression of known early lymphoid markers (CD45RA, CD10, CD7) and cytokine receptors (IL-7R: CD127) important for lymphoid development. Using multicolor flow cytometry, Lin-CD34+CD38+CD123-CD45RA+CD7+CD10+/-CD127+/- populations were sorted from human bone marrow (hBM) and umbilical cord blood (hUCB). Purified candidate NKPs were cultured on OP9, OP9DL1 stroma and in Terasaki cultures in the presence of specific cytokines and generation of CD3-CD56+CD16+NKp46+ NK cells, CD19+ B, CD3+ CD4+ CD8+ T and CD33+CD14+ myeloid cells was investigated. To study the ability of NKP candidate to generate NK cells after transplantation, purified NKPs were injected into new-born NOD/SCID γcnull (NSG) mice and at 11 weeks after transplantation the phenotype of generated progeny was evaluated. In addition, NK cells generated in stroma cultures and after transplantation were tested for their cytotoxic activity against K562 leukemic cell line in degranulation assay, where the surface expression of membrane glycoprotein LAMP-1 (CD107a) is measured after activation of specific killing receptors.The Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKP candidate population sorted from hUCB has a robust NK cell potential in vitro at the single cell level and lacked the ability to generate T, B and myeloid cells. Furthermore, NK cells generated from this candidate NKP were functionally mature: show cytotoxic activity against K562 cells and produce cytokines: IFNγ and TNFα after activation in vitro. Further phenotypic characterization showed that the candidate NKP is highly positive for lymphoid markers CD244 and CD62L, lacks the expression of mature NK cell markers NKp46 and NKG2D, and 50% of NKPs express c-kit and Flt3 receptors. At 11 weeks after transplantation, 9 out of 17 NSG mice injected with 600 Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKPs had a significant human cells engraftment and only CD3-CD56+NKp46+CD16+/- NK cells were found in the peripheral blood, bone marrow and spleen; whereas 15 out 17 NSG mice injected with CD34+ cells were positive for the presence of human B, T, NK and myeloid cells in these tissues. Human NK cells generated after transplantation into NSG mice were functionally active and able to kill K562 leukemic cells. The Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- NKP candidate was also found in adult hBM and showed NK-lineage restriction.Our results indicate that the Lin-CD123-CD34+CD38+CD45RA+CD7+CD10+CD127- cells, found in human BM and UCB, represent NK-lineage restricted progenitors that generate fully mature functional NK cells. Disclosures:No relevant conflicts of interest to declare.

Results Of a Phase 1 Study Of Quizartinib (AC220, ASP2689) In Combination With Induction and Consolidation Chemotherapy In Younger Patients With Newly Diagnosed Acute Myeloid Leukemia

FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) in acute myeloid leukemia (AML) are associated with early relapse after standard chemotherapy and poor survival. Quizartinib (AC220) is an oral FLT3 receptor tyrosine kinase inhibitor that has shown the highest level of single agent activity seen with a FLT3 targeted agent in FLT3+ relapsed AML to date. This Phase 1 dose escalation study is the first study to report data with quizartinib in combination with standard induction and consolidation chemotherapy in patients aged 18-60 years with newly diagnosed AML, regardless of FLT3-ITD status. The dose escalation was conducted using a modified 3+3 design, where 6 pts were enrolled at each dose level. The pts were given cytarabine 200 mg/m2 x 7 days and daunorubicin 60 mg/m2 x 3 days (7+3) for induction and high dose cytarabine 3 g/m2(HiDAC) q12hours on days 1, 3, and 5 for consolidation. Quizartinib was administered daily for either 7 or 14 days, starting at Day 4 of induction and/or consolidation chemotherapy. Patients were allowed to proceed directly to a stem cell transplant after achieving a response or receive further quizartinib as maintenance therapy after consolidation if they were not transplant eligible. Three dose levels were tested; dose level 1 (DL1) at 60 mg for 7 days, dose level 2 (DL2) 60 mg for 14 days, and dose level -1 (DL-1), 40 mg for 14 days. Through May 31, 2013 18 pts were enrolled in the study, and the safety information at all 3 dose levels are presented.The median age of pts was 43 years (range 22 to 60). Of the 18 patients, 16 had the FLT-ITD mutation.At DL1, one of the 6 patients had a DLT (grade 3 hyponatremia). At DL2, two of the 6 patients had a DLT (grade 3 QTc prolongation and grade 4 pericarditis) which exceeded the pre-specified criteria so DL-1 was then explored. At DL-1, one of the 6 patients had a DLT (grade 3 constrictive pericarditis).The most common (20%) treatment-related adverse events (AEs) were nausea (42%), diarrhea (32%), anemia (26%), febrile neutropenia (26%), neutropenia (21%), fatigue (21%), pyrexia (21%) and thrombocytopenia (21%). The most common (10%) Grade 3 or 4 treatment-related AEs were febrile neutropenia (26%), thrombocytopenia (21%) anemia (21%)), neutropenia (21%), leucopenia (16%), and nausea (11%).The data from this Phase 1 study demonstrates for the first time that quizartinib can be safely administered with induction and/or consolidation chemotherapy in newly diagnosed younger patients with AML. The MTD was identified as 40 mg for 14 days or 60 mg for 7 days. The efficacy results from this Phase 1 study will be available at the time of presentation. Based on these findings, multiple Phase 3 studies in newly diagnosed AML patients are planned. Disclosures:Altman:Novartis: Consultancy; Araid: Consultancy; BMS: Consultancy; Teva: Consultancy; Astellas: Consultancy. Foran:Astellas: Research Funding. Trone:Ambit: Employment. Gammon:Ambit: Employment. Cortes:Ambit: Research Funding.

Characterization Of FLT3/ITD Resistant Cells and Identification Of Novel Therapeutic Targets Utilizing High Throughput Drug Screening

The overall survival (OS) of pediatric acute myeloid leukemia (AML) exceeds 60%, however for high risk (HR) patients, including high allelic ratio FLT3/ITD+, survival remains poor. FLT3/ITD is one of the first genomic alterations in AML to be exploited for therapeutic benefit as it has greater sensitivity to the pro-apoptotic effects of FLT3-inhibitors. Children's Oncology Group (COG) phase III AML trial AAML1031 is investigating the role of sorafenib in combination with chemotherapy in HR FLT3/ITD+ patients. In vitro and in vivo studies indicate that resistance to FLT3-inhibitors can develop through varying mechanisms including up-regulation of FLT3 receptor, acquisition of secondary mutations, or activation of alternate survival mechanisms leading to apoptotic escape. For FLT3/ITD+ patients who relapse despite treatment with FLT3-inhibitors, there are often no therapeutic options and survival is very poor.In evaluation of therapeutic options for those who relapse on sorafenib, we developed an in vitro resistance model using the FLT3/ITD+ cell line MV4-11. Resistance was induced thru long-term exposure to incrementally increasing doses of sorafenib. Two distinct cell lines with resistance at 10 and 100 fold above the IC50 of naïve MV4-11 were generated for experimental evaluation. Genotypic and phenotypic characterization of the resistant cells was conducted by multidimensional flow cytometry (MDF), conventional karyotyping, and mutational profiling. MDF revealed an overall similar immunophenotype, however the resistant cells were significantly more homogeneous for expression of HLA-DR and had significantly higher CD11b expression compared to their naïve counterparts. CD135 expression was minimally increased in the resistant cells. In comparison of the karyotypes, the resistant cells were a more homogenous population with emergence of one dominant clone and disappearance of a number of pre-existing sub-clones. Mutational profiling by Sanger sequencing revealed a novel N841Y mutation in activation loop, an area implicated in TKI-resistance.Using a high throughput drug screening assay, we explored sensitivity profiles of the naïve and resistant MV4-11 cells to 163 oncology agents, including 45 FDA approved and 118 investigational agents that target a number of key pathways regulating cell growth, differentiation, and survival. The naïve MV4-11s had a sorafenib IC50 of 1.3 nM (published 1-5nM) and resistant cells had IC50 of approximately 2-log folds above the naïve, which was consistent to what we had seen in our lab-based validations. We initially assessed whether resistance to sorafenib induces cross-resistance to other TKIs. Agents in the panel with previously demonstrated efficacy for FLT3/ITD included quizartinib (AC-220), tandutinib, ponatinib, sunitinib, and midostaurin, and in all cases sorafenib-resistant cells were also more resistant to these agents. We then examined whether we could identify agents with efficacy in the resistant cells. We identified 5 novel agents to which the resistant cells retained sensitivity. Two bcl-2 inhibitors tested maintained sensitivity in the resistant cells with IC50s in the 20-100nM range. In addition, YM-155, a survivin inhibitor, also maintained sensitivity in the resistant cells with IC50s of approximately 25-50nM across the cell lines. Survivin over-expression is associated with AML stem progenitor cells and decreased OS in adults, and transcription regulation has been linked to the FLT3/STAT5 pathway. Two CRM inhibitors, a novel class of agents which inhibit nuclear export to restore tumor suppressor function, also maintained sensitivity in the resistant cell lines with an approximate 3-fold increase in IC50 from 12nM in the naïve to 32-40nM in the resistant cells.Experience with the use of directed therapy to target specific somatic events has provided evidence that leukemic evolution can continue under this selection pressure and therapeutic options for patients with emergent disease is often insufficient. Using the high throughput drug assay in a FLT3/ITD+ cell line as an in vitro model for sorafenib-resistant FLT3/ITD patients, we identified classes of targeted agents that maintain sensitivity in resistant cells. Further validation of the targets in specimens from those with resistance to such TKIs can inform on the class of agents that can be used to treat or prevent refractory disease FLT3/ITD+ patients. Disclosures:No relevant conflicts of interest to declare.

Results Of a Phase 2 Randomized, Open-Label, Study Of Lower Doses Of Quizartinib (AC220; ASP2689) In Subjects With FLT3-ITD Positive Relapsed Or Refractory Acute Myeloid Leukemia (AML)

▪FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) in acute myeloid leukemia (AML) are associated with early relapse after standard chemotherapy and poor survival. Quizartinib (AC220) is an oral FLT3 receptor tyrosine kinase inhibitor that has shown the highest level of single agent activity seen with a FLT3 targeted agent in FLT3+ relapsed AML to date. This Phase 2 study was conducted to assess the efficacy and safety of two lower doses of quizartinib monotherapy in FLT3-ITD positive (+) pts aged 18 yrs or older with AML relapsed or refractory to 2nd-line, salvage chemotherapy or relapsed after hematopoietic stem cell transplantation (HSCT) to identify the optimal dose for future studies. The co-primary endpoints of the study were the composite complete remission (CRc) rate, which included complete remission (CR), complete remission with incomplete platelet recovery (CRp), and complete remission with incomplete hematologic recovery (CRi) and the rate of grade ³2 QTc prolongation. The Phase 2 study enrolled a total of 76 pts who were randomized to either 30 mg or 60 mg daily.Preliminary data from the entire study of 76 pts are included here; updated information will be provided for presentation. Thirty eight patients each were randomized to each arm and treated continuously during 28-day cycles. 58% of the randomized patients were male. The pts had a median age of 53 yrs (range 19 to 77).For 30 mg pts the derived CRc rate was 50% (5% CR, and 45% CRi). For 60 mg pts the CRc rate was 50 % (3% CR, 3% CRp, and 45% CRi). 29% of pts at 30mg and 37% of pts at 60mg were successfully bridged to HSCT.Efficacy Results in AML Pts Relapsed/Refractory to 2nd-line Therapy or HSCTThe rate of grade 2 or higher QTc prolongation assessed by central reading was 11% and 17% in 30 mg and 60 mg arms respectively, grade 3 QT prolongation was 3% in both arms and there was no grade 4. The mean maximum prolongation from baseline was 31.5 ms and 39.7 ms in 30 mg and 60 mg arms respectively.A majority of patients (78%) had a treatment related adverse event. The most common (10%) treatment-related adverse events (AEs) were anemia (18%), nausea (15%), fatigue (12%) and diarrhea (11%). Sixteen patients (22%) had a serious adverse event (SAE) possibly related to study drug. The most common (5%) treatment-related SAE was febrile neutropenia (5%). A total of 4 pts (5%) experienced a treatment-related AE resulting in discontinuation of quizartinib.The data from this Phase 2 study confirm the high degree of activity of quizartinib monotherapy in FLT3-ITD(+) AML pts relapsed/refractory to 2nd-line treatment or HSCT, previously shown in the larger Phase 2 ACE study of 333 patients treated at higher doses (90mg, 135mg or 200mg). Importantly, 25 of 76 pts (33%) were successfully bridged to a potentially curative HSCT, an almost identical rate to that seen with higher doses. The safety profile is improved specifically decreasing QT prolongation rate at lower doses. Other safety findings were manageable, and were primarily gastrointestinal adverse events and myelosuppression. The data from this Phase 2 study confirms an improved risk:benefit profile with lower doses of quizartinib and multiple randomized Phase 3 studies with quizartinib in both relapsed and newly diagnosed AML patients are planned. Disclosures:Cortes:Ambit: Research Funding. Schiller:Astellas: Consultancy. Trone:Ambit: Employment. Gammon:Ambit: Employment. Goldberg:Astellas: Research Funding. Marie:Astellas: Travel expenses and medical writing services provided by Astellas Other. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Ariad: Consultancy; Novartis: Consultancy, Speakers Bureau. Levis:Ambit Biosciences: Consultancy, Research Funding.

Disease Characteristics and Prognostic Implications Of Cell Surface FLT3 Receptor (CD135) Expression In Pediatric Acute Myeloid Leukemia – A Report From Children's Oncology Group

FLT3 is a highly expressed cell surface receptor in a majority of acute leukemias with near universal expression in acute myeloid leukemia (AML). High FLT3 expression level in conjunction with 11q23/MLL-rearrangement has been associated with poor prognosis in pre-B acute lymphoblastic leukemia (ALL) and is considered a potential therapeutic target through FLT3 inhibition. In vitro studies have demonstrated enhanced sensitivity of ALL and AML cells with high FLT3 expression to the cytotoxic effects of FLT3 inhibition. Although the clinical impact of high allelic ratio FLT3/ITD has been demonstrated in AML, the significance of FLT3 expression in those without the mutation has not been well studied, and all previous studies have been limited to evaluation of FLT3 transcript levels for correlation with outcome. Here we present the results of a prospective evaluation of FLT3 cell surface protein expression (CD135) on the diagnostic leukemic blast population as determined by multi-dimensional flow cytometry (MDF) in specimens from patients treated on COG AAML0531. Of the 495 patients enrolled after September 25, 2008, 366 patients enrolled on the accompanying biology study and had diagnostic specimens for evaluation of central CD135 expression by MDF. All diagnostic specimens underwent central MDF analysis for CD135 expression. FLT3 mutation data was available on all 366 patients, 57 patients were FLT3/ITD (15.6%), 21 were FLT3/ALM (5.7%) and the remaining 290 were FLT3wild type (FLT3/WT).There was significant variability of CD135 expression across the population with a mean fluorescence intensity (MFI) ranging from 3-232 (median 18). Median CD135 expression for those with FLT3/ITD, FLT3/ALM, FLT3/WT was 22 vs. 19 vs. 17 respectively (p=NS). Patients were divided into quartiles based on CD135 expression and clinical characteristics and outcome were correlated with CD135 expression across the four quartiles. There was not a significant difference in CD135 expression by age, gender, race, or ethnicity across the four quartiles. Those with high CD135 expression (Q4) had similar median diagnostic WBC counts as those with lower CD135 (Q1-3), although median diagnostic blast % in those in Q4 was significantly elevated (p=0.003). Nearly half of those in Q4 were FAB M5 compared to 13% in Q1-3 (p<0.001) and all those with FAB M7 had low CD135 expression (p=0.004).Evaluation of the diagnostic cytogenetics and CD135 expression demonstrated significant correlation of CD135 expression with MLL translocations, as 22% of patients in Q4 had MLLtranslocations vs. 11% in Q1-3 (p=0.005). This was primarily driven by patients with t(9;11), p=0.001. There was an inverse correlation between inv(16) and CD135 expression as only 2% of those in Q4 had inv(16) compared to 15% in Q1-3 (p<0.001). CD135 was then correlated with response to induction and post-induction outcome. Complete remission (CR) rate was comparable between those with high or lower CD135 expression (p=0.76). Those with high or lower CD135 expression had similar 3-yr overall survival (OS) from diagnosis of 70% and 66% respectively (p=0.9) and relapse risk from CR of 42% and 36% respectively (p=0.35).Given the known prognostic and therapeutic significance of FLT3/ITD, we evaluated any prognostic implications of FLT3 expression in patients without FLT3/ITD, most of whom lack other cytogenetic risk features. Similar to the entire cohort, in the FLT3/ITD-negative cohort CD135 expression correlated with FAB M5 (p<0.001), t(9;11), (p<0.001) and inversely correlated with inv(16), p=0.001. 3-yr OS for those in Q4 was 71% vs. 67% in Q1-3 (p=0.955). Given the association of MLL and CD135 expression, we evaluated the association of CD135 expression with outcome in patients with MLLtranslocations (N=73); in these patients the 3-yr event free survival (EFS) was similar between those with high or lower CD135 expression (p=0.621).In summary, we found that CD135 surface expression did not correlate with FLT3 mutations or clinical outcomes. Although FLT3 expression in FLT3/ITD-negative patients does not appear to offer additional prognostic information with current therapy, it may provide a therapeutic target in a subset of high expressing patients. This study also demonstrates that elevated FLT3 expression is associated with MLL rearrangements and warrants further study in this population with regards to potential prognostic and therapeutic implications. Disclosures:No relevant conflicts of interest to declare.

Activating FLT3 Mutations Display a Wide Range Of Transforming Potential and a Characteristic Pathway Profile Associated With Prognosis In Acute Myeloid Leukemia

BackgroundInternal tandem duplication (ITD) and pointmutations in the tyrosine kinase domain (TKD) of the receptor tyrosine kinase FLT3 occur in about 30% of patients with acute myeloid leukemia (AML). In contrast to the negative prognostic impact of FLT3-ITD in normal karyotype AML, FLT3 pointmutations occurring in the TKD and juxtamembrane (JM) region are less frequent and of unclear clinical impact. Although TKD mutations can induce resistance to tyrosine kinase inhibitors the individual transforming potential of FLT3 pointmutations has not been analysed in detail. In this study we have performed a comprehensive analysis of various FLT3 mutants in a comparative setting in vitro and analyzed gene expression profiles, and clinical outcome with respect to FLT3mutation status. Material and MethodsWe analyzed relapse and survival in 672 cytogenetically normal AML patients and the FLT3 status at diagnosis and relapse in 156 patients. In the murine Ba/F3 cell model we analyzed the transforming potential, subcellular localization, phosphorylation status and signaling properties of eight different FLT3 mutants. The investigated FLT3 mutations include three ITD of different length and insertion site, V592A in the JM region, common FLT3-TKD mutations D835V and D835Y as well as D839G and I867S in the second TKD. FLT3-D839G and -I867S were recently found in AML patients by our group during routine diagnostics but have not been functionally characterized before. The corresponding remission samples did not express these mutations. Further a gene expression profile analysis with respect to FLT3-ITD and -TKD mutation status and evaluation of differences in activation of predefined STAT5 target gene set was performed. ResultsIn 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with an inferior relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%).In vitro, FLT3-ITD induced a fully transformed phenotype in Ba/F3 cells, whereas FLT3 pointmutations showed a weaker but clearly transformed phenotype with gradual increase in proliferation and protection from apoptosis. The transforming capacity of the investigated mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD transformed cells, further gene expression profile analyses displayed differences in predefined STAT5 target genes between FLT3-ITD and FLT3-TKD mutations. In contrast, FLT3-non-ITD mutants had an enhanced signal of AKT and MAPK activation. Further differences were found on mRNA level presenting deregulation of SOCS2, ENPP2, PRUNE2 and ART3 expression between FLT3-ITD, FLT3-TKD and FLT3-WT. ConclusionAlthough apparently divergent in response to treatment all functionally characterized mutants showed a clear gain-of-function phenotype with a wide range of transforming activity associated with clinical prognosis and signaling. Disclosures:No relevant conflicts of interest to declare.

Abstract A279: Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor.

Abstract Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard care treatment, the disease eventually relapses. Activating FLT3 mutations are found in approximately 30% of AML patients. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here preclinical evaluation of AMG 925, a potent, selective and bioavailable FLT3/CDK4 dual kinase inhibitor. The compound inhibited AML xenograft tumor growth by &amp;gt;99% without detectable body weight loss. AMG 925 was also found to inhibit FLT3 mutants (e.g, D835Y) that are resistant to the current FLT3 inhibitors (e.g., quizartinib/AC220, sorafenib). CDK4 is a cyclinD-dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4-Rb pathway in cancer development has been well established. CDK4 specific inhibitors are being developed for treating Rb positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve clinical response rates. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A279. Citation Format: Kang Dai, Kathleen Keegan, Zhihong Li, Ma Ji, Cong Li, John Eksterowicz, Coberly Suzanne, David Hollenback, Margret Weidner, Justin Huard, Lingming Liang, Grace Alba, Jessica Orf, Mei-Chu Lo, Sharon Zhao, Rachel Ngo, Ada Chen, Lily Liu, Timothy Carlson, Lawrence R. McGee, Julio Medina, Alexander Kamb, Dineli Wickramasinghe. Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A279.

Chronic carbon monoxide inhalation during pregnancy augments uterine artery blood flow and uteroplacental vascular growth in mice

End-tidal breath carbon monoxide (CO) is abnormally low in women with preeclampsia (PE), while women smoking during pregnancy have shown an increase in CO levels and a 33% lower incidence of PE. This effect may be, in part, due to lowered sFLT1 plasma levels in smokers, and perhaps low-level CO inhalation can attenuate the development of PE in high-risk women. Our previous work showed maternal chronic CO exposure (<300 ppm) throughout gestation had no maternal or fetal deleterious effects in mice. Our current study evaluated the uteroplacental vascular effects in CD-1 maternal mice that inhaled CO (250 ppm) both chronically, gestation day (GD) 0.5 to 18.5, and acutely, 2.5 h on each of GD 10.5 and 14.5. We demonstrated, using microultrasound measurements of blood velocity and microcomputed tomography imaging of the uteroplacental vasculature, that chronic maternal exposure to CO doubled uterine artery blood flow and augmented uteroplacental vascular diameters and branching. This finding may be of benefit to women with PE, as they exhibit uteroplacental vascular compromise. The ratio of VEGF protein to its FLT1 receptor was increased in the placenta, suggesting a shift to a more angiogenic state; however, maternal circulating levels of VEGF, sFLT1, and their ratio were not significantly changed. Doppler blood velocities in the maternal uterine artery and fetal umbilical artery and vein were unaltered. This study provides in vivo evidence that chronic inhalation of 250 ppm CO throughout gestation augments uterine blood flow and uteroplacental vascular growth, changes that may protect against the subsequent development of preeclampsia.

Flt3L is a novel regulator of skeletal myogenesis

Various cues initiate multiple signaling pathways to regulate the highly coordinated process of skeletal myogenesis. Myoblast differentiation comprises a series of ordered events starting with cell cycle withdrawal and ending with myocyte fusion, with each step probably controlled by multiple extracellular signals and intracellular signaling pathways. Here we report the identification of Fms-like tyrokine kinase 3 ligand (Flt3L) signaling as a novel regulator of skeletal myogenesis. Flt3L is a multifunctional cytokine in immune cells, but its involvement in skeletal muscle formation has not been reported. We found that Flt3L is expressed in C2C12 myoblasts, with levels increasing throughout differentiation. Knockdown of Flt3L, or its receptor Flt3, suppresses myoblast differentiation, which is rescued by recombinant Flt3L or Flt3, respectively. Differentiation is not rescued, however, by recombinant ligand when the receptor is knocked down, or vice versa, suggesting that Flt3L and Flt3 function together. Flt3L knockdown also inhibits differentiation in mouse primary myoblasts. Both Flt3L and Flt3 are highly expressed in nascent myofibers during muscle regeneration in vivo, and Flt3L siRNA impairs muscle regeneration, validating the physiological significance of Flt3L function in myogenesis. We have identified a cellular mechanism for the myogenic function of Flt3L, as we show that Flt3L promotes cell cycle exit that is necessary for myogenic differentiation. Furthermore, we identify Erk as a relevant target of Flt3L signaling during myogenesis, and demonstrate that Flt3L suppresses Erk signaling through p120RasGAP. In summary, our work reveals an unexpected role for an immunoregulatory cytokine in skeletal myogenesis and a new myogenic pathway.

Maternal imbalance between pro-angiogenic and anti-angiogenic factors in HIV-infected women with pre-eclampsia : cardiovascular topics

Angiogenic imbalance contributes to the development of preeclampsia. We evaluated the protein expression of the proangiogenic placental growth factor (PlGF) and transforming growth factor beta 1 (TGF-β1) compared with the anti-angiogenic soluble fms-like tyrosine kinase receptor (sFlt1) and soluble endoglin (sEng) in HIV-infected normotensive and pre-eclamptic pregnancies. Blood was obtained from 110 pregnant women, enrolled in four groups, namely, HIV-negative normotensives (27); HIV-positive normotensives (31); HIV-negative pre-eclamptics (27) and HIV-positive pre-eclamptics (25), and was used to measure PlGF, TGF-β₁, sFlt1 and sEng levels. Increased sFlt1 and sEng levels were associated with the pre-eclamptics (HIV negative and positive) compared with their counterparts. Decreased PlGF levels were observed between the HIV-negative pre-eclamptics versus HIV-negative normotensives, but levels differed significantly (p = 0.02) among the normotensives (HIV negative and positive). TGF-β₁ remained unchanged across all groups. Higher sEng/TGF-β₁ ratios were associated with the pre-eclamptics (HIV negative and positive) compared with their counterparts. This study demonstrated increased sFlt1 and sEng levels in pre-eclamptic compared with normotensive pregnancies, irrespective of the HIV status.

A review of the mechanism for poor placentation in early-onset preeclampsia: the role of autophagy in trophoblast invasion and vascular remodeling

Shallow trophoblast invasion and impaired vascular remodeling of spiral arteries have been recognized in early-onset preeclampsia. Placentation and vascular remodeling are multistep processes, and hypoxia, placental oxidative stress, excessive or atypical maternal immune response to trophoblasts, exaggerated inflammation, and increased production of anti-angiogenic factors such as the soluble form of the vascular endothelial growth factor (VEGF) receptor (sFlt-1) and soluble endoglin (sENG) may play a role in poor placentation in preeclampsia. Recent findings suggest that autophagy plays an important role in extravillous trophoblast (EVT) invasion and vascular remodeling under hypoxia, and sENG inhibits EVT invasion and vascular remodeling by the inhibition of autophagy under hypoxic conditions. In this review, we discuss the relationship between inadequate autophagy and poor placentation in preeclampsia.

Changes in the Cochlear Vasculature and Vascular Endothelial Growth Factor and Its Receptors in the Aging C57 Mouse Cochlea

Introduction. Previous work has shown a strong association between alterations in cochlear vasculature, aging, and the development of presbycusis. The important role of vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 in angiogenesis suggests a potential role for involvement in this process. The aim of this study was to characterize vascular structure and VEGF and its' receptors in young and old C57 Mice. Methods. Young (4 weeks, n = 14) and aged (32–36 weeks, n = 14) C57BL/6 mice were used. Hearing was evaluated using auditory brainstem response. Cochleas were characterized with qRT-PCR, immunohistochemistry, and gross histological quantification. Results. Old C57 mice demonstrated significantly decreased strial area, blood vessel number, luminal size, and luminal area normalized to strial area (vascularity). qRT-PCR showed a significant upregulation of Flt-1, a VEGF receptor, in older animals. No differences were found in VEGF-A or Flk-1. Immunohistochemistry did not show any differences in staining intensity or area with age or cochlear turn location. Conclusion. The marked deafness of aged C57 mice could be in part meditated by loss of vascular development and alterations in VEGF signaling.

AUF1-RGG peptides up-regulate the VEGF antagonist, soluble VEGF receptor-1 (sFlt-1)

The macrophage is essential to the innate immune response, but also contributes to human disease by aggravating inflammation. Under severe inflammation, macrophages and other immune cells over-produce immune mediators, including vascular endothelial growth factor (VEGF). The VEGF protein stimulates macrophage activation and induces macrophage migration. A natural inhibitor of VEGF, the soluble VEGF receptor (sFlt-1) is also produced by macrophages and sFlt-1 has been used clinically to block VEGF. In macrophages, we have shown that the mRNA regulatory protein AUF1/hnRNP D represses VEGF gene expression by inhibiting translation of AURE-regulated VEGF mRNA. Peptides (AUF1-RGG peptides) that are modeled on the arginine-glycine-glycine (RGG) motif in AUF1 also block VEGF expression. This report shows that the AUF1-RGG peptides reduce two other AURE-regulated genes, TNF and GLUT1. Three alternative splice variants of sFlt-1 contain AURE in their 3′UTR, and in an apparent paradox, AUF1-RGG peptides stimulate expression of these three sFlt-1 Variants. The AUF1-RGG peptides likely act by distinct mechanisms with complimentary effects to repress VEGF gene expression and over-express the endogenous VEGF blocking agent, sFlt-1. The AUF1-RGG peptides are novel reagents that reduce VEGF and other inflammatory mediators, and may be useful tools to suppress severe inflammation.