scholarly journals Design and Synthesis of 4-(Heterocyclic Substituted Amino)-1H-Pyrazole-3-Carboxamide Derivatives and Their Potent Activity against Acute Myeloid Leukemia (AML)

2019 ◽  
Vol 20 (22) ◽  
pp. 5739
Author(s):  
Yanle Zhi ◽  
Zhijie Wang ◽  
Chao Yao ◽  
Baoquan Li ◽  
Hao Heng ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Cancer Therapeutics ◽  
Strong Activity ◽  
Design And Synthesis ◽  
Proliferative Effect ◽  
Flt3 Inhibitor ◽  
Acute Myeloid

Fms-like receptor tyrosine kinase 3 (FLT3) has been emerging as an attractive target for the treatment of acute myeloid leukemia (AML). By modifying the structure of FN-1501, a potent FLT3 inhibitor, 24 novel 1H-pyrazole-3-carboxamide derivatives were designed and synthesized. Compound 8t showed strong activity against FLT3 (IC50: 0.089 nM) and CDK2/4 (IC50: 0.719/0.770 nM), which is more efficient than FN-1501(FLT3, IC50: 2.33 nM; CDK2/4, IC50: 1.02/0.39 nM). Compound 8t also showed excellent inhibitory activity against a variety of FLT3 mutants (IC50 < 5 nM), and potent anti-proliferative effect within the nanomolar range on acute myeloid leukemia (MV4-11, IC50: 1.22 nM). In addition, compound 8t significantly inhibited the proliferation of most human cell lines of NCI60 (GI50 < 1 μM for most cell lines). Taken together, these results demonstrated the potential of 8t as a novel compound for further development into a kinase inhibitor applied in cancer therapeutics.

scholarly journals The Salt-Inducible Kinase inhibitor YKL-05-099 suppresses MEF2C function and acute myeloid leukemia progressionin vivo

2019 ◽  
Author(s):  
Yusuke Tarumoto ◽  
Shan Lin ◽  
Jinhua Wang ◽  
Joseph P. Milazzo ◽  
Yali Xu ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Disease Progression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Clinical Investigation ◽  
Genetic Targeting ◽  
Acute Myeloid

AbstractLineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a Salt-Inducible Kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF MEF2C. In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. Here, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells underin vitroandin vivoconditions. Similar phenotypes were obtained when exposing cells to YKL-05-099, which caused cell cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele ofSIK3, we found that the anti-proliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated two different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progressionin vivoand extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-positive AML and provide a rationale for developing drug-like inhibitors of SIK3 for definitive pre-clinical investigation and for studies in human patients with leukemia.Key PointsAML cells are uniquely sensitive to genetic or chemical inhibition of Salt-Inducible Kinase 3in vitroandin vivo.A SIK inhibitor YKL-05-099 suppresses MEF2C function and AMLin vivo.

The Investigational Multi-Targeted Kinase Inhibitor TAK-901 Antagonizes Acute Myeloid Leukemia Pathogenesis: Results of Preclinical Studies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 581-581
Author(s):  
Patrick Griffin ◽  
Steffan T Nawrocki ◽  
Takashi Satou ◽  
Claudia M Espitia ◽  
Kevin R. Kelly ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mouse Model ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Standard Of Care ◽  
Clonogenic Survival ◽  
Preclinical Studies ◽  
Acute Myeloid

Abstract Abstract 581 The long-term prognosis for the majority of patients diagnosed with acute myeloid leukemia (AML) is very poor due, in part, to pre-existing myelodysplasia, multidrug resistance, and co-existing morbidities that limit therapeutic options. Novel strategies are essential in order to improve clinical outcomes. TAK-901 is an investigational small molecule kinase inhibitor that is currently being evaluated in Phase I trials. In preclinical studies, TAK-901 has demonstrated significant effects against a number of kinases with important roles in cancer including the Aurora kinases, which are key regulators of mitosis and whose overexpression in cancer promotes genetic instability, malignant pathogenesis, and drug resistance. We hypothesized that simultaneously targeting the activity of the Auroras and other oncogenic kinases with TAK-901 would disrupt AML pathogenesis. In order to test our hypothesis, we investigated the efficacy and pharmacodynamic activity of TAK-901 human AML cell lines, primary AML specimens, and an orthotopic bioluminescent disseminated mouse model of AML. TAK-901 potently diminished the viability of a panel of 8 AML cell lines as well as primary cells obtained from patients with AML. Acute exposure to TAK-901 ablated clonogenic survival, triggered the accumulation of polyploid cells, and induced apoptosis. The cytostatic and cytotoxic effects of TAK-901 were associated with significantly increased expression of the cyclin-dependent kinase inhibitor p27, growth arrest and DNA-damage-inducible 45a (GADD45a), and the BH3-only pro-apoptotic protein PUMA. Chromatin immunoprecipitation (ChIP) assays revealed that the elevation in the expression of these genes caused by administration of TAK-901 was due to increased FOXO3a transcriptional activity. The in vivo anti-leukemic activity of TAK-901 was investigated in a disseminated xenograft mouse model of AML established by intravenous injection of luciferase-expressing MV4-11 cells. IVIS Xenogen imaging was utilized to monitor disease burden throughout the study. In this mouse model, administration of TAK-901 was very well-tolerated and significantly more effective than the standard of care drug cytarabine with respect to suppressing disease progression and prolonging overall survival. Analysis of specimens collected from mice demonstrated that TAK-901 inhibited the homing of AML cells to the bone marrow microenvironment and induced AML cell apoptosis in vivo. Our collective findings indicate that TAK-901 is a novel multi-targeted kinase inhibitor that has significant preclinical activity in AML models and warrants further investigation. Disclosures: Satou: Takeda Pharmaceuticals: Employment. Hasegawa:Takeda Pharmaceuticals: Employment. Romanelli:Millennium Pharmaceuticals: Employment. de Jong:Takeda San Diego: Employment. Carew:Millennium Pharmaceuticals: Research Funding.

A Machine-Learning Analysis Suggests That FLX925, a FLT3/CDK4/6 Kinase Inhibitor, Is Potent Against FLT3-Wild Type Tumors Via Its CDK4/6 Activity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3520-3520
Author(s):  
Gene Cutler ◽  
Jordan S Fridman
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Rna Seq ◽  
Cell Cycle Genes ◽  
Equity Ownership ◽  
Anti Tumor Activity ◽  
Acute Myeloid

Abstract Fms-like tyrosine kinase 3 (FLT3) is universally expressed in Acute Myeloid Leukemia (AML) blast cells (Zheng R, et al. FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells. Blood. 2004) and activating mutations of FLT3 are amongst the most common genetic lesions in AML (Levis M. FLT3 mutations in acute myeloid leukemia: what is the best approach in 2013? ASH Education Program Book. 2013). FLX925, a small molecule inhibitor of FLT3 would be expected to have antitumor efficacy against FLT3-driven AML tumors. However, FLX925 also inhibits two closely related cyclin-dependent kinases (CDKs), CDK4 and CDK6 (described together as CDK4/6). Since CDK4/6 are important cell cycle genes and CDK4/6 inhibitors have demonstrated anti-tumor activity, the anti-CDK4/6 activity of FLX925 may drive anti-tumor efficacy in additional and potentially overlapping patient populations. To generate hypotheses about which tumors would be sensitive to FLX925 treatment and to understand the relative contributions of FLX925's FLT3- and CDK4/6-inhibitory activities, a panel of XXX tumor-derived cell lines were screened for sensitivity to FLX925. We performed a Gradient Boosting Machine (GBM) analysis, a machine-learning approach, to generate a model that predicts sensitivity to FLX925 based on RNA-Seq expression data. This sensitivity model can predict which cell lines and tumors are likely to be sensitive to FLX925 and also which gene expression patterns contribute to that sensitivity. The GBM analysis was performed on 133 (75%) of the assayed cell lines with a random 45 (25%) of the cell line datasets reserved for model validation. Pearson's correlations of r=0.96 on the test cell lines and r=0.68 on the validation cell lines were observed, indicating good predictive performance with modest overfitting. The two most important predictive genes in the model were Retinoblastoma 1 (RB1) and by Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A). RB1 and CDKN2A are important cell cycle genes immediately downstream and upstream, respectively, of CDK4/6, suggesting that sensitivity to FLX925 in these cell lines is driven by FLX925's CDK4/6 inhibitory activity. To strengthen this conclusion, the modeling was repeated but gene sets covering cell cycle genes or genes in the FLT3/STAT5 signaling pathway were separately excluded from the analysis. While excluding the FLT3/STAT5 pathway genes had no impact on the predictive power of the model, excluding the cell cycle gene set completely abrogated the ability of the GBM to predict sensitivity to FLX925. Thus, we conclude that in this panel of largely FLT3-wild type (FLT3-WT) cell lines, FLX925-sensitivity is driven solely by the compound's CDK4/6 activity. These results suggest that independent, but potentially overlapping, sets of tumors will be responsive to FLX925 due to both its FLT3- and its CDK4/6-inhibitory activities. To predict which tumor types would be most sensitive to FLX925 treatment, we applied our GBM model to the RNA-Seq expression data for 10,537 tumors from The Cancer Genome Atlas (TCGA) consortium. Even though this model is indifferent to FLT3/STAT5-pathway gene expression, it nevertheless identifies AML tumors as having the highest median predicted sensitivity to FLX925. This is true even when all haem/lymph malignancy-derived cell lines are excluded from the training set. Thus, both FLT3-WT and FLT3-activated tumors are expected to have sensitivity to FLX925 treatment, the former due to FLX925's CDK4/6 activity alone and the latter due to the independent FLT3 and CDK4/6 activities of the drug. The anti-tumor activity of FLX925 is currently being investigated in a Ph1/b dose-escalation study in subject with relapsed or refractory AML (NCT02335814). Disclosures Cutler: Amgen, Inc: Equity Ownership; FLX Bio: Employment, Equity Ownership. Fridman:FLX Bio: Employment, Equity Ownership.

scholarly journals The pan-class I phosphatidyl-inositol-3 kinase inhibitor NVP-BKM120 demonstrates anti-leukemic activity in acute myeloid leukemia

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Matteo Allegretti ◽  
Maria Rosaria Ricciardi ◽  
Roberto Licchetta ◽  
Simone Mirabilii ◽  
Stefania Orecchioni ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Phosphatidyl Inositol ◽  
Class I ◽  
Clinical Activity ◽  
Dependent Manner ◽  
Acute Myeloid

Abstract Aberrant activation of the PI3K/Akt/mTOR pathway is a common feature of acute myeloid leukemia (AML) patients contributing to chemoresistance, disease progression and unfavourable outcome. Therefore, inhibition of this pathway may represent a potential therapeutic approach in AML. The aim of this study was to evaluate the pre-clinical activity of NVP-BKM120 (BKM120), a selective pan-class I PI3K inhibitor, on AML cell lines and primary samples. Our results demonstrate that BKM120 abrogates the activity of the PI3K/Akt/mTOR signaling, promoting cell growth arrest and significant apoptosis in a dose- and time-dependent manner in AML cells but not in the normal counterpart. BKM120-induced cytotoxicity is associated with a profound modulation of metabolic behaviour in both cell lines and primary samples. In addition, BKM120 synergizes with the glycolitic inhibitor dichloroacetate enhancing apoptosis induction at lower doses. Finally, in vivo administration of BKM120 to a xenotransplant mouse model of AML significantly inhibited leukemia progression and improved the overall survival of treated mice. Taken together, our findings indicate that BKM120, alone or in combination with other drugs, has a significant anti-leukemic activity supporting its clinical development as a novel therapeutic agent in AML.

scholarly journals Synergistic Combination Activity of the Novel GSPT1 Degrader CC-90009 in Acute Myeloid Leukemia Models

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3330-3330
Author(s):  
Daniel W. Pierce ◽  
Tsun-Wen Sheena Yao ◽  
Emily Pace ◽  
Hongbin Wang ◽  
Pierre Flandin-Blety ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
High Throughput ◽  
Myeloid Leukemia ◽  
Publicly Traded ◽  
Pdx Model ◽  
Flt3 Inhibitor ◽  
Healthy Donors ◽  
Acute Myeloid

Abstract Introduction: CC-90009 is a novel cereblon E3 ligase modulator (CELMoD ®) agent that is a first-in-class degrader of translation termination factor G1 to S phase transition 1 (GSPT1). CC-90009 has demonstrated antileukemic activity as a single agent and is currently under investigation in patients with acute myeloid leukemia (AML; NCT02848001). Treatment with CC-90009 led to rapid reductions in peripheral and bone marrow blasts, and demonstrated preliminary promising efficacy, including several complete remissions, in patients with relapsed or refractory AML. Here, we describe the identification and preclinical activity of select anti-AML agents as potential combination partners of CC-90009 to further improve its efficacy and therapeutic index. Based on these results, the combination activity of CC-90009 with venetoclax (VEN)/azacitidine (AZA) is being evaluated in a phase 1/2 trial in patients with AML (NCT04336982). Methods: A high-throughput cell viability screen was performed to identify synergistic partners of CC-90009. AML cell lines (HL-60, HNT-34, KG-1, ML-2, NOMO-1, MOLM-13, MV4-11, F-36P, OCI-AML2, OCI-AML3) were treated with CC-90009 in combination with &gt; 70 compounds, including standard anti-AML agents, tyrosine kinase inhibitors (TKIs), unfolded protein response inducers, transcription inhibitors, and epigenetic agents. MOLM-13 and MV4-11 are fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) cell lines. Hits were validated in a colony formation (CF) assay using primary AML cells and bone marrow mononuclear cells (BMMC) from healthy donors. Synergy of the combination partners with CC-90009 was further assessed in AML patient-derived xenograft (PDX) models. Synergy between the isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib and CC-90009 was evaluated in a TF-1 erythroleukemia cell line overexpressing IDH2 R140Q mutant, and an IDH2 R140Q PDX model, AM7577. Results: Our high-throughput combination screen revealed multiple TKIs, epigenetic agents, and pro-apoptotic agents as potential combination partners of CC-90009 in AML cell lines. FLT3 inhibitors, including sunitinib, pexidartinib, midostaurin, lestaurtinib, crenolanib, and gilteritinib, synergized with CC-90009 to reduce viability in FLT3-ITD AML cell lines MV4-11 and MOLM-13. Similarly, the B-cell lymphoma 2 (BCL2) inhibitor VEN potentiated CC-90009-induced apoptosis and accelerated cell-autonomous killing. Reduction in levels of MCL-1, an anti-apoptotic factor, by CC-90009 most likely contributed to the synergy with VEN. We prioritized the evaluation of FLT3, BCL2, and IDH2 inhibitors as partners of CC-90009. In CF assays, midostaurin enhanced the inhibitory effect of CC-90009 in primary AML cells, without augmenting the effect of CC-90009 in CD34+ BMMC from healthy donors. Similarly, VEN enhanced the reduction in CF by CC-90009 in AML patient-derived BMMC without exacerbating the decrease in CF by CC-90009 in BMMC from healthy donors. We characterized FLT3 inhibitor/CC-90009 and BCL2 inhibitor/AZA/CC-90009 combinations in a FLT3-ITD PDX murine model, PDX1. The FLT3 inhibitor quizartinib significantly prolonged survival when combined with CC-90009 compared with either agent alone (P &lt; 0.001). Similarly, VEN/AZA/CC-90009 combination markedly extended survival compared with single agents or VEN/AZA doublets (P &lt; 0.001). The synergy between CC-90009 and epigenetic modulators was validated and further characterized in customized cell differentiation assays. Addition of CC-90009 to enasidenib, a mutant IDH2 inhibitor, enhanced differentiation and killing of CD34+ stem and progenitor cells, and increased differentiated CD235a+ erythroblasts, compared with enasidenib alone in a TF-1 cell line overexpressing IDH2 R140Q. Enasidenib/CC-90009 combination treatment reduced CD45+ malignant populations and increased differentiated CD14+ cells, leading to significantly prolonged animal survival in an IDH2 R140Q PDX model, AM7577, compared with either agent alone (P &lt; 0.0001). Conclusion: Using a high-throughput combination screen, we identified rational combination partners that synergize with CC-90009 in in vitro and in vivo AML models. Collectively, these results support the clinical evaluation of CC-90009 in combination with FLT3, BCL2, and IDH2 inhibitors to further improve treatment outcomes for patients with AML. Disclosures Pierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Yao: Bristol Myers Squibb: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months, Research Funding. Pace: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Wang: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flandin-Blety: Bristol Myers Squibb: Current Employment. Benitez: Bristol Myers Squibb: Current Employment. Guarinos: Bristol Myers Squibb: Current Employment. Hoffmann: Bristol Myers Squibb: Current Employment. Carrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Selective Abrogation of STAT5 Expression Impairs the Proliferation of Acute Myeloid Leukemia Cells with Various Cytogenetic Alterations

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5024-5024
Author(s):  
Youngsoo Kim ◽  
Tianyuan Zhou ◽  
Shuling Guo ◽  
Andy Siwkowski ◽  
Donna Witchell ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Significant Inhibition ◽  
Acute Myeloid

Abstract STAT5 is a key common downstream mediator of multiple signaling pathways which are often dysregulated in various hematologic malignancies, including acute myeloid leukemia (AML). Due to the heterogeneity and high relapse rate of the disease, the treatment options for AML are currently limited. Although the approach of treating the disease by inhibiting upstream kinases such as FLT3 within these signaling pathways appeared promising, the clinical efficacy of these drugs as mono-therapy have been disappointing. We hypothesized that this lack of efficacy might be due to the residual STAT5 activity that is present even in the presence of these inhibitors in vivo. Therefore, abrogating the expression of the final regulator of these pathways, STAT5, might be a much more efficient way of blocking signaling, thus inhibiting the proliferation and survival of AML cells. In this study, we first investigated the role of STAT5 in the proliferation of AML cells by selectively suppressing the expression of the gene using 2nd-Generation antisense oligonucleotides (ASOs). Suppression of STAT5 following ASO treatment (&gt;80% over control ASO) led to a significant inhibition of cell proliferation (50~70% over control ASO), a decrease in colony formation, and a modest induction of apoptosis in a range of AML lines including KG-1α, MV-4-11, and MOLM-13. STAT5 ASO treatment was highly specific for the STAT5 target and produced predictable effects on gene expression, as demonstrated by the downregulation of Pim-1 and cyclin D1, well-known STAT5 regulated genes. No changes in the expression levels of Bcl-XL, STAT1, and STAT3 were observed. Furthermore, relative anti-proliferative activity within the various AML lines correlated well with the relative levels of STAT5 activity. Interestingly, there was a strong correlation between the extent of STAT5/Pim-1 downregulation and the degree of anti-proliferation, suggesting a possible role of Pim-1 as a downstream effector of STAT5 ASO anti-proliferative activity. Studies comparing the relative effects of the STAT5 ASO inhibitor with the potent multi kinase inhibitor CEP701 in various AML cell lines demonstrated potent anti-proliferative activity for the STAT5 inhibitor in the cell lines including KG-1α that display resistance to the multi kinase inhibitor. Taken together, these results suggest that a STAT5 ASO therapeutic approach may have utility for the treatment of AML and related hematologic disorders.

Azacitidine in Combination with the Selective FLT3 Kinase Inhibitor Crenolanib Effectively Disrupts Stromal Protection of CD34+ Leukemia-Initiating Cells (LIC) in FLT3-ITD+ Acute Myeloid Leukemia (AML)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 676-676
Author(s):  
Anne-Kathrin Garz ◽  
Stefan Habringer ◽  
Saskia Wolf ◽  
Binje Vick ◽  
Marie-Theresa Weickert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Bone Marrow Niche ◽  
Next Generation ◽  
Nsg Mice ◽  
Acute Myeloid

Abstract Background: Effectively targeting the oncogenic mutation FLT3-ITD remains a crucial goal in acute myeloid leukemia (AML) therapy. Thus far, tyrosine kinase inhibitors (TKI) have not been able to eradicate the earliest leukemia-initiating cells (LIC) in FLT3-ITD+ AML, which are thought to be responsible for the frequent relapses seen in this disease. We have previously shown that LIC in FLT3-ITD+ AML persist during treatment with first-generation TKI despite effective inhibition of FLT3 phosphorylation owing to their selective protection by niche cells (Parmar et al, Cancer Res 2011). Hence, novel strategies to disrupt the protective interaction of stroma with FLT3-ITD+ LIC are urgently needed. Here, we asked whether stromal resistance of FLT3-ITD+ LIC can be overcome by the next generation TKI crenolanib alone or in combination with the hypomethylating agent azacitidine (AZA). Methods: The efficacy of crenolanib alone or in combination with AZA was assessed in the human FLT3-ITD+ cell lines MV4-11 and MOLM13 and FLT3-ITD transfected BaF3 cells as well as in primary human FLT3-ITD+ AML bone marrow samples. Cells were cultured in suspension or on the mesenchymal stromal cell line EL08-1D2, which mimics the bone marrow niche and maintains LIC in vitro (Parmar et al, Cancer Res 2011). Cultures were treated with DMSO, crenolanib and/or AZA for defined periods. Apoptosis, cell cycle and differentiation of AML cells were analyzed by flow cytometry. Clonogenic capacity and frequency of primitive FLT3-ITD+ stem/progenitors were probed by standard CFU and LTC assays. Engraftment potential of FLT3-ITD+ patient-derived xenograft (PDX) AML cells after treatment with crenolani, AZA or the combination thereof was assessed in the NSG xenograft model (Vick et al, PlosOne 2015). Treatment-induced alterations in FLT3-ITD downstream signaling were investigated by western blots. Results: Crenolanib effectively inhibited FLT3 and STAT5 phosphorylation in FLT3-ITD+ cells in suspension as well as in EL08-1D2 supported co-cultures whereas AZA had no effect on FLT3 signaling pathways. Monotherapy with crenolanib but not AZA effectively induced apoptosis and inhibited growth of FLT3-ITD+ cell lines. Primary CD34+ FLT3-ITD+ progenitor cells were also highly susceptible to inhibition by crenolanib as a single agent. However, crenolanib was completely unable to eradicate primitive CD34+ FLT3-ITD+ LIC when protected by niche cells as assessed by CFU and LTC assays as well as xenotransplantation into NSG mice. In contrast, the combination of crenolanib and AZA resulted in efficient apoptosis and dramatically impaired clonogenic capacity of FLT3-ITD+ LIC even in the presence of stroma. Pretreatment of EL08-1D2 cells with AZA before co-culture with AML cells did not influence stromal protection against crenolanib. Furthermore, soluble stromal factors did not account for TKI resistance. Successful targeting of stromally protected FLT3-ITD+ LIC by the combination of AZA and crenolanib is currently being confirmed by xenotransplantation in NSG mice. Conclusion: The combination of azacitidine and the selective next generation FLT3 kinase inhibitor crenolanib is a novel promising treatment regimen to overcome niche protection and selectively target LIC in FLT3-ITD+ AML. We hypothesize that the mechanism of action involves loss of quiescence in FLT3-ITD+ LIC leading to increased susceptibility towards TKI as well as induction of differentiation by AZA. In depth analysis of involved signaling pathways and phenotypic alterations in LIC are ongoing. Disclosures Keller: Pfizer: Consultancy; Roche: Consultancy, Honoraria. Götze:Novartis: Honoraria; Celgene Corp.: Honoraria.

scholarly journals Megakaryocytic Expansion in Gilteritinib-Treated Acute Myeloid Leukemia Patients Is Associated With AXL Inhibition

2020 ◽  
Vol 10 ◽  
Author(s):  
Kran Suknuntha ◽  
Yoon Jung Choi ◽  
Ho Sun Jung ◽  
Aditi Majumder ◽  
Sujal Shah ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Kinase Domain ◽  
Leukemia Cell Line ◽  
Tyrosine Kinase Domain ◽  
Flt3 Inhibitor ◽  
Acute Myeloid

Numerous recurrent genetic mutations are known to occur in acute myeloid leukemia (AML). Among these common mutations, Fms-like tyrosine kinase 3 remains as one of the most frequently mutated genes in AML. We observed apparent marrow expansion of megakaryocytes in three out of six patients with Flt3-mutated AML following treatment with a recently FDA-approved Flt3 inhibitor, gilteritinib which possesses activity against internal tandem duplication and tyrosine kinase domain Flt3 mutations and also inhibits tyrosine kinase AXL. To assess whether biopsy findings can be attributed to promotion of megakaryocytic (Mk) differentiation with gilteritinib, we devised a cellular assay by overexpressing double mutated Flt3-ITDY591F/Y919F in chronic myeloid leukemia cell line K562 to study Mk differentiation in the presence of Flt3 and AXL inhibitors with non-mutually exclusive mechanisms. These experiments demonstrated the lack of direct effect Flt3 inhibitors gilteritinib and quizartinib on megakaryocytic differentiation at either transcriptional or phenotypic levels, and highlighted antileukemic effects of AXL receptor tyrosine kinase inhibitor and its potential role in megakaryocytic development.

scholarly journals Apatinib Exhibits Cytotoxicity to Acute Myeloid Leukemia Cell Via Targeting VEGFR2-Mediated Pro-Survival Signaling and Angiogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5158-5158
Author(s):  
Lian Yu ◽  
Manman Deng ◽  
Zhifeng Li ◽  
Zhihong Fang ◽  
Yun Dai ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Xenograft Model ◽  
Npm1 Mutation ◽  
Survival Signaling ◽  
Acute Myeloid

Objective:Vascular permeability contributes to disease progression and drug resistance in acute myeloid leukemia (AML). Thus, targeting angiogenetic signaling is a promising strategy for the treatment of AML, especially for relapsed and resistant diseases. The aim of the present study was to evaluate the potential efficacy of Apatinib, a novel receptor tyrosine kinase inhibitor selectively targeting VEGFR-2, in AML treatment. Method: CCK8 and Annexin V/PI assay were separately utilized to determine the IC50 value and apoptosis of AML cell lines. The cytotoxic efficacy of Apatinib against primary AML cells from 57 adult patients and 11 normal counterparts were then evaluated by analysis of apoptosis. Using JC-1 kit to detect the mitochondrial membrane potential, and western bolting and mass cytometry assay as well as immunohistochemistry to explore the underlying mechanism. Finally, the anti-leukemia activity was further evaluated in in vivo xenograft models. Results: Our findings demonstrated apatinib markedly inhibited proliferation and promoted apoptosis in AML cell lines. Similarly, apatinib showed cytotoxicity against primary AML cells while harmless to normal hematopoiesis. Its effect was correlated with several clinical features such as NPM1 mutation, extramedullary infiltration, relapse/refractory disease and FAB subtypes. Additionally, apatinib suppressed AML cells growth and attenuated angiogenesis in xenograft model. Mechanistically, apatinib-induced cytotoxicity was closely associated with the inhibition of VEGFR2-meidated Src/STAT3 and AKT/mTOR pathway and the induction of mitochondrial-mediated apoptosis. Conclusion: In summary, apatinib exerts anti-leukemia function via targeting VEGFR2-triggered pro-survival signaling and angiogenesis, providing a rationale for clinical trials of apatinib in the treatment of AML. Disclosures No relevant conflicts of interest to declare.

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