scholarly journals Molecular Analysis and In Vivo Efficacy Studies on a Novel Chemical-Series of FLT3 Inhibitors in Human FLT3-ITD AML

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 36-36
Author(s):  
Katelyn Melgar ◽  
MacKenzie Walker ◽  
Jiang-kang Jiang ◽  
Kelli Wison ◽  
Kwangmin Choi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Median Survival ◽  
Kinase Inhibitors ◽  
Xenograft Model ◽  
Daily Injection ◽  
Lead Compound ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract Approximately 25% of all acute myeloid leukemias (AMLs) contain an internal tandem duplication (ITD) in the juxtamembranal region of FLT3. Even with standard chemotherapies, FLT3-ITD AML is associated with poor prognosis, with a five-year survival rate of approximately 15% as compared to 40% for wild-type FLT3 AML. Therefore, FLT3 inhibitors have been developed as a targeted treatment approach. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming, with few complete remissions and a high rate of relapse and resistance. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse in FLT3-ITD AML patients. Through chemical and structure-activity relationship studies, we have identified a novel class of FLT3 tyrosine kinase inhibitors. We have previously shown that our current lead compound (CTDS-004) has excellent selectivity against the kinase activity of FLT3-ITD and FLT-ITD D835Y at a subnanomolar concentration (IC50 <5 x 10-10 M). Additionally, CTDS-004 potently inhibits proliferation and progenitor function, and induces apoptosis of FLT3-ITD positive human AML cell lines in comparison to the second generation FLT3 inhibitors, AC220 and Crenolanib. Importantly, CTDS-004 exhibits minimal effects on the function and viability of normal human CD34+ BM cells. The most important distinction between CTDS-004 and current FLT3-ITD inhibitors is that CTDS-004 retains its efficacy and prevents subclonal recovery of FLT3-ITD AML cells that have become refractory to AC220 or Crenolanib treatment. To gain insight into the mechanistic basis for the superior response of FLT3-TID AML to CTDS-004 as compared to AC220, we performed RNA sequencing of MLL-AF9 FLT3-ITD cells after 6 and 12 hours of treatment with AC220 or CTDS-004 (IC10: 0.3 and 0.1 nM, respectively). Although both compounds target FLT3-ITD, there were striking distinctions in gene expression changes after treatment with AC220 and CTDS-004, highlighting the differences in the biological impact and cytotoxic effect of these compounds. Our lead compound has promising pharmacokinetic and pharmacodynamics properties in vivo, therefore, we tested CTDS-004 in a xenograft model of human AML. NRGS mice (NOD.Cg-Rag1tm1MomIL2rgtm1WjlTg[CMV-IL3,CSF2,KITLG]1EAv/J) were transplanted with human MLL-AF9 FLT3-ITD leukemic cells (2 x 105 cells/mouse) via tail vein injection. Starting on day 10 post-engraftment, the mice were treated with CTDS-004 (30 mg/kg; n=5), AC220 (15 mg/kg; n=5), or PBS (n=5) via intraperitoneal injection on a cycle of five days of daily injection followed by two days of rest for the duration of the experiment. Kaplan-Meier survival analysis revealed that CTDS-004 is extremely effective at prolonging survival in the human FLT3-ITD AML xenograft model. There was a significant increase in median survival for CTDS-004 and AC220 treatment groups compared to the PBS control group (Median survival post-xenograft: PBS: 28 days, AC220: 54 days, CTDS-004: 53 days; p=0.0002). Taken together, these findings suggest our novel FLT3 inhibitor shows promise for the treatment of FLT3-ITD positive AML, and particularly for patients that have intrinsic and/or acquired resistance to FLT3 tyrosine kinase inhibitors. Disclosures No relevant conflicts of interest to declare.

Identification and Characterization Of a Potent FLT3 Inhibitor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5027-5027
Author(s):  
Yun Chen ◽  
Yao Guo ◽  
Wanting Ho ◽  
Zhizhuang Joe
Keyword(s):  
Drug Development ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Kinase Assay ◽  
Cancer Drug ◽  
Protein Substrate ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract Acute myeloid leukemia (AML) is a malignant myeloid disorder for which there is no effective treatment. Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in AML patients. This makes FLT3 an attractive therapeutic target. Currently, several potent FLT3 inhibitors have been developed. However, their clinical efficacy is limited largely due to their poor effectiveness toward the FLT3-D835 mutants which are often present in AML or acquired after treatment of FLT3-ITD-positive AML with tyrosine kinase inhibitors. Needless to say, more potent FLT3 inhibitors targeting both FLT-ITD and FLT3-D835 mutants are needed. In addition, combinations of tyrosine kinase inhibitors with drugs targeting other signaling pathways represent a new trend in anti-cancer drug development. To establish an effective kinase assay for FLT3 inhibitor screening, we generated a protein substrate designated GST-FLT3S which was expressed in E. coli cells as a glutathione S-transferase fusion protein. The protein substrate together with recombinant proteins containing the catalytic domain of wild type and mutant forms of FLT3 expressed in baculovirus was used in biochemical screening of inhibitors. Several potent inhibitors were obtained. Importantly, one of the inhibitors with an oxindole core structure inhibited FLT3 and D835 FLT3 mutants equally well with nanomolar IC50 values. We further analyzed the potency of the inhibitor by performing cell-based assays. The cells used included FLT3-ITD-positive cell line MV-4-11 and an EPO-dependent erythroleukemia cell line transformed by retrovirus mediated expressions of FLT3-ITD and FLT3-D835 mutants. At nanomolar concentrations, the inhibitor blocked growth factor signaling and effectively caused apoptosis and cell cycle arrest. It showed significant advantage over the current available FLT3 inhibitor, sorafenib. Loss-of-function mutations of tumor suppressor p53 are common in solid tumors but relatively rare in AML although its expression is often suppressed. This makes p53 a potential target for anti-AML drug development. We employed MDM2 inhibitor nutlin-3 which blocks the degradation of p53. Importantly, at sub-nanomolar concentrations, FLT3 inhibitors and nutlin-3 synergistically inhibited growth of cells containing FLT3-ITD or FLT3-D835 mutants. Altogether, we developed an effective substrate for screening of FLT3 inhibitors and identified one compound with high potency toward both FLT3-ITD and FLT3-D835. We further demonstrated that targeting FLT3 and p53 simultaneously greatly increases drug potency. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Novel Small Molecule FLT3 Inhibitors for the Treatment of FLT3-ITD AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3690-3690
Author(s):  
Katelyn Melgar ◽  
MacKenzie Walker ◽  
Jian-kang Jiang ◽  
Kelli Wilson ◽  
James C. Mulloy ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Primary Concern ◽  
Hematopoietic Stem ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract FMS-like Tyrosine Kinase 3 (FLT3) is a receptor tyrosine kinase that promotes growth and survival of hematopoietic stem and progenitor cells. Mutations in the gene encoding FLT3 are found in 20-30% of acute myeloid leukemias (AML), with approximately 25% of all AMLs containing an internal tandem duplication (ITD) in the juxtamembranal region. FLT3-ITD AML is associated with poor prognosis, with a 5 year survival rate of approximately 15% as compared to 40% for wild-type FLT3 AML. As such, FLT3 inhibitors have been developed to treat FLT-ITD AML. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming. A Phase II study of quizartinib (AC220) showed few complete remissions in AML patients treated with AC220 as a single agent. Additionally, patients who relapse after FLT3 inhibitor treatment can develop FLT3-inhibitor resistant clones, many of which acquire a D835Y substitution. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse for FLT3-ITD AML patients. Through chemical and structure-activity relationship studies, we have identified a novel class of FLT3 tyrosine kinase inhibitors. After initial biochemical and functional analysis, NCGC-2327 emerged as our lead compound, and further optimized with improved solubility, stability, and permeability properties suitable for in vivo utility (NCGC-1481). Both compounds have excellent selectivity against the kinase activity of FLT3-ITD and FLT-ITD D835Y at a subnanomolar concentration (IC50 <5.08 x 10-10 M). AC220, NCGC-2327, and NCGC-1481 treatment of primary human FLT3-ITD-mutant AML cells show EC50 at subnanomolar concentrations (0.5 nM, 0.4 nM, and 0.1 nM respectively) as determined by CellTiter Glo proliferation assays. To ensure that our compounds were selectively effective against FLT3-ITD-mutant AML, primary human NRas-mutant AML cells treated with the inhibitors revealed an EC50 outside of the tested range (>30 µM). To assess the ability of the compounds to induce apoptosis, we treated FLT3-ITD-mutant AML cells with AC220, NCGC-2327, NCGC-1481, or Crenolanib (a FLT3 inhibitor that can inhibit FLT3-ITD-D835Y) for 72 hours. NCGC-148-treated cells showed the greatest levels of apoptosis (AnnexinV+) as compared to Crenolanib, AC220, and NCGC-2327 (P< 0.01). Consistent with the viability assays, NCGC-1481 treatment showed the greatest inhibition of leukemic progenitor function in methylcellulose (Vehicle: 189 ± 39, Crenolanib: 151 ± 32, AC220: 36 ± 3, NCGC-2327: 22 ± 3, and NCGC-1481: 3 ± 2) (P<0.01). Relapse and resistance is a primary concern for patients treated with AC220, therefore we investigated leukemic subclonal resistance in vitro after treatment with Crenolanib, AC220, NCGC-2327, or NCGC-1481. FLT3-ITD-mutant AML cells were treated for 72 hours, washed and then allowed to recover in the absence of the compounds for one week. Subclonal recovery was assessed by measuring cell viability (AnnexinV+) and leukemic progenitor function (methylcellulose) for up to 1 week post treatment. NCGC-2327 and NCGC-1481 delayed, and in some instances prevented, subclonal recovery as compared to AC220 or Crenolanib treatment. NCGC-2327 and NCGC-1481 show comparable potency to current FLT3 inhibitors (i.e., AC220 and Crenolanib) in regards to inhibition of FLT3 signaling, proliferation, and induction of apoptosis in FLT3-ITD-mutant AML. However, NCGC-2327 and NCGC-1481 are exquisitely effective at preventing subclonal recovery of FLT3-ITD-mutant AML as compared to both AC220 and Crenolanib. Taken together, these findings suggest our novel FLT3 inhibitors show promise for the treatment of FLT3-ITD positive AML, and particularly for patients that have intrinsic and/or acquired resistance to FLT3 tyrosine kinase inhibitors. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tyrosine Kinase Inhibitor (TKI) Combination Scheduling Impacts Secondary FLT3 Tyrosine Kinase Domain (TKD) Mutation Profiles in a Xenograft Model of FLT3-ITD+ Acute Myeloid Leukemia (AML)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3620-3620
Author(s):  
Jolieke G van Oosterwijk ◽  
Eric I Zimmerman ◽  
Yong-Dong Wang ◽  
Shelley Orwick ◽  
Aksana Vasilyeva ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Treatment Group ◽  
Tyrosine Kinase ◽  
Median Survival ◽  
Single Agent ◽  
Xenograft Model ◽  
Common Mechanism ◽  
Flt3 Inhibitor ◽  
The Impact

Abstract A common mechanism associated with clinical resistance to FLT3 inhibitors (e.g., sorafenib and quizartinib) in FLT3-ITD+ AML is the acquisition of secondary FLT3 TKD mutations. Recently, we demonstrated that crenolanib, a potent and selective FLT3 inhibitor, has preclinical activity against FLT3 inhibitor-resistant FLT3-ITD+/TKD mutants, and the combination of sorafenib and crenolanib was more efficacious than single-agent TKIs in a FLT3-ITD+ xenograft model (Zimmerman et al. Blood 2013). Here, we evaluated continuous and intermittent schedules of sorafenib (SOR) and crenolanib (CRE) and the impact on efficacy and drug resistant profiles in vivo. We hypothesized that varying selective pressure through modulation of FLT3 inhibitor exposure (e.g., single-agent vs combination, high- vslow-dose intensity) would alter drug resistance patterns. For in vivo evaluations, one million MOLM13-luciferase+ cells were administered by tail vein injection to NSG mice and treatment was started 10 days later. Mice (7-10 per treatment group) were treated with vehicle (median survival, 16 days), SOR 60 mg/kg once daily (qd) on Monday, Wednesday, and Friday (3/7); SOR 60mg/kg qd for 5 days per week (5/7); CRE 15mg/kg twice daily (bid) 5/7; CRE 15mg/kg qd 5/7 and SOR 60mg/kg qd 5/7 (combination A); or CRE 15mg/kg bid 5/7 and SOR 60mg/kg qd 3/7 (combination B). Mice were monitored daily for assessment of toxicity and serial pharmacokinetic sampling was performed on days 15 or 16 of therapy. Mice were treated continuously until leukemic progression, sacrificed and bone marrow was harvested for assessment of leukemic infiltration and FLT3 TKD mutations in exons 17 and 20 by deep amplicon sequencing. Combinations A & B were tolerated and more efficacious than single-agent TKIs: median survival combination A vs sorafenib qd 5/7 (59 days vs 45 days, p<0.001) or sorafenib qd 3/7 (59 days vs. 31 days, p<0.001); median survival combination B vs crenolanib bid 5/7 (52 days vs 31 days, p<0.001) or sorafenib qd 3/7 (52 days vs 31 days, p<0.001) or sorafenib qd 5/7 (52 days vs 45 days, p<0.001). Both combinations induced regression of leukemic burden by day 20 and delayed the outgrowth of leukemic cells compared to single-agent TKIs. SOR and CRE plasma exposure was not altered in combination therapy. Greater than 80% leukemic cell infiltration was observed in bone marrow at progression. TKD mutations were not observed in MOLM-13 cells isolated from mice treated with vehicle, crenolanib bid 5/7, or sorafenib qd 3/7, presumably due to the short duration of treatment in vivo until leukemic progression. However, TKD mutations were observed in bone marrow samples from mice treated with sorafenib qd 5/7, combination A, and combination B. In the sorafenib qd 5/7 treatment group, 4/8 (50%) mice developed clinically relevant D835Y (n=3) or F691L (n=1) mutations. In contrast, a differential pattern of TKD mutations was observed in combination A: 4/9 (44%) mice developed D835Y (n=2), D835A (n=1), D839Y (n=1), or N841K (n=1) mutations. In combination B, only 1/7 (14%) of mice developed a TKD mutation. Emergence of clinically relevant FLT3 TKD mutations during TKI treatment was modeled in a MOLM-13 bone marrow xenograft model. Although both drug combination schedules were more efficacious than single-agent TKIs, the combination incorporating more dose intensive crenolanib therapy suppressed the emergence of FLT3 TKD mutations. These results may allow for optimal clinical trial design of TKI combinations and have provided the foundation for a protocol in development for FLT3-ITD+ pediatric AML. Disclosures Schuster: AROG Pharmaceuticals: Employment. Ramachandran:AROG Pharmaceuticals: Employment. Inaba:Bayer/Onyx: Research Funding.

In vitro Measurement and In vivo Prediction of Time-Dependent Inhibitory Effects of Three Tyrosine Kinase Inhibitors on CYP3A Activity

2021 ◽  
Vol 22 ◽  
Author(s):  
Liyan Wang ◽  
Tingting Zhao ◽  
Yunxiang Wang ◽  
Banglian Hu ◽  
Jianfei Tao ◽  
...  
Keyword(s):  
Drug Interaction ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Liver Microsomes ◽  
Inhibitory Effect ◽  
Cyp3a Activity ◽  
Drug Drug Interaction ◽  
Inhibitory Potential

Background: Imatinib, sunitinib, and gefitinib are the three most common tyrosine kinase inhibitors (TKIs). However, their quantitative drug-drug interaction potentials In vivo and the relationship between their structure and inhibitory activity remain unknown. Objective: This study aimed to investigate the potential drug-drug interaction risk of three TKIs based on CYP3A. Methods: 6β-Hydroxylated testosterone formation was selected to probe the CYP3A activity in human liver microsomes. Molecular docking simulation was performed to explore the potential structural alerts. Results: Imatinib exhibited the strongest inhibitory effect towards CYP3A, while the inhibitory potential of gefitinib and sunitinib were comparable to each other but weaker than imatinib. IC50 shift assays demonstrated that the inhibitory potential of all three TKIs was significantly increased after a 30-min preincubation with NADPH. The KI and Kinact values of imatinib, sunitinib, and gefitinib were 3.75 μM and 0.055 min–1, 1.96 μM and 0.037 min–1, and 9.94 μM and 0.031 min–1, respectively. IVIVE results showed that there was a 1.3- to 43.1-fold increase in the AUC of CYP3A-metabolizing drugs in the presence of the TKIs. Conclusion: All three TKIs exhibited a typical irreversible inhibitory effect towards CYP3A. The presence of more N-heterocycles and the resulting better binding confirmation of imatinib may have been responsible for its stronger inhibitory effect than sunitinib and gefitinib. Therefore, caution should be taken when CYP3A-metabolizing drugs are co-administrated with imatinib, sunitinib, or gefitinib.

scholarly journals MEK inhibition enhances the response to tyrosine kinase inhibitors in acute myeloid leukemia

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
María Luz Morales ◽  
Alicia Arenas ◽  
Alejandra Ortiz-Ruiz ◽  
Alejandra Leivas ◽  
Inmaculada Rapado ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Resistance Pathway ◽  
Acute Myeloid

AbstractFMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.

scholarly journals Erratum: Inhibition of OATP1B1 by tyrosine kinase inhibitors: in vitro-in vivo correlations

2017 ◽  
Vol 117 (5) ◽  
pp. e3-e3
Author(s):  
S Hu ◽  
R H J Mathijssen ◽  
P de Bruijn ◽  
S D Baker ◽  
A Sparreboom
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors

scholarly journals The Discovery of Novel BCR-ABL Tyrosine Kinase Inhibitors Using a Pharmacophore Modeling and Virtual Screening Approach

2021 ◽  
Vol 9 ◽  
Author(s):  
Ting-Ting Huang ◽  
Xin Wang ◽  
Shao-Jia Qiang ◽  
Zhen-Nan Zhao ◽  
Zhuo-Xun Wu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Pharmacophore Modeling ◽  
Myelogenous Leukemia ◽  
Lipinski’S Rule ◽  
Abl Tyrosine Kinase ◽  
Pharmacophore Models

Chronic myelogenous leukemia (CML) typically results from a reciprocal translocation between chromosomes 9 and 22 to produce the bcr-abl oncogene that when translated, yields the p210 BCR-ABL protein in more than 90% of all CML patients. This protein has constitutive tyrosine kinase activity that activates numerous downstream pathways that ultimately produces uncontrolled myeloid proliferation. Although the use of the BCR-ABL tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, dasatinib, bosutinib, and ponatinib have increased the overall survival of CML patients, their use is limited by drug resistance and severe adverse effects. Therefore, there is the need to develop novel compounds that can overcome these problems that limit the use of these drugs. Therefore, in this study, we sought to find novel compounds using Hypogen and Hiphip pharmacophore models based on the structures of clinically approved BCR-ABL TKIs. We also used optimal pharmacophore models such as three-dimensional queries to screen the ZINC database to search for potential BCR-ABL inhibitors. The hit compounds were further screened using Lipinski’s rule of five, ADMET and molecular docking, and the efficacy of the hit compounds was evaluated. Our in vitro results indicated that compound ZINC21710815 significantly inhibited the proliferation of K562, BaF3/WT, and BaF3/T315I leukemia cells by inducing cell cycle arrest. The compound ZINC21710815 decreased the expression of p-BCR-ABL, STAT5, and Crkl and produced apoptosis and autophagy. Our results suggest that ZINC21710815 may be a potential BCR-ABL inhibitor that should undergo in vivo evaluation.

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