FLT3 Protein Research Articles

Declined Presentation Inhibition of carbonic anhydrase IX as a novel strategy to target FLT3/ITD mutated aml cells under hypoxic conditions

Recent data suggests that the hypoxic properties of the bone marrow niche play a key role in the evolution of drug resistance in FLT3/ITD+AML, thus indicating a role for hypoxia-specific drugs in AML therapy. However, the mechanisms by which low oxygen (O2) levels distort drug responses of FLT3/ITD+AML cells are mostly unknown. Here we investigated the cytotoxic activity of classic and novel agents under hypoxic (1%) and normoxic (21%) conditions against FLT3/ITD+AML. Molm14 (M14) and primary cells from relapsed/refractory FLT3/ITD+AML patients were treated with Cytarabine (C), Quizartinib (Q) or FC531 (FC; a selective inhibitor of the hypoxia responsive gene carbonic anhydrase IX [CA-IX]) under 21% and 1% O2. After 48h, proliferation and apoptosis induction were assessed per MTT and FACS assays. Cells were also assessed for FLT3 protein and Hypoxia inducible factor (HIF) target gene expression by western blot and PCR arrays. The growth inhibitory effects of C and Q against FLT3/ITD+ cells were significantly dampened under 1% compared to 21% O2. Similarly, hypoxia weakened the pro-apoptotic effects of either drug. In contrast, Q potently inhibited FLT3 activity under both 1% and 21% O2. We next sought to identify tractable pro-survival genes induced by hypoxia, and to assess their expression in response to therapy. Our data shows that the HIF pathway is upregulated in response to 1% O2 and remains widely functional in C or Q treated cells. CA-IX, a druggable HIF target, was significantly upregulated in FLT3/ITD+ cells under 1% O2. When we exposed M14 cells to FC, we found that cell growth was significantly inhibited under 1% but not 21% O2. Further, FC was significantly more effective than C or Q in inducing apoptosis under hypoxia. 1) Hypoxia blunts the cytotoxic effects of C and Q in FLT3/ITD+AML cells. 2) The HIF signaling pathway remains largely functional under hypoxic conditions, despite treatment. 3) FC is significantly more cytotoxic than C or Q under hypoxic conditions. 4) Further investigation of combined therapies between standard AML agents and FC in FLT3/ITD+AML is warranted.

Abstract 2364: Src-like adaptor protein 2 negatively regulates FLT3 and KIT downstream signaling

Abstract Type III receptor tyrosine kinases (RTKs) including FLT3 and KIT play a major role in cell differentiation, proliferation, and survival of hematopoietic stem cells. FLT3-ITD and KIT-D816V mutations are the most common oncogenic mutations in FLT3 and KIT found in hematological cancers. These mutations lead to constitutive activation of proliferative and survival signals. Tyrosine kinase inhibitors (TKIs) in combination with chemotherapy display promising results in a clinical setting, but patients develop resistant disease after short-term treatment,. Hence, proteins that regulate the activity of RTKs can be alternative targets for patients carrying these mutations. Activation of FLT3 and KIT results in phosphorylation on several tyrosine residues that recruit SH2 domain-containing signaling proteins. In this study we identified Src-like adaptor protein 2 (SLAP2) as a potent FLT3 and KIT interacting protein. The interaction requires an intact SH2 domain of SLAP2 as well as phosphorylation of the receptor. Overexpression of SLAP2 in murine proB Ba/F3 cells inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation. SLAP2 displayed a similar inhibitory potential in cells expressing KIT-D816V. SLAP2 partially blocked phosphorylation of several FLT3 and KIT downstream signaling proteins such as AKT, ERK and p38. Moreover, SLAP2 expression inhibited FLT3-ITD-mediated STAT5 phosphorylation and KIT-D816V-mediated STAT3 phosphorylation. SLAP2 expression significantly accelerated ubiquitination-mediated degradation of FLT3 and KIT. Collectively, these data suggest that SLAP2 negatively regulates FLT3 and KIT signaling and therefore, modulation of SLAP2 expression levels may become a potential target for anticancer therapy. Citation Format: Sausan A. Moharram, Lars Rönnstrand, Julhash U. Kazi. Src-like adaptor protein 2 negatively regulates FLT3 and KIT downstream signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2364. doi:10.1158/1538-7445.AM2017-2364

Selective Expression of Flt3 within the Mouse Hematopoietic Stem Cell Compartment.

The fms-like tyrosine kinase 3 (Flt3) is a cell surface receptor that is expressed by various hematopoietic progenitor cells (HPC) and Flt3-activating mutations are commonly present in acute myeloid and lymphoid leukemias. These findings underscore the importance of Flt3 to steady-state and malignant hematopoiesis. In this study, the expression of Flt3 protein and Flt3 mRNA by single cells within the hematopoietic stem cell (HSC) and HPC bone marrow compartments of C57/BL6 mice was investigated using flow cytometry and the quantitative reverse transcription polymerase chain reaction. Flt3 was heterogeneously expressed by almost all of the populations studied, including long-term reconstituting HSC and short-term reconstituting HSC. The erythropoietin receptor (EpoR) and macrophage colony-stimulating factor receptor (M-CSFR) were also found to be heterogeneously expressed within the multipotent cell compartments. Co-expression of the mRNAs encoding Flt3 and EpoR rarely occurred within these compartments. Expression of both Flt3 and M-CSFR protein at the surface of single cells was more commonly observed. These results emphasize the heterogeneous nature of HSC and HPC and the new sub-populations identified are important to understanding the origin and heterogeneity of the acute myeloid leukemias.

Inhibition of USP10 Induces Degradation of Oncogenic FLT3: A Novel Approach to Therapy of Leukemia

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Overall, the survival with current chemotherapy is only 20-40%, declining steadily with advancing age. Approximately 30% of AML patients have mutations that constitutively activate the FLT3 gene. The most common FLT3 mutation results in tandem duplications within the juxtamembrane domain, observed in 20-25% of AML patients (internal tandem duplication, ITD), associated with markedly decreased survival. FLT3 kinase domain inhibitors, including SU11248, SU5416, CEP-701 and PKC412 (midostaurin), have been shown to induce partial, and usually brief, remissions in clinical trials of relapsed AML patients when administered as single agents. In a large trial in newly diagnosed patients, however, midostaurin was shown to increase survival when combined with standard chemotherapy.[1] This study supports the notion that inhibition of FLT3 may be important, at least in patients with mutations in the FLT3 gene. Since drug resistance develops in some patients with newly diagnosed AML and virtually all patients with advanced disease, additional strategies to target FLT3 would be of value.We discovered that the deubiquitinating enzyme (DUB) ubiquitin specific protease 10 (USP10) removes a degradative ubiquitin tag from mutant FLT3 thereby contributing to high levels of the oncogenic protein in AML (Fig 1a). Screening of our preclinical DUB inhibitor library for ability to selectively kill growth factor-independent FLT3-ITD-positive Ba/F3 cells over IL-3-dependent parental Ba/F3 cells identified HBX19818, a reported USP7 inhibitor, as the top hit. The effects are not unique to the Ba/F3 system: when profiled against a panel of 7 leukemia cell lines, HBX19818 conferred a substantial growth suppressive effect only to those expressing the FLT3-ITD oncoprotein (Fig 1b). As an initial assessment of the mechanism of HBX19818 we confirmed that it does promote ubiquitin-mediated degradation of FLT3-ITD (Fig 1c) and that the effect is selective as HBX19818 does not impact protein levels of wt FLT3. HBX19818 is published as an irreversible USP7 inhibitor,[2] however DUBome selectivity profiling data we generated identifies USP10 as the most potently inhibited DUB of the compound (USP10 IC50 = 14 µM). We went on to validate USP10 as the DUB that stabilizes FLT3-ITD using a combination of small molecule and genetic experiments. Notably, HBX19818 binds and inhibits USP10 in cells (data not shown), small hairpin knockdown of USP10 phenocopies the antiproliferative and FLT3 degradation effects of HBX19818 (Figure 1d and data not shown), and a direct interaction between USP10 and FLT3-ITD is observed in co-immunoprecipitation experiments (Fig 1e). Additionally, SAR studies reveal correlation among USP10 IC50, FLT3-ITD degradation and anti-proliferative effects for the HBX19818 chemical series, and we identified a second chemotype that phenocopies its effects. In support of the translational potential of USP10 inhibition for FLT3 mutant AML, we observed that both USP10 inhibitor series synergize with FLT3 kinase inhibitors, suppress growth of mutant FLT3-expressing primary AML cells and primagraft AML cells and, importantly, display the ability to overcome the FLT3 inhibitor-resistant mutant FLT3-ITD-F691L among other FLT3 kinase inhibitor-resistant mutants (Fig. 1f and data not shown).Overall, our data strongly support degradation of mutant FLT3 as an alternative approach to therapeutically target FLT3. This approach, which focuses on targeting USP10, could prove more efficacious than kinase inhibitors by simultaneously blocking both enzymatic and scaffolding functions of FLT3, and blocking compensatory increases in FLT3 protein or resistant point mutations associated with some kinase inhibitors. Importantly, this is the first demonstration of stabilization of an AML mutant driver protein by a DUB enzyme and introduces a novel therapy for FLT3 mutant-positive AML.

Combination of Selinexor with High-Dose Cytarabine (HiDAC) and Mitoxantrone (Mito) for Remission Induction in Acute Myeloid Leukemia (AML) Is Feasible and Tolerable

Background: Selinexor, an exportin 1 (XPO1) inhibitor, has demonstrated anti-leukemic effect as a single agent. There are also data demonstrating synergistic killing of leukemia cells with the combination of selinexor with anthracyclines and/or DNA damaging agents. The HiDAC/Mito combination is an effective induction regimen for relapsed/refractory (R/R) AML patients with a reported overall response rate (ORR) of 55% at our institution. We hypothesize that adding selinexor to HiDAC/Mito is feasible and has synergistic anti-leukemic effects.Methods: We performed a phase I dose escalation trial with cohort expansion in patients with AML that combined increasing doses of selinexor with age-adjusted HiDAC/Mito (NCT02573363). The primary endpoint was to determine the maximum tolerated dose of selinexor when given in combination with HiDAC/Mito. Selinexor was given orally on days 2, 4, 9, and 11 during the induction phase of the study. HiDAC (1.5 to 3 gm/m2 depending on age, IV over 3 hours) followed immediately by Mito (20 to 30 mg/m2 IV over 1 hour) was administered on day 1 and 5. Initial selinexor dose was 60mg (~35mg/m2) followed by a dose escalation to a target level of 80mg (~50mg/m2). Patients who entered remission proceeded to stem cell transplantation (SCT) or consolidation chemotherapy with HiDAC/selinexor followed by maintenance therapy with weekly selinexor alone for up to one year. Dose limiting toxicity (DLT) was defined as any grade 3 or greater non-hematologic toxicity, except transient (<48 hours) nausea/vomiting or liver function abnormalities, or by persistent bone marrow aplasia lasting >56 days in the absence of disease. Once a dose level was declared tolerable, more patients could be enrolled at that level to provide additional safety, tolerability, and efficacy data.Results: As of July 2016, 12 patients had enrolled. Selinexor dose levels were 60mg (n=3) and 80mg (n=9). Median age = 61 (range 44 - 74). De novo AML = 5 (42%); secondary AML = 3 (25%), R/R AML = 4 (33%). Eight (67%) patients were previously untreated, 1 (8%) was beyond the first relapse, and 3 (25%) had primary refractory disease. Prior therapies included combination cytarabine with anthracycline, HiDAC, decitabine, and clinical trial agents. Molecular/genetic subgroup profiles by European Leukemia Net (ELN) criteria included favorable = 1 (8%), intermediate I = 5 (42%), intermediate II = 1 (8%), and adverse = 5 (42%).Only 10 patients are evaluable for safety and efficacy at the time of analysis. Myelosuppression was the most common side effect and was universal. Median and average times to count recovery for responding patients from day 1 were 38 and 47 days, respectively. Febrile neutropenia was observed in 8 patients (80%), compared to our historical experience of 64% for HiDAC/Mito alone. Other significant adverse events included line-associated thromboses (n=3), cardiac toxicity (n=3), cerebellar toxicity (n=1), arm cellulitis (n=1), and syncope (n=1). No DLTs were observed. 30 and 60-day induction mortality was 10% (1 patient with R/R AML died from progressive disease).Ten patients are evaluable for response: complete remission (CR) = 4 (40%), CR with incomplete count recovery (CRi) = 1 (10%), partial remission (PR) = 1 (10%), and treatment failure (TF) = 4 (40%). Thus, the ORR is 60%. Of those who responded, 2 patients proceeded to consolidation, 1 underwent allo-SCT, and 3 are in preparation for allo-SCT. No response was achieved in patients with R/R AML.Bone marrow samples from screening and day 12 of treatment were used for biomarker analysis in three CR patients. A marked reduction in KI67, MYC, FLT3, KIT, and DNA damage response proteins was observed in the remaining tumor cells in the post-treatment samples. Staining for P53 showed an increase in nuclear staining intensity, which is consistent with the known effect of selinexor on tumor cells.Conclusions: The selinexor/HiDAC/Mito regimen is feasible and tolerable at selinexor doses up to 80mg/day or ~50 mg/m2/day twice weekly. It yields an ORR of 60% based on currently available data. The recommended phase II dose is 80mg of selinexor. Additional molecular correlative studies are evaluating patients' marrow and blood samples for markers that predict response and relapse. DisclosuresOdenike:Spectrum Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Geron: Research Funding; Incyte: Honoraria, Membership on an entity’s Board of Directors or advisory committees; CTI/Baxter: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sanofi-Aventis: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Algeta Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Suneisis Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Stock:Sigma-Tau: Honoraria, Research Funding; ADC Therapeutics: Honoraria; Gilead Sciences: Honoraria; Amgen: Honoraria; Royalties for a chapter in Up to Date: Patents & Royalties. Liu:Karyopharm: Research Funding; BMS: Research Funding.

Reduced Chemosensitivity of FLT3/ITD Mutated Cells to Cytarabine and Quizartinib Under Hypoxic Conditions

Background: FLT3/ITD mutated AML is characterized by short remission duration and high relapse rates due to the survival of a small fraction of leukemic cells (LCs) that outlive initial therapy. There is compelling evidence that the hypoxic niche in the bone marrow (BM) provides a sanctuary where subpopulations of LCs evade cytotoxic therapy and acquire drug resistance. In order to define the mechanisms involved in this process, multiple studies have focused on the interactions between LCs and the BM microenvironment but less is known about to the role of hypoxia as a modulator of drug resistance. Hence, the effects of standard and investigational therapies under hypoxic conditions are largely unknown. Here, we investigated the cytotoxic response of FLT3/ITD-mutated cells to conventional and targeted therapy under normoxic (21% O2) and hypoxic (1% O2) conditions. Methods: Molm14 (M14) cells and primary cells from relapsed/refractory FLT3/ITD mutated AML patients were incubated in culture medium under normoxic and hypoxic conditions. Cytarabine (Cy) or Quizartinib (Quiz) were added as single agents at the indicated concentrations. After 48 hours proliferation and apoptosis assays were performed using MTT assays, annexin V/PI staining and FACS analysis (M14). Cells were also assessed for FLT3 protein and Hypoxia inducible factor (HIF) target gene expression by western blotting and PCR arrays (RT2 ProfilerTM PCR Array, Human Hypoxia Signaling Pathway Plus, Qiagen) (M14 and primary cells), respectively. Results: We found that M14 cells were significantly less susceptible to treatment under hypoxic conditions, both when exposed to Cy and targeted FLT3 inhibition with Quiz (Growth inhibition, 0.25nM Quiz: 8.5±4.2% (1% O2) vs. 22±2.5% (21% O2), p<.05; 49.6±2.5% (1% O2) vs. 58.1±2.3% (21% O2), n=8, p<.05; 0.25µM Cy: 3.7±2.5% (1% O2) vs. 14.4±1.5% (21% O2), p<.01; 1 µM Cy: 26.3±4.1% (1% O2) vs. 53.3±3.8% (21% O2), n=10-12, p<.001). In line with these findings, the apoptotic response of M14 cells to Cy treatment was significantly blunted under hypoxic conditions (Fold increase in apoptotic cells vs. untreated control, 1 µM: 5.7±0.8 fold [21% O2] vs. 2.7±0.5 fold [1% O2], n=4, p<.05). While not reaching the significance threshold, the trend was similar for the Quiz-treated cells. This effect was extended to primary, relapsed/refractory FLT3/ITD-mutated cells derived from patients (n=2) treated at our institution, where growth inhibition was consistently decreased in hypoxia at all doses of Cy and Quiz tested. Not unexpectedly, these primary cells required significantly higher doses compared to the established M14 cell line (1-10µM for Cy, and 50-200nM for Quiz). Importantly, Quiz appeared to be similarly effective in inactivating FLT3 activity under low and high oxygen tension. Therefore identifying tractable pro-survival genes induced by the low oxygen microenvironment should represent a viable strategy to increase the effectiveness of anti-leukemic agents. In order to identify such target candidates, we surveyed the effects of Quiz and Cy on a panel of 84 hypoxia-regulated genes using PCR arrays. Our preliminary studies revealed that while HIF pathway remains largely functional in the presence of Quiz or Cy, these agents severely blunt induction of several hypoxia genes (including Glucose transporter 1 [Glut1] and Carbonic Anhydrase 9 [CA9]) in both primary cells and M14 cells. Conclusions: 1.Hypoxia limits the cytotoxic effects of conventional and targeted therapeutic agents in primary and established FLT3/ITD mutated AML cells. 2. Quizartinib effectively abrogates FLT3 signaling under both normoxic and hypoxic conditions, indicating that other signaling pathways are critical for leukemic cell survival under reduced oxygen tension. 3. The induction of several potentially druggable HIF targets is disrupted by anti-AML agents, warranting the further investigation of combinational approaches between standard anti-AML agents and HIF targeted strategies. 4. Such therapeutic combinations are likely to be particularly effective undermicro-environmental stress conditions (e.g. severe hypoxia) thus addressing an unmet need in AML therapy. DisclosuresNo relevant conflicts of interest to declare.

Arsenic Trioxide Synergizes with FLT3 Tyrosine Kinase Inhibitors to Kill FLT3-ITD+ Leukemic Cells through Depressed Production and Degradation of FLT3 Protein

A number of selective FLT3 tyrosine kinase inhibitors (TKIs) have been tested for treatment of FLT3-ITD+ AML. However, monotherapy with FLT3 TKIs alone has achieved only transient and limited clinical responses due to several resistance mechanisms. Arsenic trioxide (ATO) has demonstrated significant efficacy in treating acute promyelocytic leukemia (APL). ATO has also shown some activity in treating non-APL myeloid leukemias. Recent studies have demonstrated that ATO can affect the degradation of oncogenic mutant proteins including mutant p53 and NPM through the ubiquitin-proteasome pathway (UPP). Here we investigated the feasibility of combining FLT3 TKIs with ATO in the treatment of FLT3-ITD+leukemia.Anti-leukemic effects against FLT3-ITD+ patient AML samples and leukemia cell lines (Molm14, MV4;11) by FLT3 TKIs (Sorafenib, quizartinib), ATO and the combination were examined by MTT, apoptosis, cell viability and colony forming assays. Our data revealed that the combination showed synergistic growth inhibition of the FLT3-ITD+ cell lines Molm14 and MV4;11, with combinatorial index (CI) values at ED50 below 1.0 for both cell lines (CI values were 0.46 and 0.56 for ATO + sorafenib, 0.65 and 0.57 for ATO + quizartinib in Molm14 and MV4;11 cells, respectively). In contrast, there was no synergy observed for the combination in treating leukemia cell lines that do not express mutant FLT3. Synergistic effects for the combination in inducing apoptosis and inhibiting colony formation were also observed for the FLT3-ITD+ cell lines. Furthermore, when the combination was used to treat primary FLT3-ITD+ patient samples, there was also significant reduction of viability and clonogenicity. In contrast, normal BM MNCs showed very limited responses to the combination. Western blot (WB) analysis of Molm14 and FLT3-ITD+ patient samples revealed the combination of ATO and sorefenib potently reduced phosphorylation of FLT3 and its downstream targets (STAT5, MAPK, and AKT). In vivoexperiments using the combination to treat NSG mice engrafted with Molm14 cells demonstrated a significant reduction in the level of leukemic cells.We further investigated the mechanism by which ATO contributes to an anti-leukemic effect on FLT3-ITD+ cells. Morphologic and flow cytometric analysis showed that ATO promoted the differentiation of Molm14 cells. The expression of C/EBPα and PU.1, two key regulators for myeloid differentiation, was increased in ATO-treated Molm14 cells at both the mRNA and protein levels. These data suggest ATO is capable of inducing the differentiation of leukemic cells. We also found that, in FLT3-ITD+ cells, ATO decreased expression of FLT3 protein. This could result from reduced FLT3 production and/or increased protein degradation. Further quantitative PCR analysis revealed ATO decreased expression of FLT3 and its upstream regulators HoxA9 and meis1. Co-immunoprecipitation assay showed that ATO facilitated poly-Ubiquitination and degradation of FLT3 in a dose- and time-dependent fashion. These results indicate that ATO exerts its anti-leukemic effects in FLT3-ITD+AML cell lines and primary samples at least partly through reducing the level of FLT3 protein.These studies together demonstrate that ATO has a unique activity towards FLT3-ITD+ leukemia cells. Based on these findings, ATO is a potential candidate to work in combination with FLT3 TKIs to improve the outcome of FLT3-ITD+ AML patients. DisclosuresLevis:Millennium: Consultancy, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

The RET Receptor Tyrosine Kinase Promotes Acute Myeloid Leukemia through Protection of FLT3-ITD Mutants from Autophagic Degradation

Acute myeloid leukemia (AML) is a heterogeneous disease with diverse leukemogenic driver lesions. The genetic understanding of AML has resulted in major improvements in diagnosis, classification, prognostication, and outcome prediction. However, these insights have not yet translated into molecular mechanism-based therapies in the majority of cases, because AML cells rapidly escape attempts at therapeutic targeting such as small-molecule inhibition of FLT3 internal tandem duplication (FLT3-ITD) mutants, which occur in up to 30% of AML cases and confer a poor prognosis. To identify potential new targets for combinatorial treatment approaches, we performed a series of large-scale short hairpin RNA (shRNA) screens and observed that cell lines representing various AML subtypes were dependent on expression of the RET receptor tyrosine kinase (RTK), which has not previously been implicated in AML pathogenesis. Validation experiments demonstrated that depletion of RET by shRNA knockdown or CRISPR/Cas9-mediated knockout led to cell cycle arrest in the G0/G1 phase, increased apoptosis, and reduced clonogenic activity. RTK profiling using ELISA-based antibody arrays demonstrated that RET is highly phosphorylated in RET-dependent AML cell lines. Analysis of known RET ligand/co-receptor pairs (GDNF/GFRA1, NRTN/GFRA2, ARTN/GFRA3, PSPN/GFRA4) by quantitative real-time PCR and shRNA knockdown indicated that RET signaling is facilitated mainly through NTRN/GFRA2 or ARTN/GFRA3. Interrogation of various signaling pathways known to promote myeloid leukemogenesis showed that RET knockdown resulted in decreased phosphorylation of 4E-BP1 (T37/46), p70S6K (T389), S6RP (S240/244), and ULK1 (S758), pointing to mTORC1-mediated protein synthesis and/or suppression of autophagy as important effectors of RET signaling in AML cells. Based on recent data showing that FLT3-ITD mutants can be degraded by autophagy (Larrue et al. Blood 2016), we reasoned that the RET-mTORC1 signaling axis promotes AML through protection of FLT3-ITD mutants from autophagic degradation. Consistent with this hypothesis, genetic or pharmacologic (vandetanib, danusertib) inhibition of RET predominantly affected FLT3-dependent AML cell lines and were accompanied by upregulation of autophagy and destabilization of FLT3, as evidenced by p62 degradation, LC3B turnover, increased numbers of autophagic vacuoles, and decreased FLT3 protein levels. Furthermore, we observed accumulation of STAT5, a key FLT3-ITD downstream effector, upon pharmacologic autophagy inhibition in low RET-expressing AML cells, underlining the importance of RET-mediated suppression of autophagy for leukemogenic FLT3-ITD signaling. In line with the observations in AML cell lines, preliminary data from a murine bone marrow transplantation model show that Ret is required for AML development and propagation in vivo as we observed a significant survival advantage for mice transplanted with Ret knockdown cells compared with mice transplanted with control cells. Finally, genome-wide transcriptome analysis identified elevated RET mRNA levels in 35 of 260 (13.5%) primary human AML samples. Since there are no known RET copy number alterations or mutations of the RET coding region in AML patients and cell lines, we are currently investigating whether aberrant RET expression in AML can be attributed to perturbed epigenetic regulation. To this end, we are applying chromosome conformation capture combined with high-throughput sequencing (4C-seq) technology to systematically analyze interactions of the RET promoter region with enhancer sequences in high and low RET-expressing AML cell lines. Combined, our results indicate that in a proportion of AML, RET-mTORC1 signaling promotes cell viability and proliferation through suppression of autophagy, suggesting that targeting RET or, more broadly, depletion of critical leukemogenic drivers via induction of autophagy may provide a therapeutic opportunity in this subset of patients. DisclosuresNo relevant conflicts of interest to declare.

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.

Application of High-throughput Sequencing in Acute Myeloid Leukemia Patients with Positive FLT3-ITD

OBJECTICE: To evaluate the application of high-throughput sequencing to sequence the FMS-like Tyrosine Kinase 3 internal tandem duplication (FLT3-ITD) in de novo acute myeloid leukemia (AML) patients with lower allelic ratio FLT3-ITD mutation or more than one ITD, and to analyze the feature of ITD. The genomic DNA of 23 AML patients with positive FLT3-ITD was amplified by PCR, capillary electrophoresis was used to detect the ITD mutation. Then, the FLT3 gene was amplified using primer with different barcode, and the product was analyzed by illumina Miseq, and the results were compared with UCSC database. Out of 23 AML patients, 17 had a single ITD, and 3 had 2 ITDs, and the remaining 3 had 3 ITD detected by capillary electrophoresis. The high-throughput sequencing showed that 17 ITD were the complate duplications of wild-type FLT3, and the remaining 16 ITD were partial duplications in the all 33 ITDs. The same length ITD mutation contained 2 different ITD sequences in one patient with more than one ITD, and the other patient with 2 ITD had the same ITD insertion position. The ITD occurred in the regions from p. Y572 to p. L602 of the FLT3 protein, and all the patient ITD covered one or more amino acid between p. V592 and p. E598. Illumina Miseq can analyze the sequence of ITDs precisely and accurately. ITD mutation varies widely, but the hotspots are concentrated.

Advance in treatment of pediatric leukemia through modulating signaling pathways induced by traditional Chinese medicine

Leukemia is the most common type of cancer in childhood, which is also one of the malignant tumors that can be cured by chemotherapy. However, infection and the side effects of chemotherapy drugs are the causes of death in children with leukemia. Chemotherapy combined with traditional Chinese medicine is used to treatment of pediatric leukemia in clinic. Although traditional Chinese medicine has extensive clinical experience in the treatment of leukemia, the mechanism is not clear. The targeted drugs are attractive for the advantages of the high selectivity and little side effects. Targeted therapies in pediatric leukemia are targeting BCR/ABL, TARA and FLT3 proteins, which activation results in the downstream activation of multiple signaling pathways, including the PI3K/AKT, JNK/STAT, Ras/ERK pathways. In recent years, the target site or the signaling pathways are clear for the traditional Chinese medicine in the treatment of leukemia. In this paper, we review recent studies, summary the traditional Chinese medicine prescriptions and effective ingredients which can regulate the signaling pathways. We view to provide a basis for the treatment of childhood leukemia with traditional Chinese medicine combined with chemotherapy drugs or targeted drugs.

SATB1 Expression Marks Lymphoid-Lineage-Biased Hematopoietic Stem Cells in Mouse Bone Marrow

Background: Lifelong hematopoiesis is maintained by cell differentiation in which signaling pathways and transcription factors coordinately induce step-wise maturation of hematopoietic stem cells (HSCs) toward downstream effector cells. In addition, the organization of chromatin structure that creates accessible sites of target genes is also essential so as to ensure temporally and spatially adequate control of internal gene expression. Murine HSCs can be isolated with high efficiency using surface molecules including lineage-related markers, c-Kit, Sca-1, Flt3 and SLAM family proteins. However, even the highly enriched HSC fraction is still heterogeneous regarding differentiation potential, and how the HSC diversity reflects the heterogeneity of intrinsic gene-expression in HSCs is as-yet-unknown. We previously identified Special AT-rich Sequence Binding protein 1 (SATB1), a global chromatin regulator, as a lymphoid-related gene in the HSC differentiation (Satoh and Yokota et al. Immunity 2013). Indeed, SATB1 overexpression strongly enhanced both T and B lymphopoietic potential of murine HSCs whereas SATB1 deficiency caused malfunctions of HSCs in the lymphopoietic activity. Furthermore, another report showed that SATB1-deficient HSCs were less quiescent in transplanted recipients and more prone to differentiate preferentially to myeloid-erythroid lineages (Will et al. Nat Immunol 2013). These results suggested that SATB1 is likely indispensable not only for the lymphopoietic potential but also for the integrity of HSCs. Here, to better understand the mechanism how SATB1 influences homeostatic HSC functions in adult bone marrow (BM), we have developed a new mouse model in which SATB1 expression can be precisely monitored along the HSC differentiation.Methods: The Tomato gene, coding a red fluorescent protein, was knock-in to the coding region of endogenous Satb1 gene. The heterozygous SATB1/Tomato knock-in mice in which one Satb1 allele was replaced with the Tomato were used to sort HSCs in adult BM. The sorted cells were evaluated for the differentiation potential with methylcellulose colony assays and co-cultures with MS5 stromal cells. Further, the long-term reconstitution ability was evaluated by transplantation to lethally irradiated mice. To obtain transcriptome information, total RNA was isolated from SATB1/Tomato- and SATB1/Tomato+ HSCs, and then next-generation sequencing was performed. The data were analyzed with the Ingenuity Pathway Analysis software.Results: We defined Lin- Sca1+ c-KitHi (LSK) CD150+ Flt3- cells as HSCs, especially adopting FLT3- to exclude FLT3+ lymphoid-primed multipotent progenitors from our functional analyses. We found that the LSK CD150+ Flt3- fraction contains substantial number of SATB1/Tomato+ cells. While both SATB1/Tomato- and SATB1/Tomato+ HSCs produced numerous CFU-Mix and CFU-GM/G/M colonies, the latter were less potent to produce BFU-E. In co-culture with MS5 stromal cells that support B and myeloid lineages, the output of B lineage cells from SATB1+ HSCs was more robust than that of SATB1- HSCs. Upon transplantation, enhanced B-lineage engraftment was observed in the SATB1+ HSC-transplanted recipients. Interestingly, while the two types of HSCs showed obvious difference in the differentiation potential toward lymphoid or myeloid lineage, both HSCs reconstituted the LSK CD150+ Flt3- fraction that similarly contained SATB1/Tomato- and SATB1/Tomato+ cells. With the RNA-sequencing data of SATB1- and SATB1+ HSCs, biological pathway analyses revealed that the "Hematological System Development and Function" pathway was remarkably up-regulated in the SATB1+ HSCs. Among subcategories of the "Hematological System Development and Function" pathway, the "quantity of lymphocytes" pathway was increased whereas "quantity of myeloid cells" and "quantity of granulocytes" pathways were decreased.Conclusion: We have developed a new mouse system that can be used to identify and isolate viable lymphoid-biased HSCs in the most primitive hematopoietic cell fraction of adult BM. While the SATB1- and SATB1+ HSCs differ genetically and functionally, both subtypes have displayed a self-renewal activity with mutual interconversion in transplanted recipients. These findings suggest that functional heterogeneity and variability within the HSC population is, at least in part, a manifestation of SATB1 expression. DisclosuresYokota:SHIONOGI & CO., LTD.: Research Funding.

CDC25A governs proliferation and differentiation of FLT3-ITD acute myeloid leukemia.

We investigated cell cycle regulation in acute myeloid leukemia cells expressing the FLT3-ITD mutated tyrosine kinase receptor, an underexplored field in this disease. Upon FLT3 inhibition, CDC25A mRNA and protein were rapidly down-regulated, while levels of other cell cycle proteins remained unchanged. This regulation was dependent on STAT5, arguing for FLT3-ITD-dependent transcriptional regulation of CDC25A. CDC25 inhibitors triggered proliferation arrest and cell death of FLT3-ITD as well as FLT3-ITD/TKD AC-220 resistant cells, but not of FLT3-wt cells. Consistently, RNA interference-mediated knock-down of CDC25A reduced the proliferation of FLT3-ITD cell lines. Finally, the clonogenic capacity of primary FLT3-ITD AML cells was reduced by the CDC25 inhibitor IRC-083864, while FLT3-wt AML and normal CD34+ myeloid cells were unaffected. In good agreement, in a cohort of 100 samples from AML patients with intermediate-risk cytogenetics, high levels of CDC25A mRNA were predictive of higher clonogenic potential in FLT3-ITD+ samples, not in FLT3-wt ones.Importantly, pharmacological inhibition as well as RNA interference-mediated knock-down of CDC25A also induced monocytic differentiation of FLT3-ITD positive cells, as judged by cell surface markers expression, morphological modifications, and C/EBPα phosphorylation. CDC25 inhibition also re-induced monocytic differentiation in primary AML blasts carrying the FLT3-ITD mutation, but not in blasts expressing wild type FLT3. Altogether, these data identify CDC25A as an early cell cycle transducer of FLT3-ITD oncogenic signaling, and as a promising target to inhibit proliferation and re-induce differentiation of FLT3-ITD AML cells.

Abstract 4381: High efficacy of T-LAK cell-originated protein kinase inhibitor in acute myeloid leukemia with FLT3-ITD mutation

Abstract The internal tandem duplication (FLT3-ITD) a gain-of-function mutation of FLT3, is associated with poor outcome in acute myeloid leukemia (AML). The use of FLT3 inhibitors currently undergoing clinical investigation has not yet improved overall survival. Therefore, novel therapies are needed. T-LAK cell-originated protein kinase (TOPK), a serine-threonine protein kinase, is highly expressed and associated with an aggressive cancer phenotype, but is hardly detectable in normal tissues. Here, we investigate TOPK in AML and demonstrate that it is highly expressed in most AML cell lines and some primary blasts, but is not detected in CD34+ cells of healthy donors. MV4-11 and U937 cells transfected with TOPK-siRNA showed significant decrease in cell viability (∼70%, P&amp;lt;0.001). We then examined the anti-leukemia activity of TOPK inhibitor OTS514 in AML. Primary blasts obtained from 3 patients (pts) with AML showed significant decrease in cell viability in a dose-dependent manner (IC50 values were 10-20nM). Moreover, OTS514 treatment resulted in significant decrease in number of colonies of CD34+ cells obtained from a pt with AML (41 vs 73, P = 0.01), but not of CD34+ cells obtained from healthy donors (39 vs 36, P = 0.67). Treatment of 10 AML cell lines with OTS514 showed that FLT3 mutated cell lines were significantly more sensitive (IC5020nM). Annexin/PI staining showed 80% and 70% increase in apoptosis in FLT3-ITDmut cells (MV4-11 and MOLM13) treated with OTS514 (40nM; 48hrs). In contrast, only 40% and 10% apoptosis was observed in FLT3wt cells (U937 and KG1, respectively) treated under the same conditions. Additionally, cell cycle analysis in cells treated with OTS514 (20nM; 24 and 48 hrs) showed a dramatic decrease of S phase (∼98%; P = 0.003) in MV4-11 but only 70% (P&amp;lt;0.001) and 30% (P = 0.02) in THP-1 (FLT3wt) cells compared with untreated cells. Importantly, OTS514 treatment decreased cell viability and increased cell differentiation and apoptosis in primary blasts from pts relapsed after FLT3 inhibitor treatment (AC220). Furthermore, using a MV4-11-engrafted mouse model, we found that mice treated with 7.5mg/kg IV every day for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29, P&amp;lt;0.001). Mechanistically, OTS514 or TOPK-siRNA significantly decreased FLT3 mRNA and protein levels and decreased CEBPA phosphorylation in FLT3-ITDmut cells. Furthermore, activating FLT3 in THP-1 cells via FLT3 ligand increased TOPK protein. Also, FLT3 knockdown or inhibition with MLN518 (50 and 100nM) decreased TOPK levels in MV4-11 and MOLM13 cells, but not in U937 cells, suggesting that TOPK is a downstream target of FLT3. In conclusion, TOPK inhibitor exhibits preferential activity in FLT3-ITDmut AML, partially via inhibition of FLT3 expression. Thus, OTS514 represents new targeted therapy for this adverse risk subset of AML. Citation Format: Houda Alachkar, Martin Mutonga, Jae-Hyun Park, Gregory Malnassy, Alex Woods, Gordana Raca, Olatoyosi M. Odenike, Naofumi Takamatsu, Takashi Miyamoto, Shoji Hisada, Yo Matsuo, Wendy Stock, Yusuke Nakamura. High efficacy of T-LAK cell-originated protein kinase inhibitor in acute myeloid leukemia with FLT3-ITD mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4381. doi:10.1158/1538-7445.AM2015-4381

Abstract 787: FLX925 (AMG 925) is a rationally designed FLT3, CDK4/6 inhibitor that retains potency against clinically relevant secondary resistance mutations in FLT3

Abstract Acquired secondary resistance mutations to clinically active kinase inhibitors remains a key obstacle between valid therapeutic hypotheses and meaningful patient benefit. In AML, evidence suggests that inhibition of FLT3 (particularly in FLT3-ITD mutated cancers) can be efficacious; however, relapse from complete remission is common and often rapid. As with other cancers driven by key oncogenic kinase mutations (e.g. BCR-ABL in CML), a primary mechanism of resistance is the acquisition of secondary resistance mutations in the oncogenic kinase themselves. Multiple strategies have been pursued to address such resistance, including the development of kinase inhibitors that either bind their respective targets differently or by targeting multiple important pathways simultaneously. Herein we describe a rationally conceived next generation FLT3 inhibitor, FLX925 (previously AMG 925), that was prospectively designed to address or avoid common resistance mechanism to earlier FLT3 inhibitors with its unique binding mode and potent activity against CDK4/CDK6. FLX925 is a potent and selective type 1 inhibitor of FLT3 that retains its cellular potency against clinically relevant secondary resistance mutations in FLT3 occurring with quizartinib or sorafenib treatment (FLX925 IC50: MOLM13ITD, 15 nM; MOLM13ITD/D835, 28 nM; MV4-11ITD, 16 nM; MV4-11ITD/D835, 19 nM; MV4-11ITD/N841, 16 nM; MV4-11ITD/F691, 73 nM). Indeed, while compounds currently in the clinic became more than 200-fold less potent against a number of mutants, FLX925 remained relatively equipotent (+/- 5-fold the parental cell line IC50) in these same resistant clones. This is in stark contrast to the striking cross-resistance observed with quizartinib in sorafenib resistant cells. Moreover, the few clones that grew out of a screen for resistance to FLX925 displayed a ‘persistence’ phenotype with modestly reduced sensitivity to FLX925 (∼5-fold IC50 shift) that was rapidly reversible. This persistence was associated with higher FLT3 protein levels and no detectable secondary mutations in FLT3. In addition to its suppression of FLT3 signaling, FLX925 potently inhibits CDK4/CDK6, central components of the cell cycle machinery. This unique profile may reduce the likelihood of emergent resistant clones and extends the therapeutic potential of FLX925 to other malignancies dependent on these pathways (e.g. MCL). Indeed, the addition of PD0332991 (a selective CDK4/6 inhibitor) to a relatively selective FLT3 inhibitor reduced the frequency of acquired resistance in a cell based screen, relative to a FLT3 inhibitor alone. These data suggest the unique profile of FLX925 makes it an ideal inhibitor for the treatment of cancers driven by FLT3 signaling, such as AML. A phase I clinical trial evaluating the safety, tolerability pharmacokinetics and pharmacodynamics effects of FLX925 in patients with AML is ongoing. Citation Format: Cong Li, Lingming Liang, Liqin Liu, Zhen Xia, Zhihong Li, Xianghong Wang, Lawrence McGee, Angus Sinclair, Sasha Kamb, Dineli Wickramasinghe, Sachie Marubayashi, Juan C. Jaen, Jordan S. Fridman, Kang Dai. FLX925 (AMG 925) is a rationally designed FLT3, CDK4/6 inhibitor that retains potency against clinically relevant secondary resistance mutations in FLT3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 787. doi:10.1158/1538-7445.AM2015-787

Abstract 2945: Nilotinib abrogates DNMT1-depenent DNA methylation: A novel mechanism for induction of AML leukemia regression

Abstract Nilotinib has been approved to treat newly diagnosed, imatinib-resistant or -intolerant leukemia, but the mechanisms underlying its antileukemia actions remain elusive. Here, we report that nilotinib blocks AML growth through its DNA hypomethylating activity. We showed that, in addition to inducing a reduction of KIT and FLT3 protein expression and phosphorylation, nilotinib downregulated DNMT1, 3a and 3b in Kasumi-1, MV4-11 and C1498 cells. Since DNMT1 is maintenance and de novo DNMT, we focused on DNMT1 and proved that nilotinib impaired DNMT1 expression via Sp1 dysfunction, since the luciferase activity driven by DNMT1 promoter containing Sp1 binding sites was decreased by 2.6 fold by nilotinib, while increased by 10.6 fold after Sp1 overexpression. Electrophoretic mobility shift assay disclosed that nilotinib abolished Sp1 binding on DNMT1 promoter. Dotblot using 5mC antibody revealed global DNA hypomethylation and qPCR showed p15INK4B upregulation in Kasumi-1 (3.4 fold) or MV4-11 (5.2 fold). Bisulfite sequencing found a decrease of DNA methylation on p15INK4B promoter from 100% to 89% in Kasumi-1 or 13% to 0% in MV4-11. The functional assays showed a decrease of colony formation in Kasumi-1 (60%) or MV4-11 (58%), and an increase of cell apoptosis in Kasumi-1 (6.9 fold) or MV4-11 (3.2 fold). We verified these findings in AML patient blasts (n = 3) by showing that nilotinib treatment led to the dephosphorylation of KIT, the downregulation of DNMT1, 3a, 3b and Sp1, the reduction of global DNA methylation and the re-expression of p15INK4B (4.4 fold) via promoter demethylation (from 14% to 6%), which was followed by suppressed colony formation (77%), promoted cell apoptosis (1.84 fold) and increased caspase-3 and caspase-8. To test if nilotinib induces AML regression via DNA hypomethylation in vivo, 0.1×106 C1498 cells were injected into C57BL/6 mice (n = 3 mice/group) via the tail vein. The leukemic mice were treated with 5, 10 or 15 mg/kg of nilotinib in PEG400 plus saline via intraperitoneal injection twice a week for a total of 6 doses. No toxicity was observed for the tested drug dosage and schedule, because of no evident change of mouse body weight. Compared to the control group, nilotinib-treated mice had a decrease in spleen size. H&amp;E staining showed that the control mice exhibited increased infiltration of AML cells into the spleen, lung and liver of recipients. The bone marrow (BM) histopathology identified a higher degree of mature post-mitotic cells containing metamyelocytes, bands and segmented neutrophils in nilotinib-treated mice than control. DNMT1 was downregulated (39%), p15INK4B was upregulated and global DNA methylation was reduced (60%) in BM cells from nilotinib-treated mice. Thus, our findings unveil DNA methylation machinery as an innovative target behind nilotinib's anticancer activities, and provide a new therapeutic avenue for nilotinib in overcoming aberrant epigenetics in AML. Note: This abstract was not presented at the meeting. Citation Format: Na Shen, Fei Yan, Jiuxia Pang, Naseema Gangat, Mark R. Litzow, Aref Al-Kali, Shujun Liu. Nilotinib abrogates DNMT1-depenent DNA methylation: A novel mechanism for induction of AML leukemia regression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2945. doi:10.1158/1538-7445.AM2015-2945

AMG 925 is a dual FLT3/CDK4 inhibitor with the potential to overcome FLT3 inhibitor resistance in acute myeloid leukemia.

Resistance to FLT3 inhibitors is a serious clinical issue in treating acute myelogenous leukemia (AML). AMG 925, a dual FLT3/CDK4 inhibitor, has been developed to overcome this resistance. It is hypothesized that the combined inhibition of FLT3 and CDK4 may reduce occurrence of the FLT3 resistance mutations, and thereby prolong clinical responses. To test this hypothesis, we attempted to isolate AML cell clones resistant to AMG 925 or to FLT3 inhibitors. After a selection of over 8 months with AMG 925, we could only isolate partially resistant clones. No new mutations in FLT3 were found, but a 2- to 3-fold increase in total FLT3 protein was detected and believed to contribute to the partial resistance. In contrast, selection with the FLT3 inhibitors sorafenib or AC220 (Quizartinib), led to a resistance and the appearance of a number of mutations in FLT3 kinase domains, including the known hot spot sites D835 and F691. However, when AC220 was combined with the CDK4 inhibitor PD0332991 (palbociclib) at 0.1 μmol/L or higher, no resistance mutations were obtained, indicating that the CDK4-inhibiting activity of AMG 925 contributed to the failure to develop drug resistance. AMG 925 was shown to potently inhibit the FLT3 inhibitor-resistant mutation D835Y/V. This feature of AMG 925 was also considered to contribute to the lack of resistance mutations to the compound. Together, our data suggest that AMG 925 has the potential to reduce resistance mutations in FLT3 and may prolong clinical responses.

Interleukin‐2‐mediated inhibition of dendritic cell development correlates with decreased CD135 expression and increased monocyte/macrophage precursors

We have previously shown that interleukin-2 (IL-2) inhibits dendritic cell (DC) development from mouse bone marrow (BM) precursors stimulated with the ligand for FMS-like tyrosine kinase 3 receptor (Flt3L), and have provided evidence that this inhibition occurs at the monocyte DC precursor stage of DC development. Here, we explored the mechanism of IL-2-mediated inhibition of DC development. First, we showed that these in vitro cultures accurately model DCs that develop in vivo by comparing gene and protein expression of the three main Flt3L-induced DC subsets from the BM, CD11b(+) and CD24(+) conventional DCs (cDCs) and plasmacytoid DCs (pDCs) with their respective ex vivo spleen DC subsets (CD11b(+), CD8(+) and pDCs). Next, gene expression changes were quantified in Flt3L DC subsets that developed in the presence of IL-2. These changes included increased expression of Bcl2l11, which encodes the apoptosis-inducing protein Bim, and decreased expression of Flt3 (CD135), the receptor that initiates DC development. Interleukin-2 also significantly reduced Flt3 protein expression on all three Flt3L DC subsets, and attenuated Flt3L-induced STAT3 phosphorylation in DCs. Based on these data, we hypothesized that decreased Flt3 signalling may divert BM precursors down monocyte and macrophage lineages. Indeed, addition of IL-2 led to increases in Flt3(-) cells, including cKit(+) Ly6C(+) CD11b(-) populations consistent with the recently identified committed monocyte/macrophage progenitor. Therefore, IL-2 can inhibit DC development via decreased signalling through Flt3 and increased monocyte/macrophage development.

Inhibitory Effect of Turmeric Curcuminoids on FLT3 Expression and Cell Cycle Arrest in the FLT3-Overexpressing EoL-1 Leukemic Cell Line

Leukemia is a hematologic malignancy with a frequent incidence and high mortality rate. Previous studies have shown that the FLT3 gene is overexpressed in leukemic blast cells, especially in acute myeloid leukemia. In this study, a commercially available curcuminoid mixture (1), pure curcumin (2), pure demethoxycurcumin (3), and pure bisdemethoxycurcumin (4) were investigated for their inhibitory effects on cell growth, FLT3 expression, and cell cycle progression in an FLT3-overexpressing EoL-1 leukemic cell line using an MTT assay, Western blotting, and flow cytometry, respectively. The mixture (1) and compounds 2-4 demonstrated cytotoxic effects with IC50 values ranging from 6.5 to 22.5 μM. A significant decrease in FLT3 protein levels was found after curcuminoid treatment with IC20 doses, especially with mixture 1 and compound 2. In addition, mixture 1 and curcumin (2) showed activity on cell cycle arrest at the G0/G1 phase and decreased the FLT3 and STAT5A protein levels in a dose-dependent manner. Compound 2 demonstrated the greatest potential for inhibiting cell growth, cell cycle progression, and FLT3 expression in EoL-1 cells. This investigation has provided new findings regarding the effect of turmeric curcuminoids on FLT3 expression in leukemic cells.

Single cell analysis exposes intratumor heterogeneity and suggests that FLT3-ITD is a late event in leukemogenesis

FMS-like tyrosine kinase 3 receptor-internal tandem duplication (FLT3-ITD) commonly occurs in acute myeloid leukemia and is considered rare in acute lymphocytic leukemia. Acute leukemia has poor prognosis, mainly due to relapse. Standard FLT3-ITD diagnostic techniques are based on genomic polymerase chain reaction and have recently incorporated GeneScan (Applied Biosystems, Foster City, CA) to identify variations of the FLT3 gene. As this is an average-based assay utilized in a heterogeneous leukemic cell population, we hypothesized that cells of acute leukemia, considered FLT3-ITD-negative by standard methods, could possess a fraction of FLT3-ITD-positive cells. The present study employed single cell mutation analysis to evaluate the FLT3-ITD status in newly diagnosed acute myeloid leukemia (n = 5) and acute lymphocytic leukemia (n = 3) patients. A total of 541 single leukemic cells and 36 mononuclear cells from healthy volunteers were analyzed. Seven patients, considered FLT3-ITD-negative according to bulk DNA analysis, appeared to possess a small fraction of FLT3-ITD-positive cells based on single cell analysis. Moreover, this approach revealed the heterogeneity of the tumor as evident by different FLT3-ITD mutations present in the same patient. The presence of a minor clone carrying FLT3-ITD in almost all patients tested provides evidence that this lesion is a common late event in leukemogenesis. Additionally, 3 relapsed patients demonstrated loss of heterozygosity of the normal allele, affecting 25%-100% of the cells found to be FLT3-ITD-positive. Though further clinical testing is warranted, these findings may have implications on the prognostic significance of FLT3-ITD and the use of targeted therapy.