FMS-like Tyrosine Kinase 3 Mutation Status Research Articles

The multi-kinase inhibitor CG-806 exerts anti-cancer activity against acute myeloid leukemia by co-targeting FLT3, BTK, and aurora kinases

Despite the development of several Fms-like tyrosine kinase 3 (FLT3) inhibitors that have improved outcomes in patients with FLT3-mutant acute myeloid leukemia (AML), drug resistance is frequently observed, which may be associated with the activation of additional pro-survival pathways, such as those regulated by BTK, aurora kinases (AuroK), and potentially others, in addition to acquired tyrosine kinase domain (TKD) mutations of FLT3 gene. FLT3 may not always be a driver mutation. We evaluated the anti-leukemia efficacy of the novel multi-kinase inhibitor CG-806, which targets FLT3 and other kinases, to circumvent drug resistance and target FLT3 wild-type (WT) cells. The anti-leukemia activity of CG-806 was investigated by measuring apoptosis induction and analyzing the cell cycle using flow cytometry in vitro. CG-806 demonstrated superior anti-leukemia efficacy compared to commercially available FLT3 inhibitors, both in vitro and in vivo, regardless of FLT3 mutational status. The mechanism of action of CG-806 may involve its broad inhibitory profile against FLT3, BTK, and AuroK. In FLT3 mutant cells, CG-806 induced G1 phase blockage, whereas in FLT3 WT cells, it resulted in G2/M phase arrest. Targeting FLT3 and Bcl-2 and/or Mcl-1 simultaneously results in a synergistic pro-apoptotic effect in FLT3 mutant leukemia cells. The results of this study suggest that CG-806 is a promising multi-kinase inhibitor with anti-leukemic efficacy regardless of FLT3 mutational status. A phase 1 clinical trial of CG-806 for the treatment of AML has been initiated (NCT04477291). Key points The multi-kinase inhibitor CG-806 exerts superior anti-leukemic activity in AML, regardless of its FLT3 status. CG-806 triggered G1 arrest in FLT3 mutated cells and G2/M arrest in FLT3 WT cells through the suppression of FLT3/BTK and aurora kinases. Concomitantly targeting FLT3 and Bcl-2 and/or Mcl-1 exerted synergistic pro-apoptotic effects on both FLT3 WT and mutated AML cells.

Decoding the anti-cancer potential of Pexidartinib (PLX3397), a Fms-like tyrosine kinase 3 inhibitor, using next-generation knowledge discovery methods.

Acute Myeloid Leukemia (AML) is a complex hematologic malignancy characterized by the rapid proliferation of abnormal myeloid precursor cells. The FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase, plays a pivotal role in regulating cell survival, proliferation, and differentiation within the hematopoietic system. Mutations in FLT3, particularly internal tandem duplications (ITDs) and point mutations within the tyrosine kinase domain (TKD), are prevalent in AML and are associated with poor prognosis and increased risk of relapse. The development of targeted therapies has revolutionized the landscape of cancer treatment by focusing on the inhibition of kinase signalling. Small-molecule inhibitors designed to selectively target receptor tyrosine kinases, such as PLX3397, have shown promising results in preclinical studies and early phase clinical trials. PLX3397 exerts its inhibitory effects by targeting CSF1R and KIT, leading to the disruption of receptor tyrosine kinase signalling cascades, suppression of leukemic cell growth, and induction of apoptosis. This study emphasizes the significance of FLT3 as a receptor tyrosine kinase as a therapeutic target for PLX3397. After evaluating the usefulness of PLX3397 as an enzyme inhibitor using ADMET prediction, PLX3397 was prepared for molecular docking in the FLT3 crystal structure (PDB: 4XUF). A molecular dynamics simulation was performed on PLX3397 to evaluate its binding affinity and protein stability in a simulated physiological environment. In conclusion, targeting FLT3 as a receptor tyrosine kinase with PLX3397 represents a promising therapeutic strategy for improving outcomes in patients with FLT3-mutated AML. Further clinical investigations are warranted to validate the efficacy and safety of PLX3397 and to optimize treatment strategies for AML patients based on the FLT3 mutational status.

The Clinical Utility of FLT3 Mutation Testing in Acute Leukemia: A Canadian Consensus.

FMS-like tyrosine kinase 3 (FLT3) mutations are detected in approximately 20-30% of patients with acute myeloid leukemia (AML), with the presence of a FLT3 internal tandem duplication (FLT3-ITD) mutation being associated with an inferior outcome. Assessment of FLT3 mutational status is now essential to define optimal upfront treatment in both newly diagnosed and relapsed AML, to support post-induction allogeneic hematopoietic stem cell transplantation (alloSCT) decision-making, and to evaluate treatment response via measurable (minimal) residual disease (MRD) evaluation. In view of its importance in AML diagnosis and management, the Canadian Leukemia Study Group/Groupe canadien d'étude sur la leucémie (CLSG/GCEL) undertook the development of a consensus statement on the clinical utility of FLT3 mutation testing, as members reported considerable inter-center variability across Canada with respect to testing availability and timing of use, methodology, and interpretation. The CLSG/GCEL panel identified key clinical and hematopathological questions, including: (1) which patients should be tested for FLT3 mutations, and when?; (2) which is the preferred method for FLT3 mutation testing?; (3) what is the clinical relevance of FLT3-ITD size, insertion site, and number of distinct FLT3-ITDs?; (4) is there a role for FLT3 analysis in MRD assessment?; (5) what is the clinical relevance of the FLT3-ITD allelic burden?; and (6) how should results of FLT3 mutation testing be reported? The panel followed an evidence-based approach, taken together with Canadian clinical and laboratory experience and expertise, to create a consensus document to facilitate a more uniform approach to AML diagnosis and treatment across Canada.

Development of a Fast and Quantitative FLT3-TKD Mutation Prototype Using Lab in a Cartridge™ Technology

Introduction Acute myeloid leukemia (AML) is a heterogenous hematologic malignancy characterized by the rapid proliferation of abnormal myeloid cells in the bone marrow and blood. Fast and reliable diagnostic tests are imperative for the advancement of targeted therapies in the treatment of AML. FLT3 (FMS-like tyrosine kinase3) is one of the most frequent genetic alterations in AML and is present in approximately one-third of newly diagnosed patients. Considering the targeted therapies, such as midostaurin and gilteritinib available for FLT3 mutated AML, accurate and timely diagnosis of FLT3 mutations is vital for guiding treatment decisions and improving patient outcomes. The current diagnostic methods to detect FLT3-TKD mutations require a series of complex steps including blood sample handling, plate-based PCR, enzyme digestion and capillary electrophoresis or sequencing which can be time-consuming and labor-intensive. We have developed and evaluated the performance of a FLT3-TKD prototype using Cepheid's automated cartridge technology that demonstrates significantly less hands-on time and faster turnaround. Objectives We have developed a novel, fast and simplified qPCR-based FLT3-TKD prototype using Cepheid's GeneXpert® automated platform for detecting 14 mutations, including 7 mutations present in codon D835 and another 7 mutations in I836. The FLT3-TKD prototype is an easy-to-use sample in/result out assay with minimal hands-on time and time to results in less than 3 hours, and without the use of restriction enzymes or capillary electrophoresis. This assay is designed for quantitative detection of FLT3-TKD mutated DNA (D835 & I836) using ABL1 as an endogenous control in peripheral blood (PB) of AML patients. This assay utilizes the special chemistry, special design primers, and an optimized wild-type (WT) blocker to specifically detect SNPs and indels. With the combination of WT blocker and unique design of special design primers we were able to selectively amplify the rare mutations in the presence of abundant closely related WT fragments. Results Using this prototype, we were able to detect and quantify all 14 mutations (7 of D835 and 7 of I836) with an estimated LoD of AR 0.02 (Ratio of TKD mutation copies/ABL copies in PB background) in PB samples spiked-in with positive templates. ABL1 serves as a reference gene to normalize the WT DNA present in the background. Plasmid DNA with individual mutations was generated and spiked into the PB to assess the dynamic range of the test. We observed good alignment of dCt-AR (deltaCt-Allelic ratio) regression curve among all 14 mutations within the dynamic range tested (0.02-0.33). With pre-defined thresholds, our assay specificity has been above 95% among 20 screened EDTA donor blood samples. To further assess the sensitivity of this prototype, we also evaluated a CRISPR-CAS engineered cell line for the D835 G>A mutation. Using this cell line, we were successful in detecting the mutation down to AR of 0.008 in a PB background. For quantification analysis we prepared a linear panel of the most prevalent mutation D835(G>T) in the PB background and were able to quantify all mutations within the range of AR 0.008-to 0.83. Conclusions Here we describe the development and evaluation of an automated cartridge-based FLT3-TKD prototype that can detect 14 FLT3-TKD mutations. The cartridge prototype is preloaded with sample processing reagents and qPCR reagents with a turn-around time of less than 3 hours including offboard peripheral blood processing. When tested with contrived samples generated with either plasmid DNA or engineered cell lines spiked into blood, all 14 tested FLT3-TKD mutations were detected within a dynamic range of 0.02 to 0.33 AR with a specificity of > 95% in healthy donor whole blood background. With optimized primer and WT blocker design this prototype showed high sensitivity & specificity. We tested a broader dynamic range for the most prevalent mutation, D835 G>T, and were able to detect from AR of 0.008 to 0.83 AR. Additionally, since the prototype uses the GeneXpert® platform and current universal lysate sample prep procedure, the sample can potentially be used across all Cepheid Hematology Oncology products. This prototype significantly reduces the hands-on time with future potential to provide clinicians with fast and accurate detection of FLT3 mutation status that can aid prognostic evaluation and treatment in AML patients.

Akut Miyeloid Lösemide FLT3 Mutasyonlarının Araştırılması: Hasta Sonuçları ve Tedavi Stratejileri Üzerine Tek Merkezli Gerçek Yaşam Verisi

Acute Myeloid Leukemia (AML) is a complex hematological malignancy with considerable genetic heterogeneity. Fms-like tyrosine kinase 3 (FLT3) mutations are associated with poor prognosis and occur in nearly 30% of AML cases. This study delves into the prevalence of FLT3 mutations, their impact on clinical outcomes, and the efficacy of various treatment approaches in a cohort of AML patients. We examined 157 de novo non-acute promyelocytic leukemia AML patients aged 20-95 years, screening for FLT3-ITD and FLT3-TKD mutations. We tailored chemotherapy based on age, ECOG performance status, and FLT3 mutation presence. Our research revealed that 27.3% of patients harbored FLT3 mutations, with 65% FLT3-ITD and 35% FLT3-TKD mutations. Those with FLT3 mutations exhibited higher mortality rates compared to patients without mutations. Age, FLT3 mutation status, and relapsed/refractory disease emerged as independent risk factors for mortality. Patients treated with midostaurin faced a lower mortality risk than those administered sorafenib. This study underscores the significance of FLT3 mutations in AML, their influence on clinical outcomes, and the advantages of targeted therapies. Our findings stress the urgency for further investigation aimed at enhancing the prognosis for AML patients with FLT3 mutations.

An Ex-Vivo Combination Drug Sensitivity Platform for Predicting Sensitivity to FLT3 Inhibitor-Based Combination in Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is a clonally heterogenous group of disorders with many treatment options. Despite the increase in available options including targeted therapy, challenges remain in identifying patients who may respond to a given regimen. The ability to accurately predict responses to treatment is crucial given the varying response rates to standard treatments, adverse effects and increasing drug cost. In this study, we explore the ability of an ex vivo combination drug sensitivity platform, quadratic phenotypic optimization platform (QPOP), in identifying potential responders and non-responders to possible combination therapies in FMS-like tyrosine kinase-3 (FLT3) mutant AML. We further investigate the mechanism behind selective sensitivity and resistance to FLT3 inhibitor-based combinations. Methods FLT3-ITD mutant or WT AML cell lines were treated with a set panel of drugs recommended by physicians including FLT3 inhibitors (FLT3i), midostaurin and gilteritinib. QPOP drug combination scores and ranking were derived from analysis of cell viability response in cancer cells following treatment with a series of test combinations derived from an orthogonal array composite design. Molecular analysis was conducted to determine the accuracy of QPOP predicted response and FLT3 mutant status. Subsequently, we performed a prospective cohort pilot study of QPOP analysis in AML. AML patients from the National University Hospital,Singapore, with disease amenable to blood or marrow aspiration, were recruited between 26th November 2019 and 15th July 2022 with a median follow up of 4 months. CD33 or CD34 selection was performed to enrich for malignant blasts cells, depending on the sample. Patients were treated with current standard of care treatment options as guided by clinician opinion. The primary outcomes were to determine the ability of QPOP to predict sensitivity to FLT3i-based therapies. Results QPOP analysis on 8 AML cell lines was accurately able to stratify FLT3i responders and non-responders including differential responses between an FLT3-ITD isogenic parental and FLT3i resistant line. We recruited 25 patients (2 patients having repeat QPOP evaluations post treatment) with a median age of 59 years and 2 prior lines of treatment. QPOP responses were evaluable for 18 independent treatments in 15 patients (n=3 FLT3 ITD or TKD (20%), n=12 FLT3 WT (80%)). The median turn-around time for QPOP analysis was 8 (±2.1) days. Patients with either FLT3i among the top 5 ranking 2 or 3 drug combinations were suggested to be sensitive to the use of FLT3i. QPOP analysis of FLT3 mutant patients had predicted 2 patients with the FLT3-ITD mutation to be responsive to a 2-drug combination of midostaurin-venetoclax. The third FLT3-mutant patient with a TKD point mutation was predicted to be respond to a combination of gilteritinib-dasatinib. While FLT3 WT patients did not predict sensitivity to either FLT3 inhibitor as single agents or 2-drug combinations, 4/12 (33.3%) of the WT patients also showed sensitivity to the use of gilteritinib in3-drug combination rankings. This indicates that gilteritinib may be useful for FLT3 WT patients when used in the appropriate combination therapy. Furthermore, QPOP was also able to accurately identify increased sensitivity to FLT3i-based combinations in accordance with a gain of FLT3 ITD mutation. Serial QPOP analysis in subsequent samples following midostaurin-based therapy indicated a decrease in FLT3i sensitivity that was concordant with increasing FLT3 allelic burden and development of resistance. The development of resistance to FLT3i-based combinations was correlated to an increase in phosphorylated AKT expression in both AML cell lines and the patient who developed FLT3 resistance. Conclusion QPOP analysis predicts sensitivity to drug-combinations for AML in a clinically applicable timeframe. Here we show that QPOP can be used as a tool to identify sensitivity to FLT3i-based combination therapy without prior knowledge of FLT3 mutation status. AML cell lines and patient samples can be stratified according to FLT3 inhibitor response sensitivity which parallels their mutational background and identify potential drug resistance. Further clinical study of the safety and efficacy of QPOP-derived FLT3i-based combinations as well as inquiry into the potential mechanisms of acquired resistance to these combinations will be necessary. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Potent Small Molecule Inhibitor of FLT3, PHI-101 Overcomes Resistance in Acute Myeloid Leukemia: Efficacy and PK/PD Profile in Phase 1 First in Human Study

Background: Mutations in FMS-like tyrosine kinase 3 (FLT3) are found in approximately 25-35% of acute myeloid leukemia (AML) patients and are often associated with relapse and poor prognosis. PHI-101 is an orally active, potent small molecule Type I FLT3 tyrosine kinase inhibitor (TKI) developed to overcome resistance in AML. It selectively inhibits both FLT3-ITD mutations and a wide variety of FLT3 point mutations in TKD in preclinical AML models. PHI-101 showed increased anti-leukemic activity compared to gilteritinib in both in vitro and in vivo studies with AML patient-derived primary leukemic cells. Phase 1 global clinical trials for PHI-101 (NCT04842370) against refractory and relapsed (R/R) AML are currently ongoing to assess the overall safety and efficacy. Study Design and Methods: Eligible subjects, regardless of FLT3 mutation status, were enrolled at each dose level in phase 1a to evaluate dose-limiting toxicities (DLTs) during the 28-day daily dosing period according to an accelerated 3+3 design. A dose escalation to the next dose level proceeds upon the recommendation of the safety monitoring committee. Blood samples from patients were collected to evaluate the concentration of PHI-101 in plasma during the 24-h observation period after the initial dose (cycle1, day1) and after multiple doses (cycle1, day15). A pharmacodynamic evaluation of serial plasma samples via a plasma inhibitory assay (PIA) was used to assess the FLT3-inhibitory activity of PHI-101 in FLT3-mutant cell lines. The variant allele frequency (VAF) analysis for FLT3-ITD, FLT3-TKD and other AML-related mutant in the population was performed using next-generation sequencing (NGS) technology in accordance with instructions for HEMEaccuTestTM IVD kit, with bone marrow aspirate samples collected from the patients before and after PHI-101 treatment. Results: To date, 10 enrolled patients with R/R AML have been evaluated for safety assessment at four dose levels in the dose-escalation phase 1a clinical trial of PHI-101, and they have not experienced any DLTs from daily doses of the 28-day cycle. Seventy percent of enrolled patients had more than 3 prior anti-leukemic treatment attempts, and four patients had relapsed or refractory disease after treatment with other FLT3 inhibitors including gilteritinib, quizartinib, or HM43239. The median percentage of blasts at the time of enrollment was 71%, and the median duration on study with PHI-101 was 83 days. After 1 cycle of PHI-101 treatment, bone marrow blasts were reduced by up to 98%, and the responses extended to patients who had relapsed after treatment with other FLT3 targeted therapeutics. Clinical benefits, including improvement of hematological parameters, were observed in five patients who harbored FLT3 mutations. PHI-101 exhibits a dose-proportional pharmacokinetic profile in patients. The median maximum concentration was reached 4-6h following dosing of PHI-101, and the accumulated concentration of PHI-101 in plasma on day 15 of treatment was 3-5 times higher than on day 1. Estimated T1/2 and Tmax did not appear to be dose dependent. The concomitant plasma inhibitory assay (PIA) and PK data demonstrated that exposure-related inhibition of FLT3 phosphorylation of at least 90% by plasma PHI-101 was seen within 6 hours after a single dose as long as the plasma concentration of PHI-101 reached at least 70 ng/mL. The patients with FLT3-ITD or TKD mutations showed clinical reductions of FLT3 mutation VAF of up to 77% and leukemic blast reductions of up to 87% during the same period. Notably, PHI-101 treatment significantly cleared the FLT3-N676K mutation by 96% in addition to FLT3-D835Y mutations. Conclusion: A dose-escalating phase 1a clinical data of PHI-101, which reflects up to cohort 4 of the study, indicates that PHI-101 generated potent FLT3 inhibition leading to encouraging anti-leukemic responses in R/R AML patients, including in those with prior FLT3 TKI therapeutic failure. PHI-101 showed good tolerance and favorable safety profile and reduced leukemic blasts significantly with 28-day dosing. PHI-101 sustained its activity to clear FLT3-ITD and/or FLT3-TKD mutations including D835Y or N676K identified in AML patients. Oral PHI-101 treatment results in a dose-dependent PK profile and clinically relevant efficacy of FLT3 phosphorylation inhibition. This trial indicates that PHI-101 is a next generation of FLT3 inhibitor effective for R/R AML patients.

Concomitant targeting of FLT3 and BTK overcomes FLT3 inhibitor resistance in acute myeloid leukemia through the inhibition of autophagy.

Strategies to overcome resistance to FMS-like tyrosine kinase 3 (FLT3)-targeted therapy in acute myeloid leukemia (AML) are urgently needed. We identified autophagy as one of the resistance mechanisms, induced by hypoxia and the bone marrow microenvironment via activation of Bruton tyrosine kinase (BTK). Suppressing autophagy/BTK sensitized FLT3- mutated AML to FLT3 inhibitor-induced apoptosis. Furthermore, co-targeting FLT3/BTK/aurora kinases with a novel multikinase inhibitor CG-806 (luxeptinib) induced profound apoptosis in FLT3-mutated AML by co-suppressing FLT3/BTK, antagonizing autophagy, and causing leukemia cell death in FLT3-wildtype AML by aurora kinase-mediated G2/M arrest and polyploidy, in addition to FLT3 inhibition. Thus, CG-806 exerted profound anti-leukemia activity against AML regardless of FLT3 mutation status. CG-806 also significantly reduced AML burden and extended survival in an in vivo patient-derived xenograft leukemia murine model of FLT3 inhibitor-resistant FLT3-ITD/TKD double-mutant primary AML. Taken together, these findings indicate that CG-806 has a unique mechanistic action and pre-clinical activity, which is presently undergoing clinical evaluation in both FLT3 wildtype and mutant AML.

PB1834: CLEVO: A NON-INTERVENTIONAL STUDY TO INVESTIGATE CLONAL EVOLUTION OF FMS-LIKE TYROSINE KINASE 3 (FLT3) GENE MUTATIONS DURING DISEASE PROGRESSION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

Background: Acute myeloid leukemia (AML) is clinically heterogeneous, with differing response to treatment and patient survival reflecting the diverse cytogenetic abnormalities and genetic mutations that give rise to the disease. Clonal evolution means that genetic characteristics of the leukemic cells and the proportions of individual cell clones can change because of disease progression and selection by therapy. Fms-like tyrosine kinase 3 (FLT3) gene mutations are found in approximately 30% of the general AML population and are often secondary events associated with leukemic transformation. These mutations are known to either develop or become undetectable during the disease course, and the mutant-to-wild-type allelic ratio also typically changes. This clonal evolution is not well understood but may have clinical consequences. Aims: The aim of this study is to characterize FLT3 mutations at AML diagnosis and chart the clonal evolution of FLT3 mutations (gain or loss) through treatment. Methods: This non-interventional, descriptive study will prospectively enroll approximately 650 adults (aged ≥18 years) with AML diagnosed within 30 days of enrollment at oncology or oncology/hematology specialist sites in Belgium, France, Germany, Israel, Italy, Spain, the UK and the USA, with follow-up for 36 months. Patients will be excluded if they have relapsed or refractory AML at enrollment, acute promyelocytic leukemia, or clinically active central nervous system leukemia. Patients will undergo treatment according to local standard of care and treatment guidelines, at the treating physician’s discretion. Patients will undergo routine FLT3 genetic testing at diagnosis and at relapse/refractory disease, and all AML treatments (including hematopoietic stem cell transplantation) received will be recorded. The primary objective is to determine the proportion of relapsed/refractory AML patients with FLT3 clonal evolution (gain/loss of FLT3 mutations) from diagnosis. Secondary objectives include a description of the: frequency, allelic ratio, length and location of FLT3-internal tandem duplication mutations and frequency of other FLT3 mutations at diagnosis and at relapse/refractory disease; frequency of FLT3 clonal evolution of FLT3 mutations at relapse/refractory disease by mutation type and treatment(s) received; patient survival (overall 3-year survival rate, disease-free survival and event-free survival) in relation to FLT3 mutation status at diagnosis, FLT3 clonal evolution and treatment received; frequency of composite complete remission in relation to FLT3 mutation status at diagnosis, FLT3 clonal evolution (relapsed patients only) and treatment received; and frequency and characteristics of other AML-associated genetic mutations at diagnosis and relapse/refractory disease. Interim analyses are planned for ~1 and ~3 years following study initiation to provide an estimate of FLT3 mutation frequencies in the study population, and potentially an early estimate of the rate of clonal evolution. The first patient was enrolled in September 2020. Results: The trial is in progress. Summary/Conclusion: The information generated by this study will help to better understand the clonal evolution of FLT3 throughout the course of the disease for AML patients.

The Combined Treatment With the FLT3-Inhibitor AC220 and the Complex I Inhibitor IACS-010759 Synergistically Depletes Wt- and FLT3-Mutated Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a high mortality rate and relapse risk. Although progress on the genetic and molecular understanding of this disease has been made, the standard of care has changed minimally for the past 40 years and the five-year survival rate remains poor, warranting new treatment strategies. Here, we applied a two-step screening platform consisting of a primary cell viability screening and a secondary metabolomics-based phenotypic screening to find synergistic drug combinations to treat AML. A novel synergy between the oxidative phosphorylation inhibitor IACS-010759 and the FMS-like tyrosine kinase 3 (FLT3) inhibitor AC220 (quizartinib) was discovered in AML and then validated by ATP bioluminescence and apoptosis assays. In-depth stable isotope tracer metabolic flux analysis revealed that IACS-010759 and AC220 synergistically reduced glucose and glutamine enrichment in glycolysis and the TCA cycle, leading to impaired energy production and de novo nucleotide biosynthesis. In summary, we identified a novel drug combination, AC220 and IACS-010759, which synergistically inhibits cell growth in AML cells due to a major disruption of cell metabolism, regardless of FLT3 mutation status.

Evaluation of the Impact of Minimal Residual Disease, FLT3 Allelic Ratio, and FLT3 Mutation Status on Overall Survival in FLT3 Mutation-Positive Patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) in the Chrysalis Phase 1/2 Study

Evaluation of the Impact of Minimal Residual Disease, FLT3 Allelic Ratio, and FLT3 Mutation Status on Overall Survival in FLT3 Mutation-Positive Patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) in the Chrysalis Phase 1/2 Study

A Phase (ph) 1b/2 Study of TAK-659, an Investigational Dual FLT-3 and SYK Inhibitor, in Patients (Pts) with Relapsed or Refractory Acute Myelogenous Leukemia (R/R AML)

A Phase (ph) 1b/2 Study of TAK-659, an Investigational Dual FLT-3 and SYK Inhibitor, in Patients (Pts) with Relapsed or Refractory Acute Myelogenous Leukemia (R/R AML)

A Phase II Study of Midostaurin and 5-Azacitidine for Elderly Patients with Acute Myeloid Leukemia

A Phase II Study of Midostaurin and 5-Azacitidine for Elderly Patients with Acute Myeloid Leukemia

A Phase I Study of FLX925, a Dual FLT3 and CDK4/6 Inhibitor in Patients with Relapsed or Refractory Acute Myeloid Leukemia (AML)

▪Background: Development of kinase domain or gate-keeper mutations in the FMS-like tyrosine kinase 3 (FLT3) gene limit the therapeutic benefit of Type 2 FLT3 inhibitors in internal tandem duplication mutated FLT3 (FLT3-ITD) acute myeloid leukemia (AML) and are associated with resistance to such agents. Strategies to overcome such resistance include the development of kinase inhibitors that bind FLT3 differently or targeting additional pathways simultaneously. FLX925 is a next-generation FLT3 inhibitor that was designed to address or avoid common resistance mechanisms with a unique binding mode and potent activity against CDK4/CDK6 (CDK4/6). Preclinical studies comparing FLX925 to other FLT3 inhibitors (quizartinib and gilteritinib) currently in late-stage clinical development demonstrated that FLX925 had an improved resistance profile (Li et al. Mol. Cancer Ther. 2014, 14: 375; Marubayashi et al. ASH 2016, Abstract #2323). Here we present the results of a Phase I dose escalation study of FLX925 in adult patients with relapsed or refractory AML.Methods: FLX925 was administered orally as a single agent to 51 patients at doses ranging from 40 mg twice a day (BID) to 720 mg three times a day (TID). Patients with relapsed or treatment refractory AML, regardless of FLT3 mutation status, were eligible if they were ≥18 years of age and had adequate performance status (ECOG ≤2) and organ function.Results: Median age was 60 years (range 19-81). 20 patients had FLT3-ITD+ AML, 8 patients had FLT3-D835 mutant AML, 5 subjects carried dual mutant disease, and 18 patients were FLT3-wt at study entry. Patients received a median of one 4-week cycle of treatment (range <1-6 cycles).Treatment-related adverse events (AEs) were primarily observed only in the 3 highest-dose cohorts (540 mg BID and above). The most common treatment-related adverse events were Grade 1 or 2 (CTCAE v4.03) gastrointestinal disorders, including nausea (7.8%), diarrhea (5.9%), and abdominal pain (2.0%); Grade 1 or 2 increased creatinine (7.8%); and Grade 2-4 acute or chronic renal failure (7.8%). Additional treatment-related Grade 3 and 4 AEs occurring in >1 patient included increased transaminase (3.9%) and decreased white blood cell count (5.9%). Two DLTs of elevated creatinine and acute kidney injury were observed at 720 mg TID. The maximum tolerated dose (MTD) of FLX925 was determined to be 540 mg TID.Pharmacokinetic (PK) studies demonstrated a terminal half-life of approximately 4-7 hours and dose-proportional increases in exposure (AUC). As overall exposure with BID dosing was less than predicted (~50%) in early cohorts, the protocol was amended to test TID dosing.Pharmacodynamic (PD) FACS-based assays measuring phosphorylated STAT5 and RB proteins in AML patient blasts and cell lines were used to assess the inhibition of the FLT3 and CDK4/6 pathways, respectively. A strong PK/PD correlation was observed between plasma drug levels and inhibition of both pathways (Figure).Evidence of anti-leukemic activity was observed in 8 of 21 patients treated at doses of 540 mg BID and above, including ≥50% reductions of peripheral blasts in 6 patients (2 ITD, 2 D835, 1 ITD/D835, and 1 FLT3-wt), and 2 patients who achieved a transient morphologic leukemia-free state (<5% bone marrow blasts without count recovery; 1 ITD, 1 D835). No complete or partial responses were observed.Conclusion: FLX925, the first dual FLT3 and CDK4/6 inhibitor tested in AML patients, demonstrated modest evidence of anti-leukemic activity as a single agent at the doses investigated, and a dose-limiting toxicity of increased creatinine. Lower than predicted drug exposure necessitated an increase in dosing frequency to TID to demonstrate clinical effects, but also resulted in dose-related AEs that limited prolonged dosing. [Display omitted] DisclosuresDaver:Incyte Corporation: Honoraria, Research Funding; Immunogen: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Kiromic: Research Funding; Pfizer Inc.: Consultancy, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Jazz: Consultancy; Karyopharm: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding. Pollyea:Takeda, Ariad, Alexion, Celgene, Pfizer, Pharmacyclics, Gilead, Jazz, Servier, Curis: Membership on an entity's Board of Directors or advisory committees; Agios, Pfizer: Research Funding. Rizzieri:Erytech: Research Funding; Shire: Research Funding. Kovacsovics:Seattle Genetics: Research Funding; Celgene: Consultancy; Flexus: Research Funding. Perl:Pfizer: Other: Advisory Board; Daiichi Sankyo: Consultancy; Asana Biosciences: Other: Scientific advisory board; Arog Pharmaceuticals: Consultancy; Seattle Genetics: Other: Advisory board; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Astellas: Consultancy; Novartis: Other: Advisory Board. Seitz:FLX Bio: Employment. McKinnell:FLX Bio, Inc.: Employment. Ho:FLX Bio, Inc.: Employment. Cortes:Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Sun Pharma: Research Funding; ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding.

Streamline - Retrospective Cohort Study of Relapsed or Refractory (R/R) FLT3-Mutated Acute Myeloid Leukemia (AML): Real-World Treatment, Testing Patterns, and Outcomes

Streamline - Retrospective Cohort Study of Relapsed or Refractory (R/R) FLT3-Mutated Acute Myeloid Leukemia (AML): Real-World Treatment, Testing Patterns, and Outcomes

Difference in FLT3 Mutational Status between Diagnosis and Relapsed/Refractory Acute Myeloid Leukemia: A Meta-Analysis

Introduction Research in recent years has shown that the class III receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) gene mutation was identified in 30% of Acute Myeloid Leukemia (AML) patients. The FLT3 internal tandem duplication (FLT3-ITD) is positive for 25% of AML cases, while FLT3 tyrosine kinase domain (FLT3-TKD) is present in 5% of cases. Some studies have showed that patients with AML with FLT3 mutation may maintain the alteration in the relapse/refractory (RR) disease, although this is not always the rule. This report is a review about the genetic change of FLT3 mutation in the RR AML scenario. Methods It is a retrospective meta-analysis research. A search in June/2020 was performed in PubMed to identify studies with the terms FLT3 (title/abstract) AND Acute Myeloid Leukemia without a restricted date. The papers were select based in title and abstract. Inclusion criteria were adult AML, language in English, Spanish or Portuguese. Exclusion criteria were studies in children and acute promyelocytic anemia. Data were collected from retrospective or prospective studies in which patients with RR AML had the data about FLT3-ITD or FLT-TKD in the diagnosis and relapse. Results and Discussion 1201 papers were found, in which 688 were excluded by reading the title, and 506 eliminated after the title and abstract revision. From these, 17 were eligible for full text reading. From eligible papers, 6 were rejected. All the studies used PCR as the method of evaluation of FLT3 mutational status. Six studies assessed FLT3 ITD and TKD mutations, four assessed only FLT3-ITD and one checked only FLT3-TKD mutation. Six articles described mutation status in both FLT3 alterations. The total of 272 patients with RR AML were analyzed in these studies. 74 (27%) were positive for FLT3-ITD at diagnosis and 15 (5%) for FLT3-TKD. In the relapse, 81 (29%) were FLT3-ITD positive and 11 (4%) FLT3-TKD positive. The change in mutation status occurred in 47 (17%) of patients, 3 patients of these change from FLT3-TKD to FLT3-ITD mutation. Three works had patients with acute promyelocytic leukemia, a total of 10 patients. In one work 42 children were included in the study, representing 15% of the total population. Mutational status change from negative to FLT3-ITD in 20 (7%) patients, FLT3-ITD to negative in 14 (5%). From those that were FLT3-ITD positive, 60 (22%) remained positive in the relapse. In patients with FLT3-TKD, change in mutation status from positive to negative occurred in 6 (2%) of the patients, 4 (1%) were negative and gained the mutation, 6 (2%) maintained positive in diagnosis and relapse. 159 (58%) were negative in all the disease course. When we analyzed only FLT3-ITD mutation, a total of 607 patients were studied at diagnosis and relapsed, 30% were FLT3-ITD positive in relapse. Change in mutational status occurred in 12%. Meanwhile, 392 patients had their mutational status available when considering only FLT3-TKD. 89% were negative in diagnosis and relapsed. 31 (8%) patients had their status changed. We described FLT3 mutations status in paired samples from diagnosis to relapsed/refractory AML. Our data demonstrate that 41% of patients were positive for FLT3 mutation in some point of the disease. One in six patients had their mutational status altered. Some patients may have had small clones with mutated cells that were below the limit of the detection by assay and proliferated after treatment. Another hypothesis is that ade novomutation was acquired during the disease course. From the population in the study, 7% were FLT3 positive/negative, which could represent the loss of the mutation post chemotherapy and a selection of a new clone originating from the relapsed disease. The information about FLT3 status is important due to a possible prognostic value in patients with relapse/refractory disease and because the patient can benefit from a novel therapeutic treatment with FLT3 inhibitors. Conclusion FLT3 mutation is an important mutation in AML at diagnosis and relapse/refractory scenario and the mutational status is variable during the disease. Since one in six patients had their mutational status altered in the study, reassessment should be mandatory in any case of relapse and refractoriness. More studies are needed to evaluate the prognostic value of those changes and the development of more sensitive methods could help to find small clones already present in AML at diagnosis. Disclosures No relevant conflicts of interest to declare.

An Optimized Full-Length FLT3/CD3 Bispecific Antibody Demonstrates Potent Anti-leukemia Activity and Reversible Hematological Toxicity

FLT3 (FMS-like tyrosine kinase 3), expressed on the surface of acute myeloid leukemia (AML) blasts, is a promising AML target, given its role in the development and progression of leukemia, and its limited expression in tissues outside the hematopoietic system. Small molecule FLT3 kinase inhibitors have been developed, but despite having clinical efficacy, they are effective only on a subset of patients and associated with high risk of relapse. A durable therapy that can target a wider population of AML patients is needed. Here, we developed an anti-FLT3-CD3 immunoglobulin G (IgG)-based bispecific antibody (7370) with a high affinity for FLT3 and a long half-life, to target FLT3-expressing AML blasts, irrespective of FLT3 mutational status. We demonstrated that 7370 has picomolar potency against AML cell lines invitro and invivo. 7370 was also capable of activating Tcells from AML patients, redirecting their cytotoxic activity against autologous blasts at low effector-to-target (E:T) ratio. Additionally, under our dosing regimen, 7370 was well tolerated and exhibited potent efficacy in cynomolgus monkeys by inducing complete but reversible depletion of peripheral FLT3+ dendritic cells (DCs) and bone marrow FLT3+ stem cells and progenitors. Overall, our results support further clinical development of 7370 to broadly target AML patients.

Streamline - Study of Relapse or Refractory (R/R) FLT3-Mutated Acute Myeloid Leukemia (AML) Using Electronic Medical Records (EMR): First Analysis from a Multicenter, Retrospective Cohort Study

INTRODUCTION: In AML, the ability to target disease-related mutations is an important therapeutic innovation. FMS-like tyrosine kinase 3 (FLT3) mutations (FLT3mut+) are common in AML and confer a negative impact on prognosis. Since FLT3 mutational status can change over the course of the disease and FLT3-targeting therapies may benefit FLT3mut+ patients (pts), FLT3 mutational testing is recommended for R/R AML pts, even if testing was performed at initial diagnosis. As the landscape of FLT3mut+ R/R AML evolves, it is important to understand how the utilization/sequencing of therapies and application of FLT3 mutational testing impacts pts in real-world settings. The objective of this analysis was to examine real-world data from a large, multicenter, collaborative EMR database to learn more about treatment and FLT3 testing patterns in pts with FLT3mut+ R/R AML. METHODS: This retrospective, longitudinal, observational cohort study was designed to describe treatment FLT3 testing patterns in adult (≥18 years) pts in the USA with FLT3mut+ R/R AML. For this analysis, initial diagnosis of R/R AML must have occurred between January 1, 2015 and November 30, 2018. This study focuses on data collected prior to the date of approval of gilteritinib for treatment of FLT3mut+ R/R AML (November 28, 2018). Patients were identified by confirmation of diagnosis of AML, followed by confirmation of FLT3mut+ disease, and then ≥1 R/R event. Data were derived from a consolidated EMR database, which combined data from CancerLinQ and Vector Oncology. Data were extracted through an SQL query and abstracted by clinical research nurses. Descriptive statistics were used to examine potential differences among subsets of pts. RESULTS: In the initial phase, data from 99 pts (52.5% male; n=52) with FLT3mut+ R/R AML were evaluated. The majority of pts were Caucasian (72.7%; n=72) with a median age of 62 years (range: 20-86) at first R/R episode. At the first R/R event, treatment regimens were diverse; a total of 89/99 (89.9%) pts underwent 44 different anticancer therapies and only 10.1% (n=10/99) of pts received best supportive care (BSC). At first R/R, the most common anticancer treatments were cytarabine + fludarabine + idarubicin (29.4%; n=10/34) for pts undergoing high-intensity chemotherapy (HIC) and decitabine (45.4%; n=5/11) for pts undergoing low-intensity chemotherapy (LIC). The percentage of pts receiving FLT3 inhibitors, either as single agent or in combination with chemotherapy, was 33.3% (n=33/99) of the total population (Table). Among pts aged &lt;60 years, 47.7% (n=21/44) were treated with HIC-most commonly with cytarabine + fludarabine + idarubicin (33.3%; n=7/21). Only 4.5% (n=2/44) of pts received LIC and 9.1% (n=4/44) received BSC. In the pts aged ≥60 years, 10.9% (n=6/55) received BSC. A higher proportion of pts aged ≥60 years received HIC +/- FLT3 inhibitors (38.2%; n=21/55) compared with LIC +/- FLT3 inhibitors (25.5%; n=14/55). The most common treatments included cytarabine-usually in combination with fludarabine and/or idarubicin (84.6%, n=11/13)-for HIC, and azacitidine (alone or in combination; 55.6%, n=5/9) for LIC. Approximately 38.2% (n=21/55) of pts aged ≥60 years received a FLT3 inhibitor (alone or in combination with chemotherapy), with midostaurin being the most frequently prescribed (47.6%; n=10/21), followed by sorafenib (38.1%, n=8/21). Although most pts (83.8%; n=83/99) were tested for FLT3 mutations at initial AML diagnosis, the majority of pts were not retested; retest at first R/R was performed in 29.0% (n=9/31) of pts. At first R/R, 22.2% (n=2/9) of pts had a change in FLT3 mutational status (Figure). No significant differences were observed in FLT3 retesting among pts &lt;60 years vs ≥60 years (P=0.456). CONCLUSIONS: During the study period, there was substantial heterogeneity regarding the management of FLT3mut+ R/R AML. A total of 89 pts received 44 different anticancer therapies and approximately one-third of pts received a FLT3 inhibitor (alone or in combination) at first R/R. However, during the study period, approved agents for treatment of FLT3mut+ R/R AML were not available. Despite NCCN guidelines, at first R/R, FLT3 retesting was not often performed. With recent approval of FLT3-targeted therapies, it is important to measure rates of retesting in the R/R setting to better understand how elements of pt care, such as monitoring changes in FLT3 mutational status, may impact pt outcomes. Disclosures Zeidan: Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria; Medimmune/AstraZeneca: Research Funding; ADC Therapeutics: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Otsuka: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Jazz: Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding. Gilligan:Astellas: Other: Project. Gautam:Astellas: Other: Project. Hu:Astellas: Other: Project. Grinblatt:Abbvie: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Pandya:Astellas Pharmaceuticals: Employment.

First in Human Study of SEL24/MEN1703, First in Class, Orally Available Dual PIM/FLT3 Kinase Inhibitor, in Patients with Acute Myeloid Leukemia

Introduction SEL24/MEN1703 is a potent dual inhibitor of proviral integration site for Moloney murine leukemia virus (PIM) and FMS-like tyrosine kinase 3 (FLT3) with increased activity in both primary Acute Myeloid Leukemia (AML) cells and AML cell lines, irrespectively of FLT3 mutational status, when compared to either FLT3 or PIM single agent inhibitors. PIM kinases are thought to be major drivers of resistance to FLT3 inhibitors and their inhibition in relapsed samples restores cell sensitivity to these agents. Thus, simultaneous PIM and FLT3 inhibition represents a promising strategy in AML therapy. In addition, the broader spectrum of activity of SEL24/MEN1703, which goes beyond PIM/FLT3 inhibition, warrants the compound investigation in AML patients regardless of the genetic aberrations of FLT3. Methods CLI24-001 is a First-in-Human, single-arm, phase I/II trial with a dose-escalation (DE) part followed by a cohort expansion (CE) part in patients with AML (excluding Acute Promyelocytic Leukemia) -newly diagnosed, relapsed or refractory - that are unsuitable for intensive chemotherapy and meet the main inclusion criterion of white blood count (WBC) of ≤30 x 109/L (hydroxyurea/leukapheresis permitted to lower WBC). In both study parts, patients are eligible regardless of mutational status and prior exposure to FLT3 inhibitors; however, prior treatment with PIM inhibitors is not allowed. SEL24/MEN1703 is given orally, QD, for 14 days in a 21-day cycle to be repeated until disease progression, or unacceptable toxicity or withdrawal of patient consent. Bayesian modified toxicity probability interval model was implemented in the DE part after achieving 5 incremental dose levels in order to provide the most accurate identification of the Recommended Phase 2 Dose (RP2D). Also, during the DE part, a new oral formulation was introduced and compared to the original one by repeating the 100 mg dose level in order to avoid a subsequent formal bioequivalence study in patients. The primary objective of the study is to identify the RP2D of SEL24/MEN1703 in the DE part that will be further characterized in the CE part for next steps of clinical development. Key secondary objectives include the characterization of pharmacokinetics (PK) and efficacy assessment of SEL24/MEN1703 given as single agent; exploratory objectives include the assessment of relevant biomarkers (e.g. pS6) in peripheral blood and bone marrow, and their correlation with PK at different time points. Correlation of clinical activity and the cell surface antigen CD25 will be also explored. Study update Currently, the study is enrolling at 5 US sites and will be extended both in the US and EU. The recruitment started in March 2017 and, as of July 26th, 2019, 24 patients received SEL24/MEN1703 at dose levels ranging from 25 to 150 mg. Patients had a median age of 69 (25-84) years and a median of 3 (0-8) prior treatments for AML. Adverse prognostic factors such as primary refractory AML, unfavorable cytogenetics and prior MDS history accounted for 45.8%, 37.5%, 33.% of study patients, respectively. The most frequently reported mutations were FLT3-ITD (20.8% of patients) and those of DNMT3A and IDH1 (16.7% each). Conclusions This is the first trial testing a dual PIM/FLT3 inhibitor with the potential to overcome FLT3 inhibitor resistance, but also to be active in AML regardless of FLT3 mutational status. Ongoing adjustment of the DE design, including the switch to an optimized oral formulation, has been performed to obtain more robust data on the RP2D (ClinicalTrials.gov Identifier: NCT03008187). Disclosures Nazha: Incyte: Speakers Bureau; Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Abbvie: Consultancy; Daiichi Sankyo: Consultancy. Strickland:Astellas Pharma: Consultancy; Sunesis Pharmaceuticals: Research Funding; AbbVie: Consultancy; Jazz: Consultancy; Kite: Consultancy; Pfizer: Consultancy. Walter:Boehringer Ingelheim: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Aptevo Therapeutics: Consultancy, Research Funding; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Jazz Pharmaceuticals: Consultancy; Kite Pharma: Consultancy; New Link Genetics: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy; Seattle Genetics: Research Funding; Boston Biomedical: Consultancy; Covagen: Consultancy; Daiichi Sankyo: Consultancy. Valimberti:Menarini Ricerche S.p.A: Employment. Tagliavini:Menarini Ricerche S.p.A: Employment. Mazzei:Menarini Ricerche S.p.A.: Employment. Fiesoli:Menarini Ricerche S.p.A.: Employment. Scartoni:Menarini Ricerche S.p.A.: Employment. Bellarosa:Menarini Ricerche S.p.A.: Employment. Binaschi:Menarini Ricerche S.p.A.: Employment. Chrom:Selvita S.A.: Employment. Baldini:Menarini Ricerche S.p.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Capriati:Menarini Ricerche S.p.A.: Employment. Pellacani:Menarini Ricerche S.p.A.: Employment; Amgen: Equity Ownership. Ravandi:Macrogenix: Consultancy, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclacel LTD: Research Funding; Xencor: Consultancy, Research Funding; Menarini Ricerche: Research Funding; Selvita: Research Funding.

SM09419, a Novel, Small-Molecule CDC-like Kinase (CLK) Inhibitor, Demonstrates Strong Inhibition of the Wnt Signaling Pathway and Antitumor Effects in FMS-like Tyrosine Kinase 3 (FLT3)-Mutant Acute Myeloid Leukemia Models

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD) mutation accounts for ~25% of all AMLs, carries a poor prognosis, and is prone to relapse despite targeted therapy. FLT3 mutations are associated with aberrant activation of the Wnt signaling pathway, which itself is implicated in AML initiation/progression and is required for the self-renewal and survival of leukemic stem cells. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway. These studies examined the antitumor activity of SM09419 as a single agent and in combination with targeted and standard therapies in preclinical models of FLT3-ITD AML. In MV-4-11 and MOLM-13 AML cells carrying the FLT3-ITD mutation, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt pathway-related genes (CCND1, MYC, TCF7, DVL2). The effect on cell proliferation was tested in 8 AML cell lines with varying mutation profiles as well as 26 different leukapheresis-derived primary human AML cells. Proliferation was strongly impaired by SM09419 across all tested cell lines (average EC50=0.2 + 0.048 µM]); MV-4-11 and MOLM-13 cells had EC50 of 0.049 and 0.144 µM, respectively. SM09419 also potently inhibited proliferation in all primary AML cells (average EC50=0.048 + 0.0097 µM) regardless of FLT3 mutation status, cytogenetics, or AML diagnosis (de novo or relapsed/refractory). SM09419 also induced apoptosis in MV-4-11 and MOLM-13 cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression relative to vehicle. In vivo antitumor effects and tolerability of oral SM09419 (QD) alone or combined with either midostaurin (FLT3 inhibitor) or venetoclax (BCL2 inhibitor) and/or azacitidine were assessed in FLT3-ITD xenograft models (n=5-6/group). In MOLM-13 xenografts, SM09419 (12.5 and 25 mg/kg) induced strong tumor growth inhibition (TGI) vs. vehicle at Day 14 (TGI 52% [p&amp;lt;0.05] and 74% [p&amp;lt;0.001], respectively). Midostaurin (50 mg/kg) induced significant TGI vs. vehicle (50%, p&amp;lt;0.05), which was increased when administered in combination with 12.5 mg/kg SM09419 (81%, p&amp;lt;0.001). In MV-4-11 xenografts, single-agent SM09419 (6.25, 12.5, and 25 mg/kg) induced significant TGI vs. vehicle (56% [p&amp;lt;0.05], 94%, and 95% [p&amp;lt;0.001], respectively) at Day 26 with tumor regression in all mice dosed at 12.5 mg/kg and 25 mg/kg. In a subsequent experiment, midostaurin (50 mg/kg) alone and combined with 6.25 mg/kg SM09419 for 23 days induced tumor regression in MV-4-11 xenografts (100% TGI vs. vehicle, p&amp;lt;0.0001). After treatment discontinuation, tumor regression was maintained in all mice (6/6) treated with the combination for 26 days, whereas tumor regrowth was immediately observed in midostaurin-treated mice. In another MV-4-11 xenograft study, the combination of 6.25mg/kg SM09419 with azacitidine (0.8 mg/kg QD) and/or venetoclax (25 mg/kg QD) induced significant TGI (95-98% vs. vehicle, p&amp;lt;0.001) with tumor regression at Day 26. Azacitidine + venetoclax induced 79% TGI (p&amp;lt;0.001), but no tumor regression was observed. The triple combination induced tumor regression in all mice and complete regressions in 4/6 mice (67%); it had a greater effect on slowing tumor regrowth after treatment discontinuation vs. a single agent or doublet. SM09419 alone or in combination was well tolerated in these xenograft models based on body weight measurements. In summary, SM09419 potently inhibited SRSF6 phosphorylation and Wnt signaling pathway activity and induced apoptosis in FLT3-ITD cell lines. It also inhibited proliferation in cell lines and primary AML cells regardless of FLT3 status. The strong in vivo antitumor effects observed as combination treatment suggest that SM09419 combined with standard therapies may provide a clinical benefit by slowing or preventing relapse in AML with a marker of poor prognosis such as FLT3-ITD. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.