Fetal Liver Tyrosine Kinase-3 Research Articles

MIDOSTAURIN ADDED TO CYTOSINE ARABINOSIDE PLUS IDARUBICIN (“7+3”) IN NEWLY DIAGNOSED FLT3-MUTATED ACUTE MYELOID LEUKEMIA

The combination of an anthracycline and cytosine arabinoside has been the standard induction therapy for acute myeloid leukemia (AML) for more than four decades. Fetal liver tyrosine kinase 3 (FLT3) mutations are among the most frequently found mutations in newly diagnosed AML. According to the results of RATIFY trial, midostaurin was approved for use together with intensive chemotherapy (cytarabine plus daunorubicin). Recently, idarubicin has been more frequently favored over daunorubicin. Report the outcome of patients with AML and FLT3 mutated treated with a combination of cytosine arabinoside plus anthracycline (“7+3”regimen) and midostaurin. Herein, we retrospectively analyzed the treatment outcome of three patients with newly diagnosed AML and confirmed FLT3-ITD were eligible. The treatment consisted induction with “7+3”regimen and midostaurin (administered at a dose of 50 mg orally twice daily on days 8 through 21). All were male with age between 21 and 38 years old. A normal karyotype was found in all cases. Patients received cytarabine, idarubicin and midostaurin for induction and all experienced febrile neutropenia and two necessitated preemptive liposomal amphotericin B. One patient developed neutropenic enterocolitis. A second course of induction chemotherapy was administered to 2 patients. Relapse was observed in 1 patient (after Allo-HCT). One patient died after allo-HCT due to severe acute GVHD and one patient is alive in CR1. We showed that young patients, who had AML and a FLT3-ITD mutation, can be treated with the combination of cytarabine, idarubicin and midostaurin. The RATIFY study (Stone et al., 2017) and the German-Austrian AML Study Group (Schlenk et al., 2019) used daunorubicin 60 mg/m2 for induction, in combination with cytarabine and midostaurin. The complete remission rates in these trials were 59.8% (CR – Ratify) and 77.9% (CR+Cri - German-Austrian trial). In contrast with these two studies, idarubicin was used for induction in 53.5% of cases and was favored over daunorubicin in a more recent trial (Sierra et al. 2020). Of note, the results of FLT3 are necessary before the day 8 of induction. Hence, some patients may have already received the all three planned doses of anthracycline (idarubicin) before the information on results of FLT3 mutation. The recent approvals of new drugs for treating AML will modify the treatment algorithm and our data indicate that the use of idarubicin in combination with cytarabine and midostaurin is feasible in young patients with FLT3-mutated AML.

Development of Novel Highly Specific FLT3 Targeted Inhibitors for Combination or Sequential Therapy of Acute Myeloid Leukemia

More than 25% of acute myeloid leukemia (AML) patients have an activating mutation of the fetal-liver tyrosine kinase 3 (FLT3) receptor. FLT3 mutations vary, can be multiple, are often unstable length mutations located in the receptor's juxtamembranous domain (internal tandem duplication (ITD) mutations), and represent the strongest single marker for relapse of AML. When developing novel inhibitors of FLT3, it should be taken into careful consideration that there is a high inter- and intra-patient variability in the length and sequence repeated within ITD mutations. Recently, the first FLT3 targeting kinase inhibitor midostaurin was approved for combination therapy in FLT3 mutated AML, and several other kinase inhibitors are at present also in preclinical development. These inhibitors share broad kinase specificity, in several cases co-inhibiting FLT3 with structurally related targets like c-Kit and AXL. These targets may add therapeutic effect as well as adverse effects. Highly specific kinase inhibitors against varying FLT3-ITDs are to our knowledge currently not in clinical development.We are developing a novel class of highly specific FLT3-ITD targeting kinase inhibitors based on a well-established chemical backbone previously used in well-tolerated anti-viral therapy. These inhibitors, the BiKin2 compounds, are specifically developed to have superior selectivity towards FLT3-ITD over closely related type III tyrosine kinases, minimizing the chances of adverse off-target effects. We are screening the compounds to identify analogues that target a wide variety of FLT3-ITD mutations, and that can be combined with other BiKin2 analogues and/or with other targeted therapies in order to avoid development of acquired resistance during therapy.By employing cell lines expressing wild type (wt) FLT3 and ITD mutations of varying lengths we are currently testing more than 40 analogues of the BiKin2 compounds in in vivo and in vitro settings. Among the leukemic cell lines being used are the FLT3-mutated cells MOLM-13 and MV4-11 and the FLT3-wt cells OCI-AML3. Additionally, we are using the BA/F3 cells as a model of FLT3-ITD oncogene addiction. The BA/F3 cells are stably transduced to express FLT3-ITD of different lengths cloned from an AML patient at diagnosis and relapse, in addition to specific point mutations affecting the kinase domain. These screens give us valuable information on how the BiKin2 compounds inhibit and affect ITDs of varying length and sequences. Thus far these novel compounds have shown promising results in ITD mutated cell lines, as shown in Table 1 and Figure 1A.We have piloted an in vivo toxicity test (MTD) and proof-of-principle-experiments with the first compound showing promising selectivity for FLT3-ITD, BCI-332. We found no dose-limiting toxicity at doses up to 10 mg/kg (Figure 1B), and the drug also showed significant inhibition of tumor growth in a subcutaneous model of FLT3-ITD expressing MV4-11 (ITD/null) cells (Figure 1C). The FLT3 selectivity of BCI-332 at 1 µM over 250 kinases in vitro is superior with 90% versus below 20% inhibition.In the clinic, patients often develop resistance to FLT3 inhibitors due to secondary mutations within the mutated gene as well as selection of resistant leukemic clones. Therefore, we used a high throughput in vitro drug sensitivity and resistance test (DSRT) approach to evaluate the efficacy of the model compound BCI-332 in a combination screen together with 527 clinically approved and investigational drugs. Drug combinations were tested in both FLT3-ITD expressing (MOLM-13 wt/ITD) and FLT3-wt expressing (OCI-AML3 wt/wt) cells, and we identified several potential drug candidates that showed additive effect in combination with BCI-332 in both conditions (Figure 2). Potential combination candidates need to be further validated in more AML cell lines and in primary cells from AML patients with defined ITD mutational status.In summary, we employed a novel approach to develop FLT3-ITD targeting drugs by screening inhibitors for selectivity against multiple different length ITD mutations, in an attempt to identify FLT3-ITD specific inhibitors with minimal off-target inhibition. Ultimately, our goal is to identify one or several drug candidate(s) to be tested in a clinical trial setting, in combination or in sequences combined with other targeted therapies. [Display omitted] DisclosuresAmiable:BCI Pharma: Employment. Popa:Kinn Therapeutics AS: Employment. Guillon:BCI Pharma: Employment. Heckman:Novartis: Research Funding; Orion Pharma: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; IMI2 project HARMONY: Research Funding. McCormack:Kinn Therapeutics AS: Other: Co-ownership. Surleraux:BCI Pharma: Employment, Equity Ownership.

Pre-transplant FLT3/ITD status predicts outcome in FLT3-mutated acute myeloid leukemia following allogeneic stem cell transplantation

Acute myeloid leukemia (AML) with fetal liver tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) is associated with poor prognosis, and allogeneic stem cell transplantation (Allo-SCT) seems to be the preferred therapeutic approach. However, the predictors of post-transplant outcomes were not well-defined. The aim of the study was to evaluate the significance of FLT3/ITD mutation by polymerase chain reaction as minimal residual disease (MRD) marker of outcomes after transplantation. We identified 43 patients (28 females and 15 males) with FLT3-mutated AML at the median age of 45 years who were allografted between 2009 and 2019. Hematological status at transplant was as follows: the first complete remission (CR1) in 29 patients, CR2 in 5, and 9 patients were transplanted in marrow aplasia (MA). Twenty-seven patients were FLT3 MRD negative at transplant. Median time from diagnosis to transplant was 16.7 months. Post-allograft CR rate was 88%. The relapse incidence (RI) was lower for patients who were FLT3 MRD negative at transplant when compared with those with FLT3 MRD positivity (41% vs 59%; p = 0.01). The patients who eradicated FLT3/ITD at day + 30 after transplant had lower RI than those with detectable FLT3/ITD (23% vs 76%; p = <0.001). The 2-year LFS and OS were 53% and 54%, with the median OS and LFS of 28 months and 27 months, respectively. Patients with CR1/2 and FLT3 MRD(−) had a 2-year OS of 80%. The FLT3 MRD negativity at transplant prolonged LFS in multivariate analysis (HR 5.3 95%CI 1.97–14.2); p < 0.001), whereas FLT3 MRD negativity and unrelated donor predicted favorable OS.

FLT3 internal tandem duplication does not impact prognosis after haploidentical allogeneic hematopoietic stem cell transplantation in AML patients.

Acute myelogenous leukemia (AML) patients with fetal liver tyrosine kinase 3 (FLT3) internal tandem duplications (ITDs) have poor prognoses if treated with chemotherapy only, primarily as they experience increased relapse rates. To determine whether this alteration also affects outcomes after haploidentical donor (HID) allogeneic hematopoietic stem cell transplantation (allo-HSCT), we compared 334 consecutive FLT3-ITD-positive vs -negative patients with AML (other than acute promyelocytic leukemia) who underwent HID-HSCT. FLT3-ITD was detected in 39 of 334 patients (11.7%). The 2-year relapse rates for FLT3-ITD-positive and -negative patients were 16% and 17%, respectively (P = 0.774). The 3-year disease-free survival (DFS) rates for FLT3-ITD-positive and -negative patients were 74% (95% confidence interval [CI]: 64-81) and 73% (95% CI: 70-81), respectively; P = 0.872); while the 3-year overall survival (OS) rates were 72% (95% CI: 67-81) and 77% (95% CI: 72-84), respectively (P = 0.862). FLT3-ITD mutation had no influence on non-relapse mortality (NRM 15% vs 14%, P = 0.463). Multivariate analyses showed that disease status at HSCT and white blood cell count at diagnosis were independent risk factors associated with relapse, DFS, and OS. In conclusion, FLT3 mutation status has no impact on outcomes after HID-HSCT in patients with AML. HID-HSCT is therefore a valid option for AML patients with FLT3-ITD mutation.

Molecular Dynamics Studies on D835N Mutation in FLT3-Its Impact on FLT3 Protein Structure.

Mutations in Fetal Liver Tyrosine Kinase 3 (FLT3) genes are implicated in the constitutive activation and development of Acute Myeloid Leukaemia (AML). They are involved in signalling pathway of autonomous proliferation and block differentiation in leukaemia cells. FLT3 is considered as a promising target for the therapeutic intervention of AML. There are a few missense mutations associated with FLT3 that are found in AML patients. The D835N mutation is the most frequently observed and the aspartic acid in this position acts as a key residue for the receptor activation. The present study aims to understand the structural effect of D835N mutation in FLT3. We carried out the molecular dynamics (MD) simulation for a period of 120 ns at 300 K. Root-mean square deviation, root-mean square fluctuations, surface accessibility, radius of gyration, hydrogen bond, eigenvector projection analysis, trace of covariance matrix, and density analysis revealed the instability of mutant (D835N) protein. Our study provides new insights on the conformational changes in the mutant (D835N) structure of FLT3 protein. Our observations will be useful for researchers exploring AML and for the development of FLT3 inhibitors.

Is allogeneic transplantation really the best treatment for FLT3/ITD-positive acute myeloid leukemia? A systematic review.

Fetal liver tyrosine kinase 3 (FLT3)-internal tandem duplications (ITDs) has been used as a powerful adverse prognostic indicator for acute myeloid leukemia (AML) in any age group. Evidence is mixed regarding the effects of allogeneic transplantation (allo-HSCT) in first complete remission (CR) for patients with FLT3/ITD AML. To fill this gap, this study provides a systematic review and meta-analysis of patients with FLT3/ITD AML receiving HSCT. A search of PubMed, Embase, and OVID yielded 1706 abstracts, two researchers screening the trials based on inclusion and exclusion criteria, and assessed the methodology quality independently. Meta-analysis showed that compared with chemotherapy, both allo-HSCT and autologous hematopoietic cell transplantation (auto-HSCT) can reduce the relapse rate (p < 0.01) and improve both the OS (p < 0.01) and DFS (p < 0.01). But when compared allo-HSCT with auto-HSCT, the OS (p = 0.27) and DFS (p = 0.19) have no statistical significance, and only the relapse indicator has statistical significance, p < 0.01. Based on the results, we can conclude that allo-HSCT is an efficient therapy approach for patients with FLT3/ITD AML. Chemotherapy cannot change the poor prognosis. Auto-HSCT can improve OS and DFS, but it cannot reduce the relapse rate.

Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML

AbstractSDF-1α/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow–derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1α (SDF-1α)–induced and stroma-induced chemotaxis and inhibited SDF-1α–induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor– and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.

Acute Myeloid Leukemia (AML) Patients with an Internal Tandem Duplication Mutation in the Fetal Liver Tyrosine Kinase-3 (FLT3/ITD) Gene Express CD117 (C-kit)

Acute Myeloid Leukemia (AML) Patients with an Internal Tandem Duplication Mutation in the Fetal Liver Tyrosine Kinase-3 (FLT3/ITD) Gene Express CD117 (C-kit)

Presence of FLT3 Mutations Does Not Impair Stem Cell Mobilization and Feasibility of Autologous Peripheral Blood Stem Cell Transplantation in Acute Myeloid Leukemia

Fetal liver tyrosine kinase 3 (FLT3) mutations represent a powerful prognostic indicator in acute myeloid leukemia (AML). Further, interaction between FLT3 and its ligand plays a role in normal hematopoiesis. Accordingly, FLT3 mutations may affect mobilization of peripheral blood stem cells (PBSCs) and feasibility of autologous stem cell transplantation (ASCT) in AML. We analyzed the effect of FLT3 mutations on mobilization of CD34 + cells and on PBASCT feasibility from 111 patients with AML, with a median age of 58 years and normal karyotype. Overall, 23 patients (21%) had FLT3 mutations. The complete remission rate was 74% and was not influenced by FLT3 mutations (73% for patients with FLT3 − and 78% for those with FLT3 +; P = .78). The successful mobilization rate was 79% and was comparable for patients with FLT3 − and with FLT3 + ( P = .42). Median numbers of CD34 + cells collected were 7.6 × 10 6/kg and 7.1 × 10 6/kg for patients with FLT3 − and those with FLT3 +, respectively ( P = .64). Among 73 patients evaluated for mobilization, feasibility of ASCT was 71%, and there was no difference between patients with FLT3 − (74%) and those with FLT3 + (61%), P = .43. We conclude that the FLT3 mutations have no influence on mobilization of CD34 + cells or on feasibility of PBASCT in patients with AML and normal karyotype.

Basic Fibroblast Growth Factor Supports Undifferentiated Human Embryonic Stem Cell Growth Without Conditioned Medium

Previous studies have shown that prolonged propagation of undifferentiated human embryonic stem cells (hESCs) requires conditioned medium from mouse embryonic feeders (MEF-CM) as well as matrix components. Because hESCs express growth factor receptors, including those for basic fibroblast growth factor (bFGF), stem cell factor (SCF), and fetal liver tyrosine kinase-3 ligand (Flt3L), we evaluated these and other growth factors for their ability to maintain undifferentiated hESCs in the absence of conditioned medium. We found cultures maintained in bFGF alone or in combination with other factors showed characteristics similar to MEF-CM control cultures, including morphology, surface marker and transcription factor expression, telomerase activity, differentiation, and karyotypic stability. In contrast, cells in media containing Flt-3L, thrombopoietin, and SCF, individually or in combination, showed almost complete differentiation after 6 weeks in culture. These data demonstrate that hESCs can be maintained in nonconditioned medium using growth factors.

Relation between CXCR-4 expression, Flt3 mutations, and unfavorable prognosis of adult acute myeloid leukemia

AbstractRecently it was shown that, analogous to normal hematopoietic cells, the level of CXC chemokine receptor 4 (CXCR-4) expression on acute myeloid leukemia (AML) cells correlates with stromal cell derived factor-1 alpha (SDF-1)-induced chemotaxis. As we speculated that an anomalous organ distribution of AML cells could affect cell survival and thus result in an altered fraction surviving chemotherapy, we examined a possible correlation between patient prognosis and CXCR-4 expression in AML patients. We found that patients with a high CXCR-4 expression in the CD34+ subset had a significantly reduced survival and a higher probability of relapse, resulting in a median relapse-free survival (RFS) of only 8.3 months. CXCR-4 expression was significantly higher in fetal liver tyrosine kinase-3 (Flt3)/internal tandem duplication (ITD) AML than in Flt3/wild-type (wt) AML. Covariate analysis indicated that the prognostic significance of Flt3/ITDs with respect to RFS was no more apparent when analyzed in conjunction with the expression of CXCR-4 in the CD34+ subset, suggesting that the poor prognosis of Flt3/ITD AML might be subordinate to the increased CXCR-4 expression. Using a granulocyte colony-stimulating factor receptor (G-CSF-R)-expressing 32D cell line, we observed that SDF-1/CXCR-4 interaction is required for the survival of myeloid differentiating cells, and it also induces a block in G-CSF-induced myeloid differentiation. These data suggest that the SDF-1/CXCR-4 axis may influence therapy responsiveness and defines unfavorable prognosis in AML. (Blood. 2004;104:550-557)

The sustained granulopoietic effect of progenipoietin encapsulated in multivesicular liposomes

Progenipoietin (ProGP), a dual receptor agonist of fetal liver tyrosine kinase-3 (flt3) and granulocyte colony-stimulating factor (G-CSF) receptors, has been shown to significantly enhance production of both polymorphonuclear leukocytes and dendritic cells (DCs) in the peripheral blood and spleen of mice, when administered as daily s.c. injections for about 10 days. Here, we have successfully designed a sustained-delivery formulation for this novel chimeric protein using multivesicular liposomes (DepoFoam), and studied the effects of changing both the triglyceride and phospholipid composition of the lipid matrix to modulate its delivery profile. Encapsulation of ProGP in these particles led to retention of its structural integrity, and maintenance of its biological activity in vivo. Administration of a single s.c. dose of 1 mg/kg of an optimized DepoProGP formulation in rats, led to significant elevation of absolute neutrophil counts (ANC) that were maintained at levels >10,000 μl −1 for 9–11 days, in a reproducible manner. In contrast, administration of the unencapsulated ProGP at the same dose, resulted in elevation of neutrophils by day 1, followed by a quick decline to base line levels by day 3. These data suggest the possibility of administering a single dose of DepoFoam-encapsulated ProGP to improve hematopoietic recovery time after chemotherapy, and for other indications that require multiple daily doses of ProGP.

Purification and characterization of progenipoietins produced in Escherichia. coli

The progenipoietins (ProGPs) are a family of genetically engineered chimeric proteins that contain receptor agonist activity for both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. These unique proteins have previously been shown to induce the proliferation of multiple cell lineages. The characterization of two progenipoietins, ProGP-1 and ProGP-4, refolded and purified from an Escherichia coli expression system is described. These ProGP molecules differ in the orientation of the two receptor agonists and, in addition, ProGP-4 contains a fetal liver tyrosine kinase-3 receptor agonist that has been circularly permuted to modulate its activity. Static light scattering analyses demonstrated that both ProGP molecules exist as dimers, most likely through non-covalent interaction of the fetal liver tyrosine kinase-3 receptor agonist domains. ProGP-1 and ProGP-4 have comparable secondary structures, as analyzed by circular dichroism; however, their tertiary structures, as measured by intrinsic fluorescence, were demonstrated to be different. Differential scanning calorimetry demonstrated that the thermal stability of these two proteins was indistinguishable. Interestingly, these dual agonist proteins yielded only a single melting temperature value that was intermediate between that of their individual receptor agonist components, indicating that these chimeric molecules behave as a single domain protein during thermal denaturation. This study describes the purification and physico-chemical properties of this class of proteins generated using an E. coli expression system.

Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways cooperate in mediating cytokine-induced proliferation of a leukemic cell line.

Granulocyte colony-stimulating factor (G-CSF) and fetal liver tyrosine kinase-3 (Flt3) ligand (FL) act in synergy to induce expansion and mobilization of hematopoietic progenitor cells. Regulation of mitogen activated protein (MAP) kinase pathways and gene transcription, induced by these cytokines were examined using the OCI-AML5 cell line. For this purpose, FL and G-CSF were used either alone, or in combination as the co-addition of FL and G-CSF (FL+G-CSF), or a chimeric molecule, progenipoietin-1 (ProGP-1). Both G-CSF and FL induced phosphorylation of extracellular signal-regulated kinases (ERKs) while p38 mitogen activated protein (MAP) kinase was phosphorylated only in response to G-CSF but not FL. Studies using specific kinase inhibitors suggested that both ERK and p38 MAP kinase pathways were required for the optimal cell proliferation in response to both G-CSF and FL. The magnitude of activation of the ERK pathway and induction of genes involved in cell cycle progression by G-CSF and FL exhibited a strong correlation with the degree of cell proliferation. These data suggest that OCI-AML5 cells proliferate at least in part, due to the activation of both ERK and p38 MAP kinase pathways in response to G-CSF and FL. This study represents the first report of the specific cell cycle genes induced by FL.