Potent FMS-like Tyrosine Kinase 3 Inhibitors Research Articles

Design, synthesis and biological evaluation of 2-aminopyrimidine derivatives as potent FLT3 inhibitors

Acute myeloid leukemia (AML) is an aggressive cancer, which is characterized by clonal expansion of myeloid progenitors in the bone marrow and peripheral blood. FMS-like tyrosine kinase 3 (FLT3) mutations are the most frequently identified mutations, present in approximately 25–30 % AML patients, making FLT3 inhibitors a crucial treatment option for AML. In this study, we described the design, synthesis and biological evaluation of a series of 2-aminopyrimidine derivatives as potent FLT3 inhibitors. Notably, compound 15 displayed potent kinase inhibitory activities against FLT3 (FLT3-WT IC50 = 7.42 ± 1.23 nM; FLT3-D835Y IC50 = 9.21 ± 0.04 nM) and robust antiproliferative activities against MV4-11 cells (IC50 = 0.83 ± 0.15 nM) and MOLM-13 cells (IC50 = 10.55 ± 1.70 nM). Compound 15 also possessed potent antiproliferative activities against BaF3 cells carrying various FLT3-TKD and FLT3-ITD-TKD mutations, indicating its potential to overcome on-target resistance caused by FLT3 mutations. In summary, compound 15 showed promising potential for further exploration as a treatment of AML.

Rational design of 4-((6-phenoxypyrimidin-4-yl)amino)-N-(4-(piperazin-1-yl)phenyl)-1H-pyrazole-3-carboxamide (LT-540-717) as orally bioavailable FLT3 inhibitor

In recent years, fms-like tyrosine kinase 3 (FLT3) was confirmed as an exciting target for treatment of AML. However, resistance to FLT3 inhibitors caused by acquired point mutations in tyrosine kinase domain (TKD) have limited their sustained efficacious. Thus, there remains an unmet need to develop high-efficacy FLT3 inhibitors against both FLT3 internal tandem duplication (ITD) and FLT3 (TKD) mutations. Herein, we describe the discovery of compound LT-540-717 (32), a potent FLT3 inhibitor (IC50: 0.62 nM), starting from FN-1501. Compound 32 exhibited highly inhibitory activity against several acquired FLT3 mutations including FLT3 (ITD, D835V), FLT3 (ITD, F691L), FLT3 (D835Y) and FLT3 (D835V). Additionally, 32 displayed potent antiproliferative activity against FLT3-mutation driven BaF3 and AML cells. Oral administration of 32 (25 mg/kg, QD) significantly prohibited tumor growth (tumor-inhibition rate is 94.18%), and no obvious side effect was observed even when increasing dose to 50 mg/kg (tumor-inhibition rate is 93.98%). Furthermore, 32 showed an acceptable bioavailability (F = 33.3% in rat and 72.7% in beagles), a suitable half-life time (T1/2 = 3.5 h in rat and T1/2 = 11.1 h in beagles), and a satisfactory metabolic stability. In summary, these results show the therapeutic potential of 32 to become a new anti-AML drug, especially for AML harboring dual FLT3 (ITD, TKD) mutations.

Abstract 4009: Discovery of PLM-102, a highly potent 3rd generation FLT3 inhibitor, in drug-resistant FLT3-ITD-TKD mutated acute myeloid leukemia

Abstract Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 25 ~ 30% of all acute myeloid leukemia (AML) cases and FLT3-ITD mutations are associated with a particularly unfavorable prognosis, with an increased risk of relapse and shorter overall survival (OS) compared with AML patients without the mutation. Even though multiple FLT3 inhibitors are in various stages of clinical evaluation, resistance to FLT3 inhibitors resulting from acquired point mutations in tyrosine kinase domain (TKD) have limited the sustained efficacy of treatments. Among them, a “gatekeeper” mutation (F691L) is resistant to most available FLT3 inhibitors. Here, we show the efficacy and mechanism of PLM-102, developed an orally available and highly selective small molecule, in overcoming drug-resistant FLT3-ITD-TKD mutations as a strong candidate of the 3rd generation of FLT3 inhibitor. PLM-102 displays extraordinary potency of enzyme inhibitory activities of all FLT3 WT/mutants including F691L with IC50 values of <1 nM. In FLT3-ITD-positive cell lines (MV4-11, MOLM-13, MOLM-14, Ba/F3-ITD-D835Y, Ba/F3-ITD-F691L), PLM-102 shows about 18~25-fold higher anti-proliferative activities than Gilteritinib. Also, PLM-102 potently inhibited the phosphorylation of FLT3 and its downstream signaling pathways, and induced apoptosis as evidenced by PARP-cleavage and caspase-3 activation. Moreover, in AML-related xenograft mouse models (MV4-11, MOLM-14, Ba/F3-ITD-D835Y, Ba/F3-ITD-F691L), once-daily oral dose of PLM-102 significantly suppressed the tumor growth and achieved complete tumor regression. In addition, the outstanding efficacy of our compound has been confirmed in large tumor xenograft models. These results are very encouraging because large tumors have a limited blood supply and high interstitial fluid pressure, leading to a poor absorption of anticancer drugs. Pharmacokinetics of PLM-102 displayed higher bone-marrow exposure indicating better target engagement and benefits in the clinical translation in AML. In conclusion, PLM-102 is a promising therapeutic candidate for the FLT3-ITD-mutated AML as well as the acquired resistance to current FLT3 inhibitors. Citation Format: Jin-Hee Park, Jae-Seon Lee, Su-jin Oh, So-Deok Lee, Yong June Choi, Keon Wook Kang, Miran Moon, Soo Yeon Jang, Myung Jin Kim, Yong-Chul Kim. Discovery of PLM-102, a highly potent 3rd generation FLT3 inhibitor, in drug-resistant FLT3-ITD-TKD mutated acute myeloid leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4009.

Design, synthesis, and evaluation of pyrido.[3,4-b]pyrazin-2(1H)-one derivatives as potent FLT3 inhibitors

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as driver mutations in a subgroup of AML patients. Herein, we describe the design, synthesis, and biological evaluation of a novel series of potent pyrido.[3,4-b]pyrazin-2(1H)-one derivatives as FLT3 inhibitors. The compounds exhibited moderate to potent FLT3 kinase inhibitory potency and excellent antiproliferative activities against MV4-11 cells. Among them, compound 13 demonstrated the most potent kinase activity against FLT3-D835Y (IC50 = 29.54 ± 4.76 nM) and cellular potency against MV4-11 cells (IC50 = 15.77 ± 0.15 nM). Compound 13 also efficiently inhibited the growth of multiple mutant BaF3 cells expressing FLT3-D835V/F, FLT3-F691L, and FLT3-ITD/D835Y. Furthermore, compound 13 was metabolically stable in mouse liver microsomes. Moreover, the treatment with compound 13 led to robust inhibition of FLT3 autophosphorylation on Tyr589/591 in MV4-11 cells. In summary, our data demonstrated that 13 was worthy of further study for the treatment of AML.

Rational design and synthesis of 2-(1H-indazol-6-yl)-1H-benzo[d]imidazole derivatives as inhibitors targeting FMS-like tyrosine kinase 3 (FLT3) and its mutants

Fms-like tyrosine kinase 3 (FLT3) has been verified as a therapeutic target for acute myeloid leukaemia (AML). In this study, we report a series of 2-(1H-indazol-6-yl)-1H-benzo[d]imidazol-5-yl benzamide and phenyl urea derivatives as potent FLT3 inhibitors based on the structural optimisation of previous FLT3 inhibitors. Derivatives were synthesised as benzamide 8a–k, 8n–z, and phenyl urea 8l–m, with various substituents. The most potent inhibitor, 8r, demonstrated strong inhibitory activity against FLT3 and FLT3 mutants with a nanomolar IC50 and high selectivity profiles over 42 protein kinases. In addition, these type II FLT3 inhibitors were more potent against FLT3 mutants correlated with drug resistance. Overall, we provide a theoretical basis for the structural optimisation of novel benzimidazole analogues to develop strong inhibitors against FLT3 mutants for AML therapeutics.

Molecular Modeling Studies of N-phenylpyrimidine-4-amine Derivatives for Inhibiting FMS-like Tyrosine Kinase-3.

Overexpression and frequent mutations in FMS-like tyrosine kinase-3 (FLT3) are considered risk factors for severe acute myeloid leukemia (AML). Hyperactive FLT3 induces premature activation of multiple intracellular signaling pathways, resulting in cell proliferation and anti-apoptosis. We conducted the computational modeling studies of 40 pyrimidine-4,6-diamine-based compounds by integrating docking, molecular dynamics, and three-dimensional structure–activity relationship (3D-QSAR). Molecular docking showed that K644, C694, F691, E692, N701, D829, and F830 are critical residues for the binding of ligands at the hydrophobic active site. Molecular dynamics (MD), together with Molecular Mechanics Poison–Boltzmann/Generalized Born Surface Area, i.e., MM-PB(GB)SA, and linear interaction energy (LIE) estimation, provided critical information on the stability and binding affinity of the selected docked compounds. The MD study suggested that the mutation in the gatekeeper residue F691 exhibited a lower binding affinity to the ligand. Although, the mutation in D835 in the activation loop did not exhibit any significant change in the binding energy to the most active compound. We developed the ligand-based comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) models. CoMFA (q2 = 0.802, r2 = 0.983, and = 0.698) and CoMSIA (q2 = 0.725, r2 = 0.965 and = 0.668) established the structure–activity relationship (SAR) and showed a reasonable external predictive power. The contour maps from the CoMFA and CoMSIA models could explain valuable information about the favorable and unfavorable positions for chemical group substitution, which can increase or decrease the inhibitory activity of the compounds. In addition, we designed 30 novel compounds, and their predicted pIC50 values were assessed with the CoMSIA model, followed by the assessment of their physicochemical properties, bioavailability, and free energy calculation. The overall outcome could provide valuable information for designing and synthesizing more potent FLT3 inhibitors.

First in Human (FIH) FLT3 and SYK Inhibitor HM43239 Shows Single Agent Activity in Patients (pts) with Relapsed or Refractory (R/R) FLT3 Mutated and Wild-Type Acute Myeloid Leukemia (AML)

First in Human (FIH) FLT3 and SYK Inhibitor HM43239 Shows Single Agent Activity in Patients (pts) with Relapsed or Refractory (R/R) FLT3 Mutated and Wild-Type Acute Myeloid Leukemia (AML)

Investigation of Selected Flavonoid Derivatives as Potent FLT3 Inhibitors for the Potential Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis and a high degree of relapse seen in patients. Overexpression of FMS-like tyrosine kinase 3 (FLT3) is associated with up to 70% of AML patients. Wild-type FLT3 induces proliferation and inhibits apoptosis in AML cells, while uncontrolled proliferation of FLT3 kinase activity is also associated with FLT3 mutations. Therefore, inhibiting FLT3 activity is a promising AML therapy. Flavonoids are a group of phytochemicals that can target protein kinases, suggesting their potential antitumor activities. In this study, several plant-derived flavonoids have been identified with FLT3 inhibitory activity. Among these compounds, compound 40 (5,7,4'-trihydroxy-6-methoxyflavone) exhibited the most potent inhibition against not only FLT3 (IC50 = 0.44 μM) but also FLT3-D835Y and FLT3-ITD mutants (IC50 = 0.23 and 0.39 μM, respectively). The critical interactions between the FLT3 binding site and the compounds were identified by performing a structure-activity relationship analysis. Furthermore, the results of cellular assays revealed that compounds 28, 31, 32, and 40 exhibited significant cytotoxicity against two human AML cell lines (MOLM-13 and MV-4-11), and compounds 31, 32, and 40 resulted in cell apoptosis and G0/G1 cell cycle arrest. Collectively, these flavonoids have the potential to be further optimized as FLT3 inhibitors and provide valuable chemical information for the development of new AML drugs.

PHI-101 Is a Potent Third-Generation FLT3 Inhibitor Developed to Overcome Resistance in Acute Myeloid Leukemia

Background: FMS-like tyrosine kinase 3 (FLT3), a member of the class III receptor tyrosine kinase family, plays a pivotal role in regulating cell growth and differentiation of hematopoietic cells. FLT3 is mutated in approximately 30% of AML patients either by internal tandem duplication (ITD) within the juxtamembrane portion or by point-mutations in the kinase domain (TKD). Thus, FLT3 inhibitors including quizartinib (AC220), midostaurin (PKC412) and gilteritinib (ASP2215) have been developed and undergone clinical testing for the treatment of AML. However, patients frequently relapse due to intrinsic and extrinsic resistance to these FLT3 inhibitors in spite of the initial clinical efficacy. We have developed a potent third-generation FLT3 inhibitor, PHI-101, capable of overcoming some of these mechanisms of resistance. Methods: Biochemical kinase assays for PHI-101 have been performed on 9 different FLT3 mutants and wild type FLT3. Cellular potencies of PHI-101 have also been assessed using various patient-derived AML cells as well as MV4-11, MOLM14 and BaF3 cell lines transformed with human FLT3 mutants including single mutations [FLT3(ITD), FLT3(D835Y)], double or triple mutations [FLT3(ITD/D835Y), FLT3(ITD/F691L), FLT3(ITD/F691L/D835Y)]. In order to evaluate in vivo efficacies of oral administration of PHI-101, xenograft mouse models and in vivo bioluminescence imaging have been utilized. Results: PHI-101 possessed excellent enzymatic potencies against FLT3 potential resistant mutants such as ITD/D835V and the gatekeeper ITD/F691L mutation, as well as against FLT3 single activating mutants that include ITD, D835V, D835H, and D835Y. PHI-101 inhibited the phosphorylation of FLT3 and downstream STAT5 and ERK1/2 more effectively than quizartinib and gilteritinib at equivalent doses. Moreover, oral administration of PHI-101 induced tumor regression in the xenograft mouse models developed by injection of BaF3 cells transformed with FLT3-ITD, FLT3-TKD or FLT3-ITD/TKD mutants in a dose-dependent fashion with no appreciable toxicities. In luciferase-bearing blood circulating mouse models with FLT3-double mutants (ITD/D835Y and ITD/F691L) or triple mutants (ITD/D835Y/F691L), oral administration (30 mpk, QD) of PHI-101 diminished more than 89% of bioluminescent intensity and reduced leukemic burden. PHI-101 also showed increased efficacy in extending the lifespan of xenograft mice compared to quizartinib. PHI-101 strongly suppressed proliferation and induced apoptosis in primary AML samples harboring FLT3/ITD and FLT3/TKD mutations with little effect on wild-type FLT3 samples. Conclusion: PHI-101, an orally bioavailable novel small molecule, is a potent third-generation FLT3 inhibitor able to overcome resistance to several resistance mutations based on in vitro and in vivo experiments. PHI-101 possesses excellent in vitro and in vivo activities against not only FLT3 single activating mutations (ITD or TKD mutants) but also FLT3 double (ITD/D835Y or ITD/F691L) and triple (ITD/D835Y/F691L) resistant mutations with no pronounced toxicities. Preclinical evaluation of PHI-101 showed clear evidence of antileukemic activity and improved efficacy in both in vitro and in vivo models. PHI-101 is currently under investigation in first-in-human clinical trials with relapsed or refractory AML patients. Disclosures Nam: Pharos I&BT Co., Ltd.: Current equity holder in private company. Im:Pharos I&BT Co., Ltd.: Current Employment. Han:Pharos I&BT Co., Ltd.: Current equity holder in private company. Kim:Pharos I&BT Co., Ltd.: Current Employment. Yoon:Pharos I&BT Co., Ltd.: Current equity holder in private company. Cho:Pharos I&BT Co., Ltd.: Research Funding. Choi:Pharos I&BT Co., Ltd.: Research Funding. Young:Pharos I&BT Co., Ltd.: Research Funding. Nguyen:Pharos I&BT Co., Ltd.: Research Funding. Zhu:Pharos I&BT Co., Ltd.: Research Funding. Li:Pharos I&BT Co., Ltd.: Research Funding. Small:Pharos I&BT Co., Ltd.: Consultancy, Research Funding. Sim:Pharos I&BT Co., Ltd.: Research Funding.

Discovery of Oxazol-2-amine Derivatives as Potent Novel FLT3 Inhibitors.

Internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) is the most common mutation in patients with acute myeloid leukemia (AML). FLT3-ITD+ induces constitutive activation of FLT3, causing an abnormally rapid proliferation of cancer cells. In this study, we identified novel FLT3 inhibitors and investigated 5-(4-fluorophenyl)-N-phenyloxazol-2-amine (compound 7; 7c) as candidates for the treatment of AML. The results showed that 7c inhibited the activities of FLT3 and mutated FLT3 in a cell-free kinase assay and Molm-13 and MV4-11 cells, as well as the proliferation of FLT3-ITD+ AML cells, increasing apoptosis. The anti-leukemic activity of 7c was confirmed by in vivo tumor growth inhibition in MV4-11 xenograft mice. Besides, 7c suppressed the expression of DNA damage repair genes. Combination treatment with 7c and olaparib (a poly (ADP-ribose) polymerase [PARP] inhibitor) synergistically inhibited cell proliferation in Molm-13 and MV4-11 cells. Our findings demonstrated that 7c is a therapeutic candidate targeting FLT3 for AML treatment and suggested that combination treatment with 7c and a PARP inhibitor may be an effective therapy regimen for FLT3-mutated AML.

Recent advances in FLT3 inhibitors for acute myeloid leukemia.

Fms-like tyrosine kinase-3 (FLT3) mutations occur in approximately 30% of acute myeloid leukemia (AML) cases, suggesting FLT3 as an attractive target for AML treatment. Early FLT3 inhibitors enhance antileukemia efficacy by inhibiting multiple targets, and thus had stronger off-target activity, increasing their toxicity. Recently, a number of potent and selective FLT3 inhibitors have been developed, many of which are effective against multiple mutations. This review outlines the evolution of AML-targeting FLT3 inhibitors by focusing on their chemotypes, selectivity and activity over FLT3 wild-type and FLT3 mutations as well as new techniques related to FLT3. Compounds that currently enter the late clinical stage or have entered the market are also briefly reported.

HM43239, a Novel Potent Small Molecule FLT3 Inhibitor, in Acute Myeloid Leukemia (AML) with FMS-like Tyrosine Kinase 3 (FLT3) Mutations: Phase 1 /2 Study

Background: HM43239 is an orally active small molecule inhibitor of FLT3 that selectively inhibits not only FLT3 wild type, ITD mutants or TKD mutations, but also FLT3 ITD/TKD double mutations. HM43239 directly inhibits the kinase activity of FLT3 as a reversible Type I inhibitor and effectively modulates downstream p-STAT5 and p-ERK. HM43239 also demonstrated inhibition of SYK, JAK1/2 and TAK1, known to be involved in tumor cell proliferation and/or differentiation HM43239 monotherapy induced dose-dependent regression of tumor growth in FLT3 wild-type and FLT3 mutated leukemia cancer cell lines (Poster presented at AACR 2018; Abst.804 and also prolonged survival in a FLT3 ITD/TKD double mutated xenograft mouse model (Poster presented at AACR 2019; Abst.1293). The anti-leukemic activity of HM43239 monotherapy was evaluated in in vivo studies that suggested the ability of this agent to potentially target LSCs (Poster presented at AACR 2019; Abst.1293). Preclinical studies suggest that HM43239 may be an effective agent in patients with hematologic malignancies including AML, to target various mutant types of FLT3. Herein, we present a clinical trial design to assess the potential clinical activity and safety of HM43239 in patients with AML. This phase I/II clinical trial (NCT03850574) consists of dose escalation (Part A) and dose expansion (Part B) stages to access the overall safety and efficacy findings. Trial Design: This is an ongoing, open-label, multicenter, first in human, phase I/II trial enrolling adult patient with AML who relapsed or have refractory disease after at least one prior line of therapy. Patients were treated with HM43239 once daily (QD) in 28-day cycle except for the first 30-day cycle including a single PK sampling period. This trial comprises two parts: dose escalation and dose expansion. During dose escalation, the study follows an accelerated titration design, with around 50% dose increments and 1 patient per dose level. Accelerated titration will continue until 1 patient experiences a dose-limiting toxicity (DLT) or moderate toxicity (MT), drug-related grade 2 adverse event (except for hematologic toxicities), at any dose level, after which, a 3 + 3 dose escalation design will be used. If a patient achieves a clinical response [complete remission (CR), complete remission with low platelets (CRp), complete remission with incomplete hematologic recovery (CRi) or partial remission (PR)] at any dose level in escalation cohort, then an expansion cohort will be open at that dose level. Based on the evaluation of DLTs and composite complete remissions (CRc) from the dose escalation cohorts, additional subjects may be recruited in expansion cohorts at each dose level selected for expansion. At least 10 subjects with FLT3 mutations (ITD or activating point mutations such as D835Y, D835V, I836) will be enrolled to each dose level selected for expansion (including the subjects from the dose escalation cohort). Blood samples are collected for pharmacokinetics (PK) & pharmacodynamics (PD) evaluation and for exploratory biomarker analysis in both cohorts. A 2-parameter Bayesian logistic regression will be used to model the dose-toxicity relationship on DLT in dose escalation and expansion parts and the estimated DLT rate will be provided as supportive reference for dose escalation procedure and safety monitoring. The primary endpoint is the assessment of pharmacokinetics, safety and tolerability in order to determine the recommended phase 2 dose (RP2D). Secondary endpoints include best response rate [CRc (CRc=CR+CRp=CRi)+ PR], duration of response, event free survival, overall survival, cumulative incidence of relapse of HM43239. PK-PD relationships and PD evaluated by plasma inhibitory assay (PIA) are exploratory endpoints. At first, this trial was initiated in Jan 2019 and an enrolled patient has been evaluated since May 2019. Dose level 1 (20 mg) was completed without any DLTs; enrollment to next dose level (40 mg) was initiated in June 2019 . This new cohort is currently recruiting patients in the Republic of Korea and the USA. Clinical trial information: NCT03850574. 3,800 characters allowed 3,580 characters in abstract Disclosures Daver: Pfizer: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Agios: Consultancy; Otsuka: Consultancy; Forty-Seven: Consultancy; Immunogen: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; NOHLA: Research Funding; Glycomimetics: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Hanmi Pharm Co., Ltd.: Research Funding; Celgene: Consultancy; BMS: Consultancy, Research Funding; Astellas: Consultancy; Incyte: Consultancy, Research Funding; Servier: Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding.

Design, synthesis, and biological evaluation of 4-((6,7-dimethoxyquinoline-4-yl)oxy)aniline derivatives as FLT3 inhibitors for the treatment of acute myeloid leukemia

FMS-like tyrosine kinase 3 (FLT3) was an important therapeutic target in acute myeloid leukemia (AML). We synthesized two series of 4-((6,7-dimethoxyquinoline-4-yl)oxy)aniline derivatives possessing the semicarbazide moiety and 2,2,2-trifluoro-N,N'-dimethylacetamide moiety as the linker. The cell proliferation assay in vitro against HL-60 and MV4-11 cell lines demonstrated that most series I compounds containing semicarbazide moiety had more potent than Cabozantinib. Furthermore, the enzyme assay showed that compound 12c and 12g were potent FLT3 inhibitors with IC50 values of 312 nM and 384 nM, respectively. Following that, molecular docking analysis was also performed to determine possible binding mode between FLT3 and the target compound.

Effects of CYP3A inhibitors on the pharmacokinetics of quizartinib, a potent and selective FLT3 inhibitor, and its active metabolite.

AimsQuizartinib is an oral, highly potent and selective next‐generation FMS‐like tyrosine kinase 3 (FLT3) inhibitor under investigation in patients with FLT3‐internal tandem duplication‐mutated acute myeloid leukaemia. This drug–drug interaction study assessed the pharmacokinetics (PK) of quizartinib when coadministered with strong or moderate cytochrome P450 3A (CYP3A) inhibitors.MethodsIn this parallel‐group study, subjects were randomised to receive: (i) quizartinib + ketoconazole; (ii) quizartinib + fluconazole; or (iii) quizartinib alone. On Days 1–28, subjects received ketoconazole 200 mg or fluconazole 200 mg twice daily, and on Day 8, all subjects received a single 30‐mg quizartinib dose. Blood samples were collected for PK analyses, steady‐state PK parameters were simulated by superpositioning, and safety was assessed.ResultsNinety‐three healthy subjects were randomised; 86 completed the study. When administered with ketoconazole, geometric mean ratios (90% confidence interval) for quizartinib maximum observed plasma concentration (Cmax) and area under the plasma concentration–time curve (AUC) from time 0 extrapolated to infinity were 117% (105%, 130%) and 194% (169%, 223%), respectively, vs quizartinib alone. Steady‐state PK simulation demonstrated ~2‐fold increase of both steady–state Cmax and AUC from time 0 to the end of the dosing interval when quizartinib was administered with ketoconazole due to accumulation of quizartinib at steady state. When administered with fluconazole, geometric mean ratios (90% confidence interval) for quizartinib Cmax and AUC from time 0 extrapolated to infinity were 111% (100%, 124%) and 120% (104%, 138%), respectively, vs quizartinib alone. Overall, 5.4% of subjects experienced quizartinib‐related adverse events; no serious adverse events or deaths occurred.ConclusionsThese results suggest reducing the dose of quizartinib when coadministered with a strong CYP3A inhibitor, but not with a moderate or weak CYP3A inhibitor. This dose reduction was implemented in phase 3 evaluation of quizartinib.

Abstract 4668: Therapeutic efficacy of aryl hydrocarbon receptor agonist in acute myeloid leukemia

Abstract Background The presence of leukemic stem cells (LSCs) is known to be a major cause of treatment failure and recurrence in acute myeloid leukemia (AML) patients. Therefore, it is important to develop a treatment method of targeting LSCs. Based on several research, aryl hydrocarbon receptor (AhR) antagonist, StemRegenin 1 (SR1), supports LSC activity. Thus, we speculated that the AhR agonist with the opposite of SR1 could trigger LSC differentiation which may result in the anti-cancer effects. We also hypothesize that the combination of the AhR agonist and ASP2215 targeting FMS-like tyrosine kinase 3(FLT3) mutation, which is known to be present at the LSC level shows the augmented effect on cancer therapy. Here we show the biological significance of AhR and its contribution to leukemic cells, and the therapeutic applicability of this approach. Methods and Results The effects of AhR agonists of 6-formylindolo[3,2-b]carbazole(FICZ), and (2′Z)-Indirubin, and AhR antagonist, SR1 were tested on AML cell lines (MV4-11, MOLM14, HL60, NB4, KG-1) using western blot and flow cytometry. AhR modulation was confirmed by changes in AhR and CYP1B1 protein expression levels at 24hours. . When AML cells were exposed to FICZ for 72hours, surface markers for myeloid differentiation, increased significantly. Mononuclear cells isolated from AML patients’ leukapheresis blood samples(n=5) were cultured with stem culture medium containing growth factors with agonists or antagonist of AhR to determine the effect on LSC differentiation and stem-ness. The cell surface markers Lin-CD34+CD38- represent the stem/progenitor cell fraction were analyzed by FACS, FICZ and indirubin reduces this cell proportion by 2.4-fold 1.8-fold respectively compared to the control. In addition, multipotential progenitors-like LSCs(Lin- CD34+CD38-CD90-CD45RA-) and lymphoid-primed multipotential progenitors-like LSCs (Lin- CD34+CD38-CD90-CD45RA+) was reduced by 2.8 and 2.6-fold in the FICZ treatment group and 1.8 and 2.2-fold in the Indirubin treatment group, respectively. Colony formation assay was performed to verify the effect of AhR modulating on LSC function. FICZ and indirubin reduced the stem-ness of cultured leukemic cells compared with control and SR1. We investigated whether the AhR agonist, FICZ, has an anti-leukemia effect through a cell viability assay. As a single agent, FICZ did not affect AML cells. However, FICZ improves the cytotoxicity of ASP2215, a potent FLT3 inhibitor, in FLT3 mutant AML cells. And more, the combination enhances the effect of ASP2215 on cancer cell signaling pathways AKT-mTOR and MEK-ERK. Conclusion We found that FICZ, an AhR agonist not only results in the LSCs differentiation, but also enhances the inhibitory effect of ASP2215 on cancer cell survival through the AKT-mTOR and MEK-ERK signaling. This cross-talk has the multiple meaning on cancer therapy in that it can simultaneously target cancer cells as well as LSCs. Citation Format: Hee Joo Han, Dong-Yeop Shin, Sung-Soo Yoon. Therapeutic efficacy of aryl hydrocarbon receptor agonist in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4668.

Clinical considerations for the use of FLT3 inhibitors in acute myeloid leukemia.

Internal tandem duplications and tyrosine kinase mutations in the fms-like tyrosine kinase 3 (FLT3) receptor can occur in acute myeloid leukemia (AML) and portend a poor prognosis. Midostaurin, a multikinase inhibitor that targets FLT3, demonstrated a survival benefit in FLT3-mutated AML in combination with front-line chemotherapy. Despite this advancement, the use of FLT3 inhibitors in clinical practice is complicated by significant drug-drug interactions and uncertainty about optimal timing, duration, and sequencing of therapy. As monotherapy, the utility of FLT3 inhibitors was initially limited by incomplete and transient clinical responses and the development of acquired resistance. This led to the development of more potent and selective FLT3 inhibitors designed to overcome common resistance mechanisms. One of these second generation FLT3 inhibitors, gilteritinib, is now FDA-approved for the treatment of relapsed or refractory AML. Now that multiple FLT3 inhibitors are commercially available, it is important to further delineate the role of these agents in the AML population. This review aims to provide a comprehensive overview of the role of FLT3 inhibitors in AML and apply the current literature to clinical practice.

Discovery of LDD-1075 as a potent FLT3 inhibitor.

Fms-like tyrosine kinase 3 (FLT3) is a valuable pharmacological target in the treatment of acute myeloid leukemia (AML). LDD-1075 and LDD-1076 are indirubin derivatives, and LDD-1075 is the ester form of LDD-1076. LDD-1076 exhibited a potent in vitro FLT3 kinase activity inhibition with an IC50 of 7.89 nM, whereas, LDD-1075 demonstrated a relatively weak activity against FLT3 (IC50 of 3.19 µM). In contrast with the results of the FLT3 kinase activity inhibition assay, the LDD-1076 did not affect the growth of the MV4-11 cell line, which harbors the constitutively activated form of the FLT3 mutation. Notably, LDD-1075 exhibited a strong cytotoxic effect against the MV4-11 cells. When LDD-1075 was incubated with the MV4-11 cell lysate, the formation of LDD-1076 was observed. Treatment with LDD-1075 inhibited the FLT3 phosphorylation along with the phosphorylation of the signal transducer and activator of transcription 5 protein, which is a downstream signal transducer of FLT3. Treatment with LDD-1075 induced apoptosis and cell cycle arrest at the G1 phase. The present study demonstrated that the LDD-1076 formed by the bioconversion of LDD-1075 is a potent FLT3 inhibitor with anti-leukemic activity.

Comprehensive structure-activity-relationship of azaindoles as highly potent FLT3 inhibitors.

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.

Abstract 804: Antitumor activity of the potent and novel FLT3 inhibitor HM43239 in acute myeloid leukemia

Abstract Introduction: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutations is associated with poor prognosis with a high risk of relapse after therapy and reduced overall survival. Activating mutations of internal tandem duplication (ITD) and tyrosine kinase domain point mutations (TKD) of FLT3 have been reported in approximately 30% of AML as oncogenic driver mutations. Currently, FLT3 inhibitors showed clinical benefits in the corresponding AML patients. In this study, we have characterized a novel FLT3 inhibitor, HM43239, and assessed the potential as a novel therapy for AML patients. Materials and Methods: HM43239 is a novel, highly potent FLT3 kinase inhibitor. This compound showed tight binding to FLT3 kinase in in silico docking model as a reversible Type I inhibitor. In vitro kinase assay was performed to identify kinase selectivity of HM43239. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM43239 in AML cell lines. HM43239 was evaluated in MV-4-11 and MOLM-13 xenograft mice models. Tumor sizes were measured and tumor samples were analyzed of the mechanisms of action. Results: Among 191 kinases biochemically assayed, HM43239 showed the high selectivity toward FLT3 and AML associated other kinases (e.g. SYK, JAK and TAK1). IC50s' of HM43239 against FLT3 WT, FLT3 ITD and FLT3 D835Y kinases were 1.1 nM, 1.8 nM and 1.0 nM, respectively. HM43239 potently inhibited the growth of AML cell lines harboring FLT3 ITD mutation, such as MV4-11 (IC50: 1.3 nM), MOLM-13 (5.1 nM) and MOLM-14 (2.9 nM). Furthermore, HM43239 effectively inhibited the phosphorylation levels of FLT3 and of downstream kinases related with cell proliferation. In addition, caspase 3/7-dependent apoptosis was induced by HM43239 in AML cell lines expressing FLT3 ITD mutant. And HM43239 inhibited proliferation and induced apoptosis of leukemic stem cell (LSC) marker-expressing KG1a cells (CD34+/CD38- cells) suggesting that the possibility for targeting LSC. HM43239 showed the excellent dose proportional antitumor activity in mouse models xenografted with both MV4-11 and MOLM-13 cell line without any significant toxicity. Moreover, we identified in vivo modulation of related targets (p-FLT3 & p-STAT5) in AML cell with FLT3 mutant. Conclusion: Taken together, HM43239 has demonstrated the potential therapeutic efficacy for the treatment of AML patients and implicated the mechanism of overcoming resistance and preventing relapse. Citation Format: Miyoung Lee, Young Eun Ha, Mi Jin Moon, Joo-Yun Byun, HyunKyung Yu, SeokJong Kang, JaeHo Lee, Kyuhang Lee, Eunkyung Kim, Eunyoung Kim, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. Antitumor activity of the potent and novel FLT3 inhibitor HM43239 in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 804.

Abstract 806: KI2011 is a novel potent FLT3 tyrosine kinase inhibitor in acute myeloid leukemia cells with FLT3-mutation

Abstract More than 30% of patients with acute myeloid leukemia (AML) harbor a mutation with internal tandem duplication (ITD) in the juxtamembrane domain and point mutations in the tyrosine kinase domain of FMS-like tyrosine kinase-3 (FLT3), which results in aberrant activation FLT3 by auto-phosphorylation, and confers poor prognosis. To develop novel FLT3 inhibitors, ten novel oxazole amine derivatives were designed and synthesized based on FLT3 protein structure. Among the synthesized small molecule compounds, KI2011 exhibited selectively inhibitory activities of the class III receptor tyrosine kinases, especially FLT3, wile-type, FLT3-D835Y mutant and FLT3-internal tandem duplication (ITD) mutant isoforms with IC50 values of 25, 9, and 1.2 nM, respectively, determined by an in vitro kinase activity assay. The compound decreased FLT3 phosphorylation and its downstream signaling in MV4-11 cells harboring FLT3-ITD mutation. Treatment of KI2011 selectively inhibited cell proliferation and induced apoptosis of MV411 cells in a concentration-dependent manner, but not in K562 (FLT3 negative) cells. In addition, we found that KI2011 enhanced radiosensitivity of MV4-11 cells. The results suggest that KI2011 is a novel potent FLT3 inhibitor that may be further developed in the preclinical studies as a therapeutic agent in AML treatments. Citation Format: Hwani Ryu, Jie-Young Song, Jiyeon Ahn. KI2011 is a novel potent FLT3 tyrosine kinase inhibitor in acute myeloid leukemia cells with FLT3-mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 806.