FLT3-ITD transduces autonomous growth signals during its biosynthetic trafficking in acute myelogenous leukemia cells.

FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), then transduces growth signals. FLT3 gene alterations that lead the kinase to assume its permanently active form, such as internal tandem duplication (ITD) and D835Y substitution, are found in 30-40% of acute myelogenous leukemia (AML) patients. Thus, drugs for molecular targeting of FLT3 mutants have been developed for the treatment of AML. Several groups have reported that compared with wild-type FLT3 (FLT3-wt), FLT3 mutants are retained in organelles, resulting in low levels of PM localization of the receptor. However, the precise subcellular localization of mutant FLT3 remains unclear, and the relationship between oncogenic signaling and the mislocalization is not completely understood. In this study, we show that in cell lines established from leukemia patients, endogenous FLT3-ITD but not FLT3-wt clearly accumulates in the perinuclear region. Our co-immunofluorescence assays demonstrate that Golgi markers are co-localized with the perinuclear region, indicating that FLT3-ITD mainly localizes to the Golgi region in AML cells. FLT3-ITD biosynthetically traffics to the Golgi apparatus and remains there in a manner dependent on its tyrosine kinase activity. Tyrosine kinase inhibitors, such as quizartinib (AC220) and midostaurin (PKC412), markedly decrease FLT3-ITD retention and increase PM levels of the mutant. FLT3-ITD activates downstream in the endoplasmic reticulum (ER) and the Golgi apparatus during its biosynthetic trafficking. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. We provide evidence that FLT3-ITD signals from the early secretory compartments before reaching the PM in AML cells.

FLT3-ITD and CD135 Over-Expression are Frequent Findings of Poor Survival in Adult Patients with Acute Leukemias.

BACKGROUND: Fms-like tyrosine kinase 3 (FLT3) expression and mutation have been considered a poor prognostic factor in acute myeloid leukemia (AML). FLT3-ITD mutation is present in 30% of adult patients with AML and 2-5% in childhood acute lymphoblastic leukemia (ALL). The impact of these mutations on the prognosis of ALL patients, has not yet been established. Moreover, a limited number of publications regarding the level of expression of the FLT3 receptor (CD135) in both leukemias exist. This study aimed to analyze the clinical outcomes associated to the presence of FLT3-ITD mutation and the expression of CD135. METHODS: 82 adult patients with newly diagnosed acute leukemia (39 with AML and 43 with ALL) were included. Flow cytometry and RT-PCR were done to analyze the expression of CD135 and the presence of FLT3 ITD mutation, respectively. RESULTS: FLT3-ITD was present in 14 (36%) of AML and 15 (35%) of ALL patients. Disease free survival (DFS) and overall survival (OS) were lower in ALL patients having a CD135 expression >3000 cells/µL. There was a trend for poor OS in AML patients expressing FLT3 ITD. OS was worse in AML patients with high expression of CD135. CONCLUSION: A higher (35%) frequency of FLT3-ITD was found in adult ALL patients. The presence of FLT3-ITD was associated with a trend of poor OS in AML cases, and overexpression of CD135 was correlated with poor DFS in ALL cases and poor OS in both acute leukemias.

Synergistic targeting of FLT3 mutations in AML via combined menin-MLL and FLT3 inhibition.

The interaction of menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and provides a potential opportunity for treatment of NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. In this study, transcriptional profiling after pharmacological inhibition of the menin-MLL complex revealed specific changes in gene expression, with downregulation of the MEIS1 transcription factor and its transcriptional target gene FLT3 being the most pronounced. Combining menin-MLL inhibition with specific small-molecule kinase inhibitors of FLT3 phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream of FLT3 signaling. The drug combination induced synergistic inhibition of proliferation, as well as enhanced apoptosis, compared with single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary acute myeloid leukemia (AML) cells harvested from patients with NPM1mutFLT3mut AML showed significantly better responses to combined menin and FLT3 inhibition than to single-drug or vehicle control treatment, whereas AML cells with wild-type NPM1, MLL, and FLT3 were not affected by either of the 2 drugs. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared with results in the single-drug and vehicle control groups. Our data suggest that combined menin-MLL and FLT3 inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.

AAV-Vectored Fms-Related Tyrosine Kinase 3 Ligand Inhibits CD34+ Progenitor Cell Engraftment in Humanized Mice.

Humanized mice have become useful animal models for HIV/AIDS. Since NOD.Cg-Prkdc scid Il2rgtm1Wjl/SzJ (NSG) mice allow the engraftment of primary human immune cells, we aim to determine the role of human Fms-related tyrosine kinase 3 ligand (hFlt3L), a major growth factor for dendritic cells (DCs), in regulating the differentiation of cord blood-derived CD34+ progenitor cells in this murine species. Soluble recombinant hFlt3L protein and AAV-vectored hFlt3L were administrated before or after human CD34+ progenitor cell transplantation, respectively. We then measured the peripheral levels of hFlt3L by ELISA. Meantime, reconstituted human immune cells were analyzed by flow cytometry over time. We found that without hFlt3L there were significantly increased types of human immune cells in NSG-huCD34 compared with NSG-huPBL mice but the frequency of human DCs remains low. Transient treatment with recombinant hFlt3L expanded human conventional CD1c+ and CD141+ DCs as well as plasmacytoid DCs in humanized NSG-huCD34 mice. Surprisingly, however, the prolonged in vivo expression of AAV-vectored hFlt3L resulted in significant suppression of total human CD34+ cell engraftment and differentiation. The suppression occurred within 2 weeks when AAV-vectored hFlt3L was administered either before or after the transplantation of CD34+ progenitor cells, which was likely associated with the induction of murine myeloid-derived immune suppressive cells and reactive oxygen species in NSG-huCD34 mice. Since chronic  HIV-1 patients displayed significantly high levels of hFlt3L expression, our findings may have implication to explore the role of prolonged hFlt3L in regulating  the differentiation of human CD34+ progenitor cells in both NSG-huCD34 mice and infected people. Graphical Abstract ᅟ.

Internal tandem duplication mutations in the tyrosine kinase domain of FLT3 display a higher oncogenic potential than the activation loop D835Y mutation.

Acute myeloid leukemia (AML) remains the most common form of acute leukemia among adults and accounts for a large number of leukemia-related deaths. Mutations in FMS-like tyrosine kinase 3 (FLT3) is one of the most prevalent findings in this heterogeneous disease. The major types of mutations in FLT3 can be categorized as internal tandem duplications (ITD) and point mutations. Recent studies suggest that ITDs not only occur in the juxtamembrane region as originally described, but also in the kinase domain. Although the juxtamembrane ITDs have been well characterized, the tyrosine kinase domain ITDs have not yet been thoroughly studied due to their recent discovery. For this reason, we compared ITD mutations in the juxtamembrane domain with those in the tyrosine kinase domain, as well as with the most common activating point mutation in the tyrosine kinase domain, D835Y. The purpose of this study was to understand whether it is the nature of the mutation or the location of the mutation that plays the main role in leukemogenesis. The various FLT3 mutants were expressed in the murine pro-B cell line Ba/F3 and examined for their capacity to form colonies in semisolid medium. The size and number of colonies formed by Ba/F3 cells expressing either the internal tandem duplication within juxtamembrane domain of the receptor (JMD-ITD) or the tyrosine kinase domain (TKD)-ITD were indistinguishable, while Ba/F3 cells expressing D835Y/FLT3 failed to form colonies. Cell proliferation and cell survival was also significantly higher in TKD-ITD expressing cells, compared to cells expressing D835Y/FLT3. Furthermore, TKD-ITD is capable of inducing phosphorylation of STAT5, while D835Y/FLT3 fails to induce tyrosine phosphorylation of STAT5. Other signal transduction pathways such as the RAS/ERK and the PI3K/AKT pathways were activated to the same level in TKD-ITD cells as compared to D835Y/FLT3 expressing cells. Taken together, our data suggest that TKD-ITD displays similar oncogenic potential to the JMD-ITD but a higher oncogenic potential than the D835Y point mutation.

Predictive value of pretransplantation molecular minimal residual disease assessment by WT1 gene expression in FLT3-positive acute myeloid leukemia.

The FMS-like tyrosine kinase 3 (FLT3) mutation in acute myeloid leukemia (AML) is a negative prognostic factor and, in these cases, allogeneic stem cell transplantation (allo-SCT) can represent an important therapeutic option, especially if performed in complete remission (CR). However, it is increasingly clear that not all cytological CRs (cCRs) are the same and that minimal residual disease (MRD) before allo-SCT could have an impact on AML outcome. Unfortunately, FLT3, due its instability of expression, is still not considered a good molecular MRD marker. We analyzed the outcome of allo-SCT in a population of FLT3-positive AML patients according to molecular MRD at the pretransplantation workup, assessed by the quantitative expression evaluation of the panleukemic marker Wilms' tumor (WT1) gene. Sixty-two consecutive AML FLT3-positive patients received allo-SCT between 2005 and 2016 in our center. The median age at transplantation was 55 years. The quantitative analysis of the WT1 gene expression (bone marrow samples) was available in 54 out of 62 (87%) cases, both at diagnosis (100% overexpressing WT1 with a mean of 9747 ± 8064 copies) and before allo-SCT (33 WT1-negative and 21 WT1-positive cases at the pretransplantation workup). Of these cases, 33/54 (61%) were both in cCR and molecular remission (WT1-negative) at the time of transplantation, 13/54 (24%) were in cCR but not in molecular remission (WT1-positive), and 8/54 (15%) showed a cytological evidence of disease (relapsed or refractory). Both post-allo-SCT overall survival (OS) and disease-free survival (DFS) were significantly better in patients who were WT1-negative (WT1 <250 copies) at the time of transplantation compared with those who were WT1-positive (WT1 >250 copies), with a median OS and DFS not reached in the WT1-negative group and 10.2 and 5.5 months, respectively, in the WT1-positive group (OS log-rank p = 0.0005; hazard ratio [HR] = 3.7, 95% confidence interval [95% CI] = 1.5-9; DFS log-rank p = 0.0001; HR = 4.38, 95% CI = 1.9-10). Patients with cCR who were WT1-positive had the same negative outcome as those with a cytological evidence of disease. The relapse rate after allo-SCT was 9% (3/33) in pre-allo-SCT WT1-negative cases and 54% (7/13) in WT1-positive cases (p = 0.002). At multivariate analysis, WT1 negativity before allo-SCT and grade <2 acute graft versus host disease were the only independent prognostic factors for improved OS and DFS. These data show that pre-allo-SCT molecular MRD evaluation through WT1 expression is a powerful predictor of posttransplantation outcomes (OS, DFS, relapse rate). Patients with both cCR and a WT1-negative marker before allo-SCT have a very good outcome with very low relapse rate; conversely, patients with positive molecular MRD and refractory/relapsed patients have a negative outcome. The WT1 MRD stratification in FLT3-positive AML is a valuable tool with which to identify patients who are at high risk of relapse and that could be considered from post-allo-SCT prophylaxis with FLT3 inhibitors or other strategies (donor lymphocyte infusion, tapering of immunosuppression, azacitidine).

Stable curcumin-loaded polymeric micellar formulation for enhancing cellular uptake and cytotoxicity to FLT3 overexpressing EoL-1 leukemic cells.

The present study aims to develop a stable polymeric micellar formulation of curcumin (CM) with improved solubility and stability, and that is suitable for clinical applications in leukemia patients. CM-loaded polymeric micelles (CM-micelles) were prepared using poloxamers. The chemical structure of the polymers influenced micellar properties. The best formulation of CM-micelles, namely CM-P407, was obtained from poloxamer 407 at drug to polymer ratio of 1:30 and rehydrated with phosphate buffer solution pH 7.4. CM-P407 exhibited the smallest size of 30.3±1.3nm and highest entrapment efficiency of 88.4±4.1%. When stored at -80°C for 60days, CM-P407 retained high protection of CM and had no significant size change. In comparison with CM solution in dimethyl sulfoxide (CM-DMSO), CM kinetic degradation in both formulations followed a pseudo-first-order reaction, but the half-life of CM in CM-P407 was approx. 200 times longer than in CM-DMSO. Regarding the activity against FLT3 overexpressing EoL-1 leukemic cells, CM-P407 showed higher cytotoxicity than CM-DMSO. Moreover, intracellular uptake to leukemic cells of CM-P407 was 2-3 times greater than that of CM-DMSO. These promising results for CM-P407 will be further investigated in rodents and in clinical studies for leukemia treatment.

Development and Characterization of FLT3-Specific Curcumin-Loaded Polymeric Micelles as a Drug Delivery System for Treating FLT3-Overexpressing Leukemic Cells.

This study aimed at developing a curcumin (CM) nanoparticle targeted to Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FLT3) protein on the surface of leukemic cells and at evaluating their properties, specificity, cytotoxicity, and inhibitory effect on FLT3 protein level in FLT3-overexpressing leukemic cells, EoL-1, and MV-4-11 cells. FLT3-specific peptides were conjugated onto modified poloxamer 407 using the copper-catalyzed azide-alkyne cycloaddition reaction. The thin film hydration method was performed for FLT3-specific CM-loaded polymeric micelles (FLT3-CM-micelles) preparation. Flow cytometry and fluorescence microscopy were used to determine rate of cellular uptake. 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to test the cytotoxicity of the micelles on leukemic cells. FLT3-CM-micelles demonstrated a mean particle size less than 50 nm, high entrapment efficiency, and high rate of CM uptake by leukemic cells. The intracellular CM fluorescence is related to FLT3 protein levels on the leukemic cell surfaces. Moreover, FLT3-CM-micelles demonstrated an excellent cytotoxic effect and decreased FLT3 protein expression in the leukemic cells. The FLT3-CM-micelles could enhance both solubility and cytotoxicity of CM on FLT3-overexpressing leukemic cells. These promising nanoparticles may be used for enhancing antileukemic activity of CM and developed as a targeted drug delivery system in the future.

High fms-like tyrosine kinase-3 (FLT3) receptor surface expression predicts poor outcome in FLT3 internal tandem duplication (ITD) negative patients in adult acute myeloid leukaemia: A prospective pilot study from India.

BACKGROUND & OBJECTIVES: Mutations in fms-like tyrosine kinase 3 (FLT3) receptor have significant role in assessing outcome in patients with acute myeloid leukaemia (AML). Data for FLT3 surface expression in relation to FLT3 internal tandem duplication (ITD) status and outcome are not available from India. The objective of the current study was to investigate adult patients with AML for FLT3 expression and FLT3 ITD mutation, and their association with long-term outcome. METHODS: Total 51 consecutive de novo AML patients aged 18-60 yr were enrolled in the study. FLT3 ITD was detected by polymerase chain reaction (PCR); flowcytometry and qPCR (Taqman probe chemistry) were used for assessment of FLT3 protein and transcript, respectively. Kaplan Meier curves were obtained for survival analysis followed by log rank test. RESULTS: FLT3 ITD was present in eight (16%) patients. Complete remission was achieved in 33 (64.6%) patients. At 57.3 months, event free survival (EFS) was 26.9±6.3 per cent, disease free survival (DFS) 52.0±9.2 per cent, and overall survival event (OS) 34.5±7.4 per cent. FLT3 surface expression was positive (>20%) by flow-cytometry in 38 (88%) of the 51 patients. FLT3 surface expression and transcripts were not associated with FLT3 ITD status. FLT3 expression was significantly associated with inferior EFS (P=0.026) and OS (P=0.018) in those who were negative for FLT3 ITD. INTERPRETATION & CONCLUSIONS: This study evaluated FLT3 ITD mutation along with FLT3 expression in AML patients, and associated with survival. Negative impact of FLT3 surface expression on survival was observed in AML patients who were FLT3 ITD negative.

Novel anticancer compound [trifluoromethyl-substituted pyrazole N-nucleoside] inhibits FLT3 activity to induce differentiation in acute myeloid leukemia cells.

Anticancer properties of chemically synthesized compounds have continuously been optimized for better efficacy and selectivity. Derivatives of heterocyclic compounds are well known to have selective antiproliferative effect against many types of cancer. In this study, we investigated the ability of an indigenously synthesized anticancer molecule, G-11 [1-(2",3",4",6"-Tetra-O-acetyl-ß-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one], to cause selective cytotoxicity and induce differentiation in the acute myeloid leukemia HL-60 cells. G-11 was able to exert cytotoxic effect on hematological (Jurkat, U937, K562, HL-60, CCRF-SB) and solid tumor (MCF-7, HepG2, HeLa, Caco-2) cell lines, with IC50 values significantly lower than noncancerous cells (HEK-293, BJ and Vero) and normal peripheral blood mononuclear cells. G-11 induced differentiation of HL-60 cells to granulocytes and monocytes/macrophages by inhibiting the activation of FLT3 (CD135 tyrosine kinase). ITD-FLT3 mutation found in many acute myeloid leukemia patients could also be targeted by G-11 as exhibited by its inhibitory effect on MOLM-13 and MV4-11 cell lines. Molecular docking studies suggest the involvement of Leu616, Asp698, Cys694 and Cys828 residues in binding of G-11 to FLT3. The ability of G-11 to cause selective cytotoxicity and induce differentiation in cancer cells could be clinically relevant for therapeutic gains.

Purification of dendritic cell and macrophage subsets from the normal mouse small intestine.

Mononuclear phagocytes are essential for protecting against pathogens breaching the intestinal mucosa and maintaining the integrity of the gastrointestinal tract. The mononuclear phagocyte family of the healthy intestine is represented by a small population of hematopoietic cells including dendritic cells and macrophages. Distinct mononuclear phagocyte subsets strategically accumulate within and below the mucosal epithelium and are distributed in the submucosa and muscularis externa. Shaped by its unique microenvironment, each mononuclear phagocyte subset is developmentally and functionally unique and phenotypically distinct. Here we summarize our recent advances on identifying and purifying various intestinal mononuclear phagocyte subsets by flow cytometry in the context of their developmental properties and location within the intestinal tissue.

High receptor tyrosine kinase (FLT3, KIT) transcript versus anti-apoptotic (BCL2) transcript ratio independently predicts inferior outcome in pediatric acute myeloid leukemia.

OBJECTIVE: In acute myeloid leukemia (AML), simultaneous expression of proliferative (FLT3, KIT) and anti-apoptotic genes (BCL2) is unknown. The aim of the study was to prospectively evaluate proliferative and anti-apoptotic gene transcripts, their interrelationship and impact on the outcome in pediatric AML patients. METHODS: We assessed proliferative and anti-apoptotic gene transcripts by Q-polymerase chain reaction (TaqMan probe) in 64 consecutive pediatric AML patients. Survival data was analyzed by Kaplan-Meier curves followed by log rank test to compare statistical significance between groups. Stepwise multivariable Cox regression method was used to evaluate independent prognostic factors. RESULTS: In univariate analysis, transcript ratio of FLT3/BCL2 and FLT3+KIT/BCL2 significantly predicted event free survival (EFS) (<0.01 and <0.01 respectively) and overall survival (OS) (<0.01 and<0.01 respectively). In stepwise Cox-regression model, high white blood cell count and high FLT3+KIT/BCL2 ratio predicted EFS (HR: 2.2 and 2.3); high hemoglobin and high FLT3+KIT/BCL2 ratio predicted OS (HR: 0.45 and 3.85). Prognostic index (PI) was calculated using the hazard coefficient of independent prognostic factors; at 57.3 months, predicted OS of patients with the highest PI of 1.8 was 8% versus 73% for the lowest PI of -0.3. The mean PI of patients who died was 1.8±0.72 versus 0.54±0.70 for those who are alive, P=0.004. CONCLUSIONS: This first study showed that individual expression of proliferative and anti-apoptotic transcripts is not as important in AML patients, rather their interrelationship and relative level probably determines the outcome.

BET protein antagonist JQ1 is synergistically lethal with FLT3 tyrosine kinase inhibitor (TKI) and overcomes resistance to FLT3-TKI in AML cells expressing FLT-ITD.

Recently, treatment with bromodomain and extraterminal protein antagonist (BA) such as JQ1 has been shown to inhibit growth and induce apoptosis of human acute myelogenous leukemia (AML) cells, including those expressing FLT3-ITD. Here, we demonstrate that cotreatment with JQ1 and the FLT3 tyrosine kinase inhibitor (TKI) ponatinib or AC220 synergistically induce apoptosis of cultured and primary CD34(+) human AML blast progenitor cells (BPC) expressing FLT3-ITD. Concomitantly, as compared with each agent alone, cotreatment with JQ1 and the FLT3-TKI caused greater attenuation of c-MYC, BCL2, and CDK4/6. Simultaneously, cotreatment with JQ1 and the FLT3-TKI increased the levels of p21, BIM, and cleaved PARP, as well as mediated marked attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels in AML BPCs. Conversely, cotreatment with JQ1 and FLT3-TKI was significantly less active against CD34(+) normal bone marrow progenitor cells. Knockdown of BRD4 by short hairpin RNA also sensitized AML cells to FLT3-TKI. JQ1 treatment induced apoptosis of mouse Ba/F3 cells ectopically expressing FLT3-ITD with or without FLT3-TKI-resistant mutations F691L and D835V. Compared with the parental human AML FLT3-ITD-expressing MOLM13, MOLM13-TKIR cells resistant to AC220 were markedly more sensitive to JQ1-induced apoptosis. Furthermore, cotreatment with JQ1 and the pan-histone deacetylase inhibitor (HDI) panobinostat synergistically induced apoptosis of FLT3-TKI-resistant MOLM13-TKIR and MV4-11-TKIR cells. Collectively, these findings support the rationale for determining the in vivo activity of combined therapy with BA and FLT3-TKI against human AML cells expressing FLT3-ITD or with BA and HDI against AML cells resistant to FLT3-TKI.

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

Increased coexpression of c-KIT and FLT3 receptors on myeloblasts: independent predictor of poor outcome in pediatric acute myeloid leukemia.

BACKGROUND: Significance of mutations in FLT3 and c-KIT genes in AML has been well established, but role of their coexpression has not been evaluated. The aim of this study was to evaluate clinical significance of FLT3 (CD135) and c-KIT (CD117) coexpression on myeloblasts in AML. METHODS: Using flow-cytometry, we prospectively observed in 115 AML patients that CD135, CD117, and CD135+CD117 coexpression was expressed in 95 (82%), 104 (90%), and 81 (70%) patients respectively. Median expression of CD135, CD117, and their co expression was used as cut off for high and low expression. RESULTS: FLT3 ITD (internal tandem duplication) was present in 20 (17%) patients. High coexpression did not correlate with FLT3 ITD (P = 0.432) and cytogenetics (P = 0.244). Out of 115 patients, 86 (74.7%) achieved remission. At median followup of 15.5 months, EFS and OS was 29% and 35%, respectively for the entire cohort. Patients with high coexpression of CD135 and CD117 in comparison to those with low coexpression had significantly inferior EFS (20% vs 38% P < 0.001) and OS (27% vs 44% P = 0.001). In step wise Cox regression multivariable analysis, hazard ratio for high hemoglobin, WBC count, and coexpression of CD135 and CD117 was 0.63, 1.73, and 2.46 respectively for EFS, and for OS only CD135+CD117 coexpression emerged as an independent predictor (hazard ratio 2.25). CONCLUSIONS: This is the first study to show that high coexpression of CD135+CD117 is an independent predictor of poor outcome in AML and is easily measurable by routine diagnostic flow-cytometry.

Inhibitors of DNA binding proteins restrict T cell potential by repressing Notch1 expression in Flt3-negative common lymphoid progenitors.

Lineage commitment is regulated during hematopoiesis, with stepwise loss of differentiation potential ultimately resulting in lineage commitment. In this study we describe a novel population of B/NK bipotent precursors among common lymphoid progenitors in the fetal liver and the bone marrow. The absence of T cell precursor potential, both in vivo and in vitro, is due to low Notch1 expression and secondary to inhibition of E2A activity by members of the inhibitor of DNA binding (Id) protein family. Our results demonstrate a new, Id protein-dependent, molecular mechanism of Notch1 repression, operative in both fetal and adult common lymphoid progenitors, where T cell potential is selectively inhibited without affecting either the B or NK programs. This study identifies Id proteins as negative regulators of T cell specification, before B and NK commitment, and provides important insights into the transcriptional networks orchestrating hematopoiesis.

Expression of FMS-like tyrosine kinase 3 ligand by oncolytic herpes simplex virus type I prolongs survival in mice bearing established syngeneic intracranial malignant glioma.

BACKGROUND: Glioblastoma is a fatal brain tumor in needing urgent effective therapy. Treatments with both oncolytic viruses and immunotherapy have shown preclinical efficacy and clinical promise. We sought to exploit possible synergies between oncolytic herpes simplex virus type 1 (oHSV-1) infection of intracranial gliomas and delivery of immune-stimulating fms-like tyrosine kinase 3 ligand (Flt3L) by engineering a herpes vector to express the cytokine. OBJECTIVE: To construct an oHSV-1 vector that expresses high levels of Flt3L and examine its antiglioma efficacy in an immunocompetent murine model. METHODS: G47Δ and a bacterial artificial chromosome system were used to generate a novel oHSV-1, termed G47Δ-Flt3L, expressing Flt3L. Cytokine expression was confirmed, and G47Δ-Flt3L was injected intratumorally into established intracranial CT-2A gliomas in syngeneic C57/Bl6 mice. Animals were followed for survival and assessed by the Kaplan-Meier method. RESULTS: G47Δ-Flt3L expressed high levels of Flt3L in culture. Expression of Flt3L affected neither viral replication nor had a cytotoxic effect on CT2A glioma cells. Direct inoculation into intracerebral CT2A glioma cells resulted in high levels of detectable Flt3L in mouse blood and was superior to parental G47Δ in prolonging survival in glioma-bearing animals. CONCLUSION: Treatment with G47Δ-Flt3L improves survival of glioma-bearing mice.

Ectopic expression of Flt3 kinase inhibits proliferation and promotes cell death in different human cancer cell lines.

Stable ectopic expression of Flt3 receptor tyrosine kinase is usually performed in interleukin 3 (IL-3)-dependent murine cell lines like Ba/F3, resulting in loss of IL-3 dependence. Such high-level Flt3 expression has to date not been reported in human acute myeloid leukemia (AML) cell lines, despite the fact that oncogenic Flt3 aberrancies are frequent in AML patients. We show here that ectopic Flt3 expression in different human cancer cell lines might reduce proliferation and induce apoptotic cell death, involving Bax/Bcl2 modulation. Selective depletion of Flt3-expressing cells occurred in human AML cell lines transduced with retroviral Flt3 constructs, shown here using the HL-60 leukemic cell line. Flt3 expression was investigated in two cellular model systems, the SAOS-2 osteosarcoma cell line and the human embryonic kidney HEK293 cell line, and proliferation was reduced in both systems. HEK293 cells underwent apoptosis upon ectopic Flt3 expression and cell death could be rescued by overexpression of Bcl-2. Furthermore, we observed that the Flt3-induced inhibition of proliferation in HL-60 cells appeared to be Bax-dependent. Our results thus suggest that excessive Flt3 expression has growth-suppressive properties in several human cancer cell lines.

[Design and expression of an inhibitor for HIV-1 targeting dendritic cell].

Human immunodeficiency virus (HIV) infects the host cells by the fusion of viral and cell membranes. Blocking the combining between HIV and the receptors can prevent HIV from entering the host cells. We designed an invasion-inhibitor for HIV-1 targeting dendritic cell (DC), including 2 important HIV-1 receptors CD4 and CCR5, and 2 molecules Flt3-L and Mip-3alpha. With the synthetic gene of the inhibitor, 2 eukaryotic expression vectors pABK-CKR5-CD4/Flt3L-Mip3alpha (pABK-HIV-MF) and pABK-CKR5-CD4 (pABK-HIV-MT) were constructed and transfected into HEK 293 cells for expression. Results from RT-PCR, immunofluorescent assay, ELISA and Western blot approved that the invasion-inhibitor for HIV-1 was successfully and exactly expressed in the eukaryotic cells. Current study formed a solid base for the further research on the function of inhibitors for HIV-1 and elimination targeting DC.

Enrichment of functionally distinct mouse hematopoietic progenitor cell populations using CD62L.

The details of the bifurcation of the lymphoid and myeloid lineages following commitment by multipotent progenitor cells (MPP) remain a topic of controversy. We report that the surface glycoprotein CD62L can be characterized as a novel marker of this and other stages of early hematopoietic differentiation. Cell isolation and transplant studies demonstrated CD62L(neg/low) long-term hematopoietic stem cells and CD62L(high) MPP within the traditionally defined c-kit(pos)Lin(neg/low)Sca-1(pos) stem/progenitor cell population. Within the MPP population, previously defined as c-kit(pos)Lin(neg/low)Sca-1(pos)-Thy-1.1(neg)Flt3(pos), Sca-1 and CD62L resolved four populations and segregated Sca-1(high)CD62L(neg/low) MPP from Sca-1(high)CD62L(high) leukocyte-biased progenitors. Using a novel transplantation method that allows tracking of erythroid and platelet engraftment as an alternative to the classical method of in vitro colony formation, we characterized Sca-1(high)CD62L(neg/low) cells as MPP, based on transient engraftment of these lineages. These data establish CD62L as a useful tool in the study of early hematopoiesis and emphasize the power of trilineage-engraftment studies in establishing the lineage potential of MPP subsets.