Selective FMS-like Tyrosine Kinase 3 Inhibitor Research Articles

Enhancing Therapeutic Efficacy of FLT3 Inhibitors with Combination Therapy for Treatment of Acute Myeloid Leukemia.

FMS-like tyrosine kinase 3 (FLT3) mutations are genetic changes found in approximately thirty percent of patients with acute myeloid leukemia (AML). FLT3 mutations in AML represent a challenging clinical scenario characterized by a high rate of relapse, even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The advent of FLT3 tyrosine kinase inhibitors (TKIs), such as midostaurin and gilteritinib, has shown promise in achieving complete remission. However, a substantial proportion of patients still experience relapse following TKI treatment, necessitating innovative therapeutic strategies. This review critically addresses the current landscape of TKI treatments for FLT3+ AML, with a particular focus on gilteritinib. Gilteritinib, a highly selective FLT3 inhibitor, has demonstrated efficacy in targeting the mutant FLT3 receptor, thereby inhibiting aberrant signaling pathways that drive leukemic proliferation. However, monotherapy with TKIs may not be sufficient to eradicate AML blasts. Specifically, we provide evidence for integrating gilteritinib with mammalian targets of rapamycin (mTOR) inhibitors and interleukin-15 (IL-15) complexes. The combination of gilteritinib, mTOR inhibitors, and IL-15 complexes presents a compelling strategy to enhance the eradication of AML blasts and enhance NK cell killing, offering a potential for improved patient outcomes.

Abstract 1096: FMS-like tyrosine kinase 3 (FLT3) inhibition by olverembatinib (HQP1351) downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 in preclinical models of FLT3-mutant acute myeloid leukemia (AML)

Abstract AML accounts for 80% of acute leukemias in adults. FLT3 gene mutations are observed in approximately 30% of patients with AML and augur a poor prognosis. Despite antitumor effects of selective FLT3 inhibitors, resistance to these agents continues to pose a formidable clinical challenge. The expression of pro-survival protein BCL-2 is frequently dysregulated, conferring resistance to FLT3 inhibitors, in AML. In this study, we explored the combination of clinical stage multikinase inhibitor HQP1351 (which also targets FLT3) and BCL-2-selective inhibitor APG-2575 in preclinical models of FLT3-mutant AML. Antileukemic activity of HQP1351 was first examined using AML cell lines MV-4-11 and MOLM-13, which harbor FLT3-internal tandem duplication (FLT3/ITD) mutations. In both cell lines, HQP1351 dose-dependently induced cellular apoptosis, APG-2575 also induced apoptosis, and their combination synergistically induced this process. In particular, treatment of subcutaneous MV-4-11 xenograft tumors in nude mice with HQP1351 10 mg/kg significantly suppressed tumor growth, with a tumor growth inhibition (TGI) of 72.4%. This antitumor effect was potentiated when APG-2575 was combined with HQP1351, resulting in a TGI of 97.2%. The combination also improved survival of tumor-bearing mice with a systemic MOLM-13 model compared to either agent alone. Mechanistically, HQP1351 likely inhibited phosphorylation of FLT3 and its downstream signaling pathways, including phosphorylation of AKT, ERK1/2 and STAT5. Taken together, these findings indicate that the antileukemic activity of olverembatinib can be ascribed to on-target inhibition. Single-agent APG-2575 agent triggered cellular apoptosis, as evidenced by cleavage of caspase-3 cleavage and poly(ADP-ribose) polymerase-1 (PARP-1) activation, which are considered hallmarks of apoptosis. Consequently, APG-2575 treatment also increased expression of another antiapoptotic protein (MCL-1), upregulation of which is a pivotal mechanisms of resistance to BCL-2 inhibitors. Notably, HQP1351 substantially decreased cellular MCL-1 expression when combined with APG-2575, further enhancing apoptosis.Taken together, our data suggest that FLT3 inhibition by HQP1351 downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 to potentiate cellular apoptosis in FLT3-mutant AML. The results provide scientific rationale for clinical development of HQP1351 combined with APG-2575 in patients with FLT3-ITD-mutant AML. Citation Format: Douglas D. Fang, Hengrui Zhu, Qiuqiong Tang, Qixin Wang, Na Li, Xu Fang, Ping Min, Guangfeng Wang, Dajun Yang, Yifan Zhai. FMS-like tyrosine kinase 3 (FLT3) inhibition by olverembatinib (HQP1351) downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 in preclinical models of FLT3-mutant acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1096.

Concordance between Bone Marrow and Peripheral Blood Samples for Assessment of FLT3 Internal Tandem Duplication (ITD) Mutations: Data from Patients Screened for Participation in Quantum-R, a Global, Randomized, Open-Label, Phase 3 Study Examining the Effect of Quizartinib Monotherapy Vs Salvage Chemotherapy on Overall Survival in Patients with FLT3 - ITD - Mutated AML Who Are Refractory to or Have Relapsed

Concordance between Bone Marrow and Peripheral Blood Samples for Assessment of FLT3 Internal Tandem Duplication (ITD) Mutations: Data from Patients Screened for Participation in Quantum-R, a Global, Randomized, Open-Label, Phase 3 Study Examining the Effect of Quizartinib Monotherapy Vs Salvage Chemotherapy on Overall Survival in Patients with FLT3 - ITD - Mutated AML Who Are Refractory to or Have Relapsed

Cytokine Activation of JAK Kinases Rescues FLT3-Mutant AML from FLT3 Inhibition and Requires PIM Kinases

Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase occur in ~30% of acute myeloid leukemias (AMLs) and are associated with a poor prognosis. Activating mutations in FLT3 predominantly occur as internal tandem duplications in the juxtamembrane region (FLT3-ITD), resulting in constitutive activation of canonical signaling pathways including STAT5, RAS/MAPK, and PI3K/mTORC1. Selective, potent 2nd and 3rd generation FLT3 inhibitors have shown significant clinical activity in patients with relapsed FLT3-ITD+ AML; however, clearance of FLT3 mutant clones does not consistently occur and most patients will eventually progress after an initial response. This challenges the model of FLT3 mutant AML being oncogene addicted and suggests that redundant signaling pathways may regulate AML cell survival after FLT3 inhibition. We hypothesized that these cells are resistant to FLT3 inhibition due to cytokines within the bone marrow microenvironment, resulting in low levels of primary resistant cells that will ultimately result in relapse. We have shown that primary FLT3-ITD+ AML cells escape FLT3 inhibition in vitro in the presence of cytokines produced normally in the bone marrow and that this effect is abrogated in patient cells by co-treatment with a JAK kinase inhibitor. To understand the mechanism of this rescue, we have studied a FLT3-ITD+ AML cell line (MV4;11). GM-CSF and IL-3 in particular are able to rescue cells from apoptosis following FLT3 inhibition. Interestingly, despite reactivating the canonical signaling pathways, GM-CSF and IL-3 maintain cell survival without rescuing proliferation, consistent with the clinical scenario. Both GM-CSF and IL-3 signal through JAK kinases. Co-treatment with the JAK1/2 inhibitor, ruxolitinib, or STAT5 knockdown re-sensitized AML cells to FLT3 inhibition when grown in the presence of cytokines. Inhibition of MEK or mTORC1 was unable to fully abrogate cytokine-mediated rescue of cell survival. These data demonstrate that JAK-dependent cytokine signaling mediate rescue from FLT3 inhibition through STAT5, but is not dependent on reactivation of MAPK or mTORC1 pathways. PIM kinases are a non-canonical target downstream of FLT3-ITD and downstream of STATs in cytokine signaling. PIM kinases are transcriptionally regulated. All three PIM isoforms were downregulated upon FLT3 inhibition. Both GM-CSF and IL-3 rescued PIM1 and PIM2 expression versus IL-6, a cytokine that is unable to protect from FLT3 inhibition. Co-treatment with a novel pan-PIM kinase inhibitor (INCB053914), was sufficient to block IL-3 rescue of cell survival. Altogether, these data demonstrate that FLT3-ITD+ AML cells utilize redundant signaling pathways activated by bone marrow cytokines through a JAK-STAT5-PIM axis to escape cell death from FLT3 inhibition. Pre-clinical testing with patient-derived xenografts is planned to validate these findings in an in vivo model and assess the feasibility of these combination approaches to improve clinical responses to FLT3 inhibitor therapy. DisclosuresSung:Radius Health Inc.: Equity Ownership. Koblish:Incyte Corporation: Employment, Equity Ownership. Perl:Seattle Genetics: Other: Advisory board; Astellas: Consultancy; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Daiichi Sankyo: Consultancy; Novartis: Other: Advisory Board; Arog Pharmaceuticals: Consultancy; Pfizer: Other: Advisory Board; Asana Biosciences: Other: Scientific advisory board. Carroll:Incyte Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding.

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.

Cage Transcriptome Analysis Reveals BCL2A1 Upregulation in FLT3-ITD/D835 Dual Mutated AML Cells Harboring Complex Co-Mutations

Cage Transcriptome Analysis Reveals BCL2A1 Upregulation in FLT3-ITD/D835 Dual Mutated AML Cells Harboring Complex Co-Mutations

The Relationship between Transplant Status and Patient-Reported Outcomes in Patients with FLT3-Mutated Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML): Results from the Phase 3 Admiral Study

The Relationship between Transplant Status and Patient-Reported Outcomes in Patients with FLT3-Mutated Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML): Results from the Phase 3 Admiral Study

Isoliquiritigenin, an Orally Available Natural FLT3 Inhibitor from Licorice, Exhibits Selective Anti–Acute Myeloid Leukemia Efficacy In Vitro and In Vivo

Licorice is a medicinal herb widely used to treat inflammation-related diseases in China. Isoliquiritigenin (ISL) is an important constituent of licorice and possesses multiple bioactivities. In this study, we examined the selective anti-AML (acute myeloid leukemia) property of ISL via targeting FMS-like tyrosine kinase-3 (FLT3), a certified valid target for treating AML. In vitro, ISL potently inhibited FLT3 kinase, with an IC50 value of 115.1 ± 4.2 nM, and selectively inhibited the proliferation of FLT3-internal tandem duplication (FLT3-ITD) or FLT3-ITD/F691L mutant AML cells. Moreover, it showed very weak activity toward other tested cell lines or kinases. Western blot immunoassay revealed that ISL significantly inhibited the activation of FLT3/Erk1/2/signal transducer and activator of transcription 5 (STAT5) signal in AML cells. Meanwhile, a molecular docking study indicated that ISL could stably form aromatic interactions and hydrogen bonds within the kinase domain of FLT3. In vivo, oral administration of ISL significantly inhibited the MV4-11 flank tumor growth and prolonged survival in the bone marrow transplant model via decreasing the expression of Ki67 and inducing apoptosis. Taken together, the present study identified a novel function of ISL as a selective FLT3 inhibitor. ISL could also be a potential natural bioactive compound for treating AML with FLT3-ITD or FLT3-ITD/F691L mutations. Thus, ISL and licorice might possess potential therapeutic effects for treating AML, providing a new strategy for anti-AML.

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 CT184: Gilteritinib significantly prolongs overall survival in patients with FLT3-mutated (FLT3mut+) relapsed/refractory (R/R) acute myeloid leukemia (AML): Results from the Phase III ADMIRAL trial

Abstract Introduction: Gilteritinib is a potent/selective oral inhibitor of FMS-like tyrosine kinase 3 (FLT3). Based upon interim analysis response rates from the ADMIRAL Phase III study of gilteritinib vs salvage chemotherapy (SC) in patients (pts) with R/R FLT3mut+ AML (NCT02421939), gilteritinib became the first FLT3 inhibitor approved as single agent therapy in this population. Here we present the final results of this pivotal trial. Methods: Adults with confirmed FLT3mut+ AML (FLT3-ITD and/or FLT3-TKD D835 or I836 mutations) refractory to induction chemotherapy or in untreated first relapse were randomized (2:1) to receive continuous 28-day cycles of 120 mg/day gilteritinib or pre-randomization selected SC: low-dose cytarabine (LoDAC), azacitidine (AZA), mitoxantrone/etoposide/cytarabine (MEC), or fludarabine/cytarabine/granulocyte colony-stimulating factor/idarubicin (FLAG-IDA). Prior FLT3 inhibitor use, other than midostaurin or sorafenib, was excluded. Overall survival (OS) and the combined rate of complete remission/complete remission with partial hematologic recovery (CR/CRh) were co-primary endpoints. Secondary endpoints were event-free survival (EFS) and CR rate; safety/tolerability was also examined. Results: A total of 371 pts were randomized: 247 to gilteritinib and 124 to SC (MEC, 25.7%; FLAG-IDA, 36.7%; LoDAC, 14.7%; AZA, 22.9%). Median age was 62 years (range, 19-85). Baseline FLT3 mutations were: FLT3-ITD, 88.4%; FLT3-TKD, 8.4%; both FLT3-ITD and FLT3-TKD, 1.9%; unconfirmed, 1.3%. Overall, 39.4% of pts had refractory AML and 60.6% had relapsed AML. Patients randomized to gilteritinib had significantly longer OS (9.3 months) than SC (5.6 months; hazard ratio [HR] for death = 0.637; P=0.0007); 1-year survival rates were 37.1% and 16.7%, respectively. The CR/CRh rates for gilteritinib and SC were 34.0% and 15.3%, respectively (P=0.0001); CR rates were 21.1% and 10.5% (2-sided P=0.0106). Median EFS was 2.8 months and 0.7 months in the gilteritinib and SC arms, respectively (HR 0.793, P=0.0830). Common adverse events (AEs) in all randomized pts were febrile neutropenia (43.7%), anemia (43.4%), and pyrexia (38.6%). Common grade ≥3 AEs related to gilteritinib were anemia (19.5%), febrile neutropenia (15.4%), thrombocytopenia (12.2%), and decreased platelet count (12.2%). Adjusted for exposure duration, serious treatment-emergent AEs per patient year were less common with gilteritinib (7.1%) than SC (9.2%). Conclusions: In patients with R/R FLT3mut+ AML, the potent, selective FLT3 inhibitor gilteritinib resulted in significantly longer OS and higher response rates compared with chemotherapy and had a favorable safety profile. These results change the treatment paradigm for salvage therapy of R/R FLT3mut+ AML and establish gilteritinib as the new standard of care. Citation Format: Alexander E. Perl, Giovanni Martinelli, Jorge E. Cortes, Andreas Neubauer, Ellin Berman, Stefania Paolini, Pau Montesinos, Maria R. Baer, Richard A. Larson, Celalettin Ustun, Francesco Fabbiano, Antonio Di Stasi, Robert Stuart, Rebecca Olin, Margaret Kasner, Fabio Ciceri, Wen-Chien Chou, Nikolai Podoltsev, Christian Recher, Hisayuki Yokoyama, Naoko Hosono, Sung-Soo Yoon, Je-Hwan Lee, Timothy Pardee, Amir T. Fathi, Chaofeng Liu, Xuan Liu, Erkut Bahceci, Mark J. Levis. Gilteritinib significantly prolongs overall survival in patients with FLT3-mutated (FLT3mut+) relapsed/refractory (R/R) acute myeloid leukemia (AML): Results from the Phase III ADMIRAL trial [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 CT184.

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 the selective and efficacious inhibitors of FLT3 mutations

Fms-like tyrosine kinase 3 (FLT3) is among the most frequently mutated protein in acute myeloid leukemia (AML), which has been confirmed as an important drug target for AML chemotherapy. Starting from the lead compound LT-106-175, a series of 1-H-pyrazole-3-carboxamide derivatives were synthesized to improve the FLT3 inhibitory potency and selectivity. Among them, compound 50 was identified as a highly potent and selective FLT3 inhibitor (IC50 = 0.213 nM), which showed equal activities against various mutants of FLT3 including FLT3 (ITD)-D835V and FLT3 (ITD)-F691L that is resistant to quizartinib. Compound 50 also exhibited efficacy against the human AML cell line MV4-11 (IC50 = 16.1 nM) harboring FLT3-ITD mutants. Inversely, compound 50 displayed no cytotoxicity to FLT3-independent cells, and the biochemical analyses showed that its effects were related to the inhibition of FLT3 signal pathways. Additionally, compound 50 induced apoptosis in MV4-11 cell as demonstrated by flow cytometry. Moreover, compound 50 showed enhanced metabolic stability. Altogether, it was concluded that compound 50 could be a promising FLT3 inhibitor for further developing therapeutic remedy of AML.

A novel irreversible FLT3 inhibitor, FF-10101, shows excellent efficacy against AML cells with FLT3 mutations

AbstractAn activating mutation of Fms-like tyrosine kinase 3 (FLT3) is the most frequent genetic alteration associated with poor prognosis in acute myeloid leukemia (AML). Although many FLT3 inhibitors have been clinically developed, no first-generation inhibitors have demonstrated clinical efficacy by monotherapy, due to poor pharmacokinetics or unfavorable safety profiles possibly associated with low selectivity against FLT3 kinase. Recently, a selective FLT3 inhibitor, quizartinib, demonstrated favorable outcomes in clinical studies. However, several resistant mutations emerged during the disease progression. To overcome these problems, we developed a novel FLT3 inhibitor, FF-10101, designed to possess selective and irreversible FLT3 inhibition. The co-crystal structure of FLT3 protein bound to FF-10101 revealed the formation of a covalent bond between FF-10101 and the cysteine residue at 695 of FLT3. The unique binding brought high selectivity and inhibitory activity against FLT3 kinase. FF-10101 showed potent growth inhibitory effects on human AML cell lines harboring FLT3 internal tandem duplication (FLT3-ITD), MOLM-13, MOLM-14, and MV4-11, and all tested types of mutant FLT3-expressing 32D cells including quizartinib-resistant mutations at D835, Y842, and F691 residues in the FLT3 kinase domain. In mouse subcutaneous implantation models, orally administered FF-10101 showed significant growth inhibitory effect on FLT3-ITD-D835Y- and FLT3-ITD-F691L–expressing 32D cells. Furthermore, FF-10101 potently inhibited growth of primary AML cells harboring either FLT3-ITD or FLT3-D835 mutation in vitro and in vivo. These results indicate that FF-10101 is a promising agent for the treatment of patients with AML with FLT3 mutations, including the activation loop mutations clinically identified as quizartinib-resistant mutations.

A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220.

Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD-positive and -negative AML patients and as maintenance therapy. To understand the mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR-pooled library to screen new genes whose loss of function confers resistance to AC220. We identified SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated reactivation of downstream FGF/Ras/ERK and Wnt signaling as major mechanisms of resistance to AC220. We confirmed these findings in primary AML patient samples. Expression of SPRY3 and GSK3A was dramatically reduced in AC220-resistant AML samples, and SPRY3-deleted primary AML cells were resistant to AC220. Intriguingly, expression of SPRY3 was greatly reduced in GSK3 knockout AML cells, which positioned SPRY3 downstream of GSK3 in the resistance pathway. Taken together, our study identified novel genes whose loss of function conferred resistance to a selective FLT3 inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in AML. Cancer Res; 77(16); 4402-13. ©2017 AACR.

Design, synthesis and biological evaluation of indolin-2-one-based derivatives as potent, selective and efficacious inhibitors of FMS-like tyrosine kinase3 (FLT3)

Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in approximately one third of acute myeloid leukemia (AML) patients, which has been proposed as a promising drug target for AML therapy. A series of indolin-2-one derivatives bearing different groups at the solvent interface position based on sunitinib as FLT3 inhibitors were designed, synthesized and evaluated in FLT3-dependent human AML cell line MV4-11. Structure-activity relationship (SAR)analysis showed that heterocyclic alkane at the solvent interface position could significantly increase the potency for the inhibition of proliferation of MV4-11 cell line. Compound 10a and 10d exhibited better efficacy (MV4-11, IC50: 14.7 nM for 10a and 24.8 nM for 10d) than positive control sunitinib (MV4-11, IC50: 38.5 nM). The kinase and cellular inhibition assay exhibited that 10d (FLT3, IC50: 5.3 nM) was a potent and selective FLT3 inhibitor. Furthermore, the pharmacokinetic experiments showed that 10d had good properties of oral bioavailability, Cmax, Tmax, T1/2 and AUC in mice, respectively. The in vivo study indicated that 10d could significantly suppress tumor growth in MV4-11 xenografts nude mice model and occupied with a commendable therapeutic window compared to sunitinib.

Discovery of 3-phenyl-1H-5-pyrazolylamine derivatives containing a urea pharmacophore as potent and efficacious inhibitors of FMS-like tyrosine kinase-3 (FLT3)

Preclinical investigations and early clinical trials suggest that FLT3 inhibitors are a viable therapy for acute myeloid leukemia. However, early clinical data have been underwhelming due to incomplete inhibition of FLT3. We have developed 3-phenyl-1H-5-pyrazolylamine as an efficient template for kinase inhibitors. Structure–activity relationships led to the discovery of sulfonamide, carbamate and urea series of FLT3 inhibitors. Previous studies showed that the sulfonamide 4 and carbamate 5 series were potent and selective FLT3 inhibitors with good in vivo efficacy. Herein, we describe the urea series, which we found to be potent inhibitors of FLT3 and VEGFR2. Some inhibited growth of FLT3-mutated MOLM-13 cells more strongly than the FLT3 inhibitors sorafenib (2) and ABT-869 (3). In preliminary in vivo toxicity studies of the four most active compounds, 10f was found to be the least toxic. A further in vivo efficacy study demonstrated that 10f achieved complete tumor regression in a higher proportion of MOLM-13 xenograft mice than 4 and 5 (70% vs 10% and 40%). These results show that compound 10f possesses improved pharmacologic and selectivity profiles and could be more effective than previously disclosed FLT3 inhibitors in the treatment of acute myeloid leukemia.

506 p53 Activation of Mesenchymal Stromal Cells (MSC) Partially Abrogates Microenvironment-Mediated Resistance to FLT3 Inhibition in FLT3/ITD AML

Abstract 2151 Fms-like tyrosine kinase-3 (FLT3) inhibitors have recently been introduced to overcome the dismal prognosis of acute myeloid leukemia (AML) with FLT3/ITD mutations. However, while ciculating blasts are rapidly eliminated, bone marrow (BM) responses are in general less impressive (Zhang et al., JCI 2009). One potential explanation for the reduced bone marrow response compared to the striking activity against circulating blast cells may be microenvironmental resistance to FLT3 inhibitors, including protection of FLT3/ITD+ blasts through the SDF-1 (CXCL12)/CXCR4 axis (Zeng et al., BLOOD 2009) and novel strategies to overcome microenvironmental resistance may further enhance the clinical benefit of FLT3 inhibitors. Here we investigated the role of p53 in bone marrow stromal cells in stromal cell-mediated resistance to FLT3 inhibition in FLT3/ITD AML. We confirmed that Mdm2-p53 interaction and mutant FLT3 signaling in leukemic blasts were inhibited by the Mdm2 inhibitor Nutlin-3a and the selective FLT3 inhibitor FI-700, respectively, as previously reported (Kojima et al., BLOOD 2004; Kojima et al., LEUKEMIA 2009). BM samples were obtained from normal individuals or from FLT3/ITD AML patients with more than 70% leukemia cells after informed consent. BM-derived stromal cells were seeded in 12-well plates in MEM-alpha medium and exposed to Nutlin-3a for 24 hours, gamma-irradiated (2 Gy), or treated with 100 nM doxorubicin. The wells were then washed three times and AML cells were added. Cell cycle distribution was determined by propidium iodide staining and apoptosis by Annexin V binding. In FLT3/ITD AML cell lines and primary cells, apoptosis was induced by FI-700, but apoptosis induction was diminished under stromal coculture conditions (43.2 ± 0.5% versus 17.0 ± 1.2% in MOLM-13; p Disclosures: No relevant conflicts of interest to declare.

Selective FLT3 inhibitor FI-700 neutralizes Mcl-1 and enhances p53-mediated apoptosis in AML cells with activating mutations of FLT3 through Mcl-1/Noxa axis.

Treatment using Fms-like tyrosine kinase-3 (FLT3) inhibitors is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. We found that FI-700 immediately reduced antiapoptotic Mcl-1 levels and enhanced Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/internal tandem duplication cells through the Mcl-1/Noxa axis. FI-700 induced proteasome-mediated degradation of Mcl-1, resulting in the reduced ability of Mcl-1 to sequester proapoptotic Bim. Nutlin-3 induced Noxa, which displaced Bim from Mcl-1. The FI-700/Nutlin-3 combination profoundly activated Bax and induced apoptosis. Our findings suggest that FI-700 actively enhances p53 signaling toward mitochondrial apoptosis and that a combination strategy aimed at inhibiting FLT3 and activating p53 signaling could potentially be effective in AML.

Oncogenic CBL Mutants Have Transforming Potential in Cells Expressing the FLT3 Tyrosine Kinase.

CBL is a E3-ubiquitin ligase, that negatively regulates many receptor tyrosine kinases (RTKs). Translocations (t(4;11); t(11;14) involving CBL in humans and two oncogenic CBL deletion mutants, CBL-70Z and v-CBL, inducing B-cell lymphomas in mice, were described. The RTK Fms-like tyrosine kinase 3 (FLT3) is expressed by the leukemic cells of 70–90% of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) and is highly implicated in the malignant transformation by harbouring activating mutations, aberrant autocrine stimulation or overexpression.We hypothesized that CBL could play a role in regulating FLT3 activity and if mutated, could cause aberrant FLT3 signaling in hematopoietic cells. To analyze if CBL mutants could lead to transformation of FLT3-expressing cells we stably transduced Ba/F3 cells with CBL-WT, CBL-70Z or v-CBL alone or together with FLT3-WT. Coexpression of FLT3-WT and a CBL mutant, CBL-70Z or v-CBL, but not CBL-WT or expression of a CBL-construct alone, conferred IL-3 independent growth to Ba/F3 cells. In presence of FLT3 ligand FLT3-WT/CBLmutant-expressing cells showed hyperproliferation. To determine whether the proliferative advantage of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells is mediated by FLT3 we cultivated the cells in presence of selective FLT3 inhibitors, SU5614 and PKC412. Both inhibitors were able to abrogate the IL-3 independent and FL-stimulated growth of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells. The FLT3-WT receptors and the downstream signaling pathway, STAT5, were constitutively active in FLT3-WT/CBLmutant-expressing cells in contrast to control cells. To clarify the mechanism of transformation we performed internalization assays. The rate and speed of internalization of the FLT3-WT receptors were not altered in FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells compared to FLT3-WT cells. However, we could show that CBL-WT promotes FLT3 degradation. To investigate if CBL mutations play a role in acute leukemias we screened the cDNA of 62 AML and 6 ALL cell lines and found a CBL deletion mutant in MOLM-13 and MOLM-14 cells lacking exon 8, which comprises parts of the linker and RING finger domain. Hence, this mutation targets the same functionally important regions of the CBL protein than the CBL-70Z deletion.In summary, CBL-70Z and v-CBL, but not CBL-WT, confer a transforming potential to cells expressing FLT3. The pro-proliferative phenotype of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells is mediated by an increase in FLT3 tyrosine kinase activity. The CBL-70Z and v-CBL mutations were hypothesized to act in a dominant negative manner abrogating the negativ-regulatory function of CBL-WT. We identified a CBL deletion mutant in the MOLM-13 and MOLM-14 cells, that is reminiscent of CBL-70Z, indicating that CBL mutations might play a role in the pathogenesis of acute leukemias.

FLT3/ITD Expression Increases Expansion, Survival and Entry into Cell Cycle of Human Hematopoietic Stem Cells.

FMS-like tyrosine kinase-3 (FLT3) is a Class III receptor tyrosine kinase that is important for normal hematopoiesis. Activating mutations of FLT3 by internal tandem duplications (ITDs) in the juxtamembrane region are the most common molecular aberrations found in acute myeloid leukemia (AML). The contributions of FLT3 activating mutations in the leukemic transformation of normal human hematopoietic stem/progenitor cells (HSCs) have not yet been fully elucidated. In this study, using a single lentiviral vector containing two promoters, we achieved consistent and efficient coexpression of FLT3/ITD and green fluorescent protein (GFP) in transduced human CD34+ HSCs. When cultured in medium containing SCF, TPO and FLT3 ligand (FL), FLT3/ITD-transduced cells survived with enhanced self-renewal and survival potential, which was not affected by the withdrawal of FL. These cells retained a surface immunophenotype typical of HSCs (CD34+CD38−). Compared to cells transduced with a vector expressing GFP alone, FLT3/ITD-transduced HSCs had a higher fraction of cells in cell cycle. Clonogenic assays showed that FLT3/ITD-transduced HSCs produced fewer CFU-GM, implying that they were at least partially blocked in their ability to differentiate along the myeloid lineage. FLT3/ITD-transduced HSCs were more sensitive to the induction of cytotoxicity by CEP-701, a selective FLT3 inhibitor. In the FLT3/ITD-transduced HSCs, we detected increased expression of Pim-1, a serine/threonine kinase with an important role in cell survival, proliferation and differentiation, c-myc, a transcription factor involved in cell proliferation and cell cycle regulation, and Cyclin D3, a key factor in cell cycle regulation, each of which may contribute to the altered genetic program instituted by FLT3/ITD signaling.These results together indicate that FLT3/ITD mutations may contribute to leukemic transformation of normal HSCs by prolonging survival, promoting proliferation, and blocking differentiation. CEP-701 may act as a potent agent for AML stem cells harboring FLT3/ITD mutations.