FLT3-mutated AML Research Articles

Role of Biomarkers in FLT3 AML.

Simple SummaryGenetically heterogeneous disorder acute myeloid leukemia (AML) is marked by recurring mutations in FLT3. Current FLT3 inhibitors and other emerging inhibitors have helped in the improvement of the quality of standard of care therapies; however, the overall survival of the patients remains static. This is due to numerous mutations in FLT3, which causes resistance against these FLT3 inhibitors. For effective treatment of AML patients, alternative approaches are required to overcome this resistance. Here, we will summarize the biomarkers for FLT3 inhibitors in AML, as well as the alternative measures to overcome resistance to the current therapies.Acute myeloid leukemia is a disease characterized by uncontrolled proliferation of clonal myeloid blast cells that are incapable of maturation to leukocytes. AML is the most common leukemia in adults and remains a highly fatal disease with a five-year survival rate of 24%. More than 50% of AML patients have mutations in the FLT3 gene, rendering FLT3 an attractive target for small-molecule inhibition. Currently, there are several FLT3 inhibitors in the clinic, and others remain in clinical trials. However, these inhibitors face challenges due to lack of efficacy against several FLT3 mutants. Therefore, the identification of biomarkers is vital to stratify AML patients and target AML patient population with a particular FLT3 mutation. Additionally, there is an unmet need to identify alternative approaches to combat the resistance to FLT3 inhibitors. Here, we summarize the current knowledge on the utilization of diagnostic, prognostic, predictive, and pharmacodynamic biomarkers for FLT3-mutated AML. The resistance mechanisms to various FLT3 inhibitors and alternative approaches to combat this resistance are also discussed and presented.

Identification of 2-Aminopyrimidine Derivatives as FLT3 Kinase Inhibitors with High Selectivity over c-KIT.

Herein, we report two promising compounds 30 and 36 possessing nanomolar FLT3 inhibitory activities (IC50 = 1.5-7.2 nM), high selectivity over c-KIT (>1000-fold), and excellent anti-AML activity (MV4-11 IC50 = 0.8-3.2 nM). Furthermore, these two compounds efficiently inhibited the growth of multiple mutant BaF3 cells expressing FLT3-ITD, FLT3-D835V/F, FLT3-F691L, FLT3-ITD-F691L, and FLT3-ITD-D835Y. Oral administration of 30 and 36 at 6 mg/kg/d could significantly suppress tumor growth in the MV4-11 cell-inoculated xenograft model, exhibiting tumor growth inhibitory rates of 83.5% and 95.1%, respectively. Importantly, 36 could prolong the mouse survival time in the FLT3-ITD-TKD dual mutation syngeneic mouse model (BaF3-FLT3-ITD-D835Y) at a dose of 6 mg/kg p.o. bid/4W. No clear myelosuppression was observed in the treated group of 36 in the MPO strain of zebrafish, even at 10 μM. In summary, our data demonstrated that 36 may represent a promising candidate for the treatment of FLT3 mutant AML.

Multi-Modal Targeting of FLT3 with Chimeric Antigen Receptor T Cell Immunotherapy and Tyrosine Kinase Inhibition in High-Risk Pediatric Leukemias

Background:Clinical outcomes for children with FLT3-mutant AML and infants with KMT2A-rearranged (KMT2A-R) B-ALL remain dismal. These leukemias share a common feature of aberrant activation of FLT3 kinase signaling, which occurs by activating FLT3 mutations in AML and by overexpression of wild-type FLT3 in KMT2A-R ALL. Several FLT3 tyrosine kinase inhibitors (FLT3i) are approved for adults with FLT3-mutant AML, but potential efficacy against KMT2A-R ALL remains incompletely characterized and may differ from responses in AML. We previously developed and preclinically validated chimeric antigen receptor (CAR) T cells directed against FLT3 (FLT3CART), which importantly showed potent anti-leukemia activity in preclinical models of both childhood FLT3-mutant AML and infant KMT2A-R ALL (Chien CD et al. ASH 2016). In the current studies, we hypothesized that combinatorial targeting of these two high-risk leukemia subtypes with FLT3CART and the selective next-generation FLT3i gilteritinib would have superior activity and potentially mitigate therapeutic resistance now known to occur with kinase inhibitors or CAR T cell immunotherapy.Methods and Results:We first assessed in vitro sensitivity of human FLT3-mutant AML and KMT2A-R ALL cell lines to gilteritinib, a second-generation selective FLT3i with established clinical activity in FLT3-mutant AML and unknown activity in KMT2A-R ALL. As detrimental effects of kinase inhibitors (e.g., dasatinib, ruxolitinib) upon CAR T cells have been reported, we evaluated for similar effects with gilteritinib co-incubated in vitro with CD3/CD28-bead activated healthy human donor T cells. However, we observed minimal deleterious effects of gilteritinib on normal T cell viability, immunophenotype, and IL-2 and interferon-gamma (IFNg) production. We validated combinatorial effects of gilteritinib and FLT3CART-induced cytotoxicity against FLT3-mutant AML and KMT2A-R ALL cell lines in vitro without impairment of IL-2/IFNg production. We then assessed this dual therapy approach in luciferase+ FLT3-mutant AML (MOLM14) and KMT2A-R ALL (SEM) cell line murine xenograft models. As predicted, both FLT3CART and gilteritinib monotherapies transiently inhibited in vivo leukemia proliferation, although leukemia progression eventually occurred. Conversely, FLT3CART and gilteritinib combination therapy strikingly induced enhanced and sustained leukemia clearance in all assessed AML and ALL cell line xenograft models (Figure 1). Confirmatory studies in our established childhood FLT3-mutant AML and KMT2A-R ALL patient-derived xenograft (PDX) models have also demonstrated potent anti-leukemia efficacy of combined FLT3CART and gilteritinib therapy.Earlier-generation FLT3i have been reported to increase cell surface FLT3 expression on FLT3-mutant AML cells. Given the known importance of target antigen site density for CAR T cell efficacy, we reasoned that a sequential approach to dual therapy with FLT3i ‘priming’ followed by FLT3CART may be superior to a simultaneous treatment strategy. In vitro studies with leukemia cell lines and in vivo studies with PDX models indeed confirmed gilteritinib-induced increases in FLT3 surface antigen density in FLT3-mutant AML cells. Intriguingly, we observed contrasting effects in KMT2A-R ALL cell lines and PDX with decreased surface FLT3 expression upon gilteritinib exposure. Ongoing studies are currently validating gilteritinib priming for FLT3CART given these initial data suggesting potentially divergent sequencing approaches in FLT3-mutant AML versus KMT2A-R ALL.Conclusions:Taken together, our preclinical studies demonstrate that dual targeting with FLT3CART immunotherapy and gilteritinib is a promising therapeutic strategy in FLT3-mutant AML and, importantly, also in KMT2A-R ALL. Notably, we also report minimal negative effects of gilteritinib on FLT3CART, suggesting that FLT3i may be used to enhance CAR T cell immunotherapy without inhibiting T cell function. Phase 1 clinical trials of FLT3CART will open soon for adults and children with FLT3-mutant AML and/or KMT2A-R ALL. [Display omitted] DisclosuresFry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company; ElevateBio: Research Funding. Tasian: Kura Oncology: Consultancy; Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

Hypomethylating Agent (HMA) Therapy and Venetoclax (VEN) with FLT3 Inhibitor “Triplet” Therapy Is Highly Active in Older/Unfit Patients with FLT3 Mutated AML

Hypomethylating Agent (HMA) Therapy and Venetoclax (VEN) with FLT3 Inhibitor “Triplet” Therapy Is Highly Active in Older/Unfit Patients with FLT3 Mutated AML

RNA Polymerase II Is a Therapeutic Target to Overcome FLT3 Inhibitor Resistance Mediated By the Bone Marrow Microenvironment

Background: Gilteritinib is a clinically active FLT3 tyrosine kinase inhibitor (TKI) approved for relapsed/refractory FLT3-mutant AML, but nearly all patients treated with gilteritinib and other FLT3 TKIs eventually develop clinical resistance. Activating RAS/MAPK pathway mutations are a predominant non-FLT3 dependent resistance mechanism in patients treated with gilteritinib. AML blasts can also develop FLT3 TKI resistance secondary to paracrine MAPK activation stimulated by FLT3 Ligand, FGF2, or other protective cytokines within the bone marrow microenvironment (BME). To identify potential targets that sensitize AML cells to gilteritinib-induced apoptosis in a model of the BME, we performed a genome-wide CRISPR/Cas9 death screen in MOLM-14 FLT3-ITD+ human AML cells cultured in bone marrow stromal cell conditioned media. We hypothesize that identified genes represent promising combinatorial therapeutic targets that can enhance clinical efficacy of FLT3 TKIs in AML.Methods: To model stroma-mediated TKI resistance, we used the HS5 human bone marrow stromal cell line that secretes multiple cytokines (G-CSF, GM-CSF, FGF2) and supports myeloid progenitor proliferation in co-culture. MOLM-14 CRISPRi cells transduced with CRISPRi-v2 genome-wide sgRNA library were cultured in HS5 conditioned media for 24 hours and then treated with gilteritinib 250 nM. Cells were stained with a fluorogenic caspase 3/7 reagent and then fixed after 24 hours of drug treatment. Caspase-3 positive cells were sorted from the entire drug-treated cell population by FACS and guide RNAs enriched or depleted in this sample as compared to an untreated T0 sample were determined by NGS.Results: We identified several gene-level hits that were enriched in the apoptotic population (FDR <0.2). Among these, we identified multiple transcription factors or regulators of transcriptional activation. The latter included multiple components of RNA pol II machinery (POLR2G, RTF1) and multiple subunits of Mediator (MED12, MED30, MED21, MED11), a complex that regulates RNA pol II activity and has been shown to modulate super-enhancer-associated genes in AML cells. To validate select hits, we transduced MOLM-14 and MV411 CRISPRi cells with a tetracycline-inducible sgRNA expression vector. Using this system, we found that conditional knockdown of MED12, the top scoring Mediator subunit in our screen, significantly sensitized MOLM-14 (FgH1) cells to gilteritinib while modestly augmenting the cytotoxicity of gilteritinib in MV411 (FgH1) cells when compared to a non-targeting sgRNA. Functionally, MED12 associates with MED13, Cyclin C, and CDK8 to form the CDK8 kinase module of Mediator. MED12 knockdown, as expected, led to suppression of STAT1 S7272 and STAT5 S726 phosphorylation, known targets of CDK8. Based on these results, we hypothesized that CDK8 inhibition would augment apoptosis induced by gilteritinib in HS5 conditioned media. Using SEL120, a novel CDK8 inhibitor already in early phase AML clinical trials, we performed an 8 x 8 dose matrix drug synergy analysis of gilteritinib and SEL120 in multiple FLT3-mutant AML cell lines using Bliss independence modeling. We found that SEL120 was synergistic with gilteritinib in inducing apoptosis in MOLM-14 and MV411 cells in HS5 conditioned media (Bliss synergy scores of 2.44 and 11.45 with most synergistic area scores of 10.91 and 26.85, respectively). We also found combinatorial activity against MOLM-14 cells harboring secondary NRAS activating mutations (G12C and Q61K), suggesting the therapeutic combination could potentially overcome cell intrinsic and extrinsic MAPK-activating resistance mechanisms. Lastly, we found that gilteritinib and SEL120 combined to impart greater cytotoxicity than either drug alone in a primary sample (AML #1) from a patient with newly diagnosed AML possessing a FLT3-ITD mutation at high mutant allele ratio.Conclusions: The results and validation of our CRISPRi screen suggest that combined CDK8 and FLT3 inhibition is a novel strategy for augmenting gilteritinib cytotoxicity. Assessment of the activity of the combination in additional primary AML samples and in vivo murine models of AML is planned. Additional candidate targets already described and other Mediator and RNA pol II subunits from our screen are also being further evaluated to precisely define the transcriptional programs that influence FLT3 inhibitor resistance. DisclosuresLogan: Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding; Amgen, Pfizer, AbbVie: Consultancy. Gilbert: Denali Therapeutics: Ended employment in the past 24 months, Other: Spouse/Significant Other's Employment; GSK: Consultancy, Research Funding; AstraZeneca: Research Funding; Chroma Medicine: Consultancy, Other: Co-founder. Smith: Revolutions Medicine: Research Funding; AbbVie: Research Funding; Daiichi Sankyo: Consultancy; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Momelotinib Is a Highly Potent Inhibitor of FLT3-Mutant AML

Approximately one-third of AML patients harbor kinase activating mutations in FLT3. Several small-molecule first generation FLT3 tyrosine kinase inhibitors (TKIs) have been evaluated in the last two decades, but none could induce a durable response possibly due to poor pharmacokinetics and target selectivity. Second generation FLT3 inhibitors such as, quizartinib, gilteritinib, and crenolanib, were designed for greater selectivity with a narrow kinome-profile and better pharmacokinetic properties, but they failed to induce a durable response. Possibly due to intrinsic resistance conferred by growth factor signaling in the bone marrow resident leukemic cells, which serve as a reservoir to develop resistance resulting to disease relapse. Activation of Jak2 signaling by chemokines and cytokines from the stroma have been reported to confer TKI refractoriness. Recent studies from Martin Carroll's group showed that both GMCSF and IL-3 confer resistance by activating JAK2 signaling, which can be suppressed by combined FLT3 and JAK2 inhibition. However, so far, it is not established whether upfront FLT3/Jak2 inhibition will provide durable response.For instance, AML patients who achieved complete remission lacking FLT3-ITD clones showed better overall survival than patients with measurable MRD, suggesting that eradicating the FLT3-ITD clones will have a deeper response with better overall survival. Thus, an ideal FLT3 inhibitor should be able to suppress FLT3-ITD resistant mutants, and growth factor activated JAK2 signaling while sparing c-KIT receptor and hERG to avoid myelosuppression and cardiotoxicity, respectively. Here we show that Jak2 inhibitor, momelotinib, is an equipotent type-I FLT3 inhibitor (Fig 1A-D). Biochemical and structural modeling revealed that it binds to an active conformation of FLT3 kinase. Therefore, like gilteritinib, it efficiently suppresses the resistance conferred by activation loop mutations (Fig1 E-H). Moreover, its lack of activity against c-KIT and inhibition of ACVR1 provides additional benefit in alleviating myelosuppression and anemia, which is commonly observed with currently used JAK2 and FLT3 inhibitors (ruxolitinib, fedratinib, and quizartinib). Perhaps more interestingly, momelotinib efficiently suppressed the disease in a preclinical model of AML using NSGS mice which recapitulates cytokine induce refractoriness as usually observed in clinical setting (Fig1 I-L). Our preclinical data provide evidence that momelotinib is an equipotent dual JAK2/FLT3 inhibitor and suppresses resistance conferred by both activation-loop mutations and growth-factor signaling. These data provide evidence that momelotinib treatment will have clinical activity in FLT3-mutated AML. Thus, warrants its clinical evaluation. [Display omitted] DisclosuresStarczynowski: kurome Inc: Consultancy.

TP-0184, a Novel FLT3-ALK2 Dual Inhibitor, Targets Amino Acid Transport and Biosynthesis in AML Cells and Sensitizes AML Cells to Chemotherapy and BCL2 Inhibition

Background FMS-like tyrosine kinase 3 (FLT3), a transmembrane receptor tyrosine kinase that is frequently mutated in AML, is associated with poor prognosis. Inhibitors of FLT3 internal tandem duplication (ITD) mutants and wild-type (WT) FLT3 have been studied, but their clinical usefulness is limited owing to treatment resistance. However, the molecular factors contributing to this resistance are unknown. We reported that AML cells induce osteogenic differentiation of bone marrow-derived mesenchymal stromal cells through the bone morphogenetic protein (BMP)-mediated signaling pathway, promoting leukemic growth. However, the effects of targeting BMP signaling in patients with FLT3-mutated AML are unexplored. Here, we hypothesized that the BMP type 1 receptor ALK2 is a key biomarker and a therapeutic target in AML patients with FLT3-ITD mutations and that the FLT3-ALK2 dual inhibitor TP-0184 inhibits leukemia growth.Methods: To determine whether ALK2 is a potential target in AML patients with FLT3-ITD mutations, we analyzed gene expression datasets (OHSU and TCGA). We treated 9 AML cell lines with FLT3-WT or ITD mutations with varying doses of ALK2 inhibitors (LDN-212854 or TP-0184) and measured their effect on cell proliferation and the cell cycle. To determine the mechanism of TP-0184-mediated cell cycle arrest, we measured activation of FLT3 downstream signaling by using Western blotting and RNA sequencing. IncuCyte live-cell imaging was used to determine the apoptotic effects of TP-0184 in combination with chemotherapy or targeted therapy agents. Further, we performed human RTK kinase binding assay to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. Finally, the effect of TP-0184 on AML growth in vivo was investigated using an FLT3-ITD positive AML xenograft model (MOLM13).Results: Analysis of AML datasets showed that ALK2 is significantly upregulated in AML patients with FLT3 mutations compared to those with WT-FLT3 (p < 0.00001) and predicts poor overall survival (p = 0.05). Validating these findings, we found higher ALK2 mRNA expression in AML cell lines with FLT3-ITD mutations than in those with WT-FLT3 (p = 0.039). This suggests that ALK2 could serve as a therapeutic target in AML with FLT3-ITD mutation. Treatment of FLT3-WT and -mutated AML cell lines with the ALK2 inhibitors LDN-212854 and TP-0184 resulted in significant inhibition of FLT3-ITD-mutant cell growth at low concentrations (IC50<25nM), while WT-FLT3 cells were affected only at high concentrations (IC50>100nM). Interestingly, TP-0184 was 10-fold more potent in inhibiting AML cell proliferation than was LDN-212854. TP-0184 induced G1/G0 arrest in AML cell lines with FLT-ITD mutations but had minimal to no effect in FLT3-WT AML cells, suggesting that AML cell lines with FLT3-ITD mutations depend on ALK2 for their survival. Further, we observed that treatment with TP-0184 in AML cell lines significantly inhibited multiple signaling proteins downstream of FLT3, such as p-STAT5, p-MKK3, and p-ERK, as well as p-PI3K, p-AKT, p-mTOR, p-4E-BP1, and p-S6K. Gene expression analysis revealed that treatment with TP-0184 in FLT3-ITD cell lines significantly downregulated the serine biosynthesis pathway, which is essential in these cells (Bjelosevic S. et al., Cancer Discov, 2021). Moreover, molecular docking and kinase-binding studies revealed that TP-0184 is bound to wild-type FLT3 as well as most of the FLT3 mutants with dissociation constants (KD) less than 5nM. These data suggest that TP-0184 inhibits both mutant FLT3 and ALK2 in AML cells. Interestingly, TP-0184 plus chemotherapy showed a synergistic effect only in FLT3-ITD cell lines, whereas TP-0184 plus the BCL2 inhibitor, venetoclax was synergistic in both FLT3-ITD and FLT3-WT cell lines. Lastly, treatment with TP-0184 inhibited AML growth and significantly prolonged survival of FLT3-ITD-mutated AML-bearing mice in a dose-dependent manner (p <0.0001).Conclusion: Our data indicate that ALK2 is a prognostic marker for AML patients with FLT3-ITD mutations. TP-0184 significantly inhibits cell proliferation by inhibiting signaling pathways downstream of FLT3, including serine biosynthesis, in AML cells. Kinase assays confirmed that TP-0184 is a highly specific FLT3 inhibitor as well as an ALK2 inhibitor. TP-0184 sensitizes AML cells to chemotherapeutic agents and targeted therapy and inhibits AML growth in vivo. DisclosuresFoulks: Sumitomo Dainippon Pharma Oncology: Patents & Royalties: WO2021102343A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: CA3103995A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US11040038B2. Warner: Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US11040038B2; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US10752594B2; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: CA3103995A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: WO2021102343A1. Battula: Tolero Pharmaceuticals: Research Funding.

Combination Therapy of FLT3 Tyrosine Kinase Inhibitors and BH3 Mimetics Targeting Antiapoptotic MCL-1 Synergistically Eliminates FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and In Vivo

Combination Therapy of FLT3 Tyrosine Kinase Inhibitors and BH3 Mimetics Targeting Antiapoptotic MCL-1 Synergistically Eliminates FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and In Vivo

A Phase-Ib/II Clinical Evaluation of Ponatinib in Combination with Azacitidine in FLT3-ITD and CBL-Mutant Acute Myeloid Leukemia (PON-AZA study)

A Phase-Ib/II Clinical Evaluation of Ponatinib in Combination with Azacitidine in FLT3-ITD and CBL-Mutant Acute Myeloid Leukemia (PON-AZA study)

AML-027: Menin Inhibition Decreases Bcl-2 and Bcl-xL and Enhances Venetoclax Activity in NPM1/FLT3-Mutated AML

<h3>Context:</h3> MLL1-menin interaction in NPM1-mutated (NPM1c) AML shares a common HOX gene signature and dependencies as that of MLL-rearrangements (MLL1-r) with menin. Menin inhibition has demonstrated anti-leukemia activity in both NPM1c and MLL-r AML. NPM1c frequently occurs in AML with FLT3-ITD/FLT3-TKD mutations. Co-inhibition of menin and FLT3 has shown enhanced anti-leukemia activity in MLL-r/FLT3- and NPM1c/FLT3-mutated AML. Targeting Bcl-2 has emerged as a promising therapeutic option for AML patients. However, despite the major improvement of combining the Bcl-2 inhibitor venetoclax with hypomethylating agents, most patients develop resistance and ultimately relapse. <h3>Objective:</h3> Investigate anti-leukemic activities, potential synergism, and mechanisms of menin-MLL1 inhibitor SDNX-50469, an equipotent surrogate of the clinical compound SNDX-5613 and venetoclax combination <i>in vivo</i> in an NPM1c/FLT3-ITD/TKD PDX model. <h3>Design:</h3> The PDX cell-engrafted NSG mice were treated with 0.05 or 0.1% SNDX-50469-spiked chow, venetoclax (50 mg/kg), or 0.1% SNDX-50469 plus venetoclax (one month). Engraftment and disease progression were assessed by flow cytometric measurement of circulating human CD45+ cells. The treatment effects on leukemia cell populations and protein levels were determined by CyTOF mass cytometry. <h3>Results:</h3> Menin inhibition exhibited strong anti-leukemia activity, which was enhanced by venetoclax, while venetoclax alone had minimal effects. The combination most effectively extended survival (143 d for 0.1% SNDX-50469 plus venetoclax, P=0.0003; 131 and 125 d for 0.1% or 0.05% SNDX-50469, respectively, both P=0.0001; 69 d for venetoclax, P=0.025; <i>vs</i> 61 d for controls). At the end of treatment, CyTOF analysis of bone marrow cells demonstrated that menin inhibition preferentially targeted CD34+CD38+ cells, while venetoclax targeted CD34+CD38- cells. Only the combination effectively eliminated bulk and CD34+CD38+/CD34+CD38-stem/progenitor cells. Menin inhibition increased the percentage of CD11b+ myeloid cells. Mechanistically, menin inhibition decreased Bcl-2 and Bcl-xL and concomitantly increased Bax and Bim, which seemingly enhanced venetoclax activity. However, we observed increased p-FLT3 in the surviving leukemia cells, particularly in the combination group, which may contribute to the regrowth of leukemia cells. <h3>Conclusions:</h3> Our study validated menin as a therapeutic target and demonstrated that its inhibition synergizes with venetoclax in NPM1/FLT3-mutated AML, which warrants clinical evaluation. Inhibition of FLT3 may further enhance menin and Bcl-2 co-targeting efficacy in NPM1- and FLT3-mutated AML.

AML-347: A Preliminary Analysis of FLAM (Italian Non-Interventional Multi-Center Study of FLT3-Mutated AML Patients): FLT3 Receptor Gene Mutational Analysis and FLT3 Inhibitor Administration in Real-Life Clinical Practice

AML-347: A Preliminary Analysis of FLAM (Italian Non-Interventional Multi-Center Study of FLT3-Mutated AML Patients): FLT3 Receptor Gene Mutational Analysis and FLT3 Inhibitor Administration in Real-Life Clinical Practice

Poster: AML-347: A Preliminary Analysis of FLAM (Italian Non-Interventional Multi-Center Study of FLT3-Mutated AML Patients): FLT3 Receptor Gene Mutational Analysis and FLT3 Inhibitor Administration in Real-Life Clinical Practice

Poster: AML-347: A Preliminary Analysis of FLAM (Italian Non-Interventional Multi-Center Study of FLT3-Mutated AML Patients): FLT3 Receptor Gene Mutational Analysis and FLT3 Inhibitor Administration in Real-Life Clinical Practice

Differentiation Response to Quizartinib, a Potent 2nd Generation FLT3 Inhibitor, in Relapsed FLT3 -Mutant AML Patients Correlates with Specific DNA Methylation Signature Prior to Therapy

Activating mutations in Fms-like tyrosine kinase 3 receptor (FLT3 ) gene occur in 30% of AML patients, and are correlated with a high risk for early relapse. The second generation FLT3 inhibitor AC-220 (quizartinib), has shown significant responses in patients with relapsed and refractory FLT3 mutant AML. For those patients who respond, two different types of pathologic responses have been observed: a cytotoxic response or a differentiation response. Given these two different responses to therapy, we hypothesized that pre-existing epigenetic programs may be responsible for determining response type, and that DNA methylation analysis of AML blasts before and after treatment may help better understand the molecular underpinnings of the varied roles of FLT3 in AML pathogenesis. For this purpose, we analyzed a cohort of 13 FLT3 -ITD mutant AML patients relapsed after standard chemotherapy and treated with quizartinib as part of the Phase II ACE trial (NCT00989261). Patients were evaluated prior to therapy and after at time of maximal response and classified as having either cytotoxic (6 patients) or differentiation (7 patients) responses as previously described. Bone marrow or peripheral blood specimens were collected from 13 patients at baseline, prior to the administration of quizartinib, and 10 matched, post-treatment specimens were available for 9 patients. DNA was extracted from mononuclear cell fractions and analyzed using multiplexed Enhanced Reduced Representation Bisulfite Sequencing (mERRBS), which captures quantitative DNA methylation status at ~3 million CpG sites across the human genome. Supervised analysis between the different groups was performed using a beta binomial model as implemented in MethylSig, with significant differentially methylated regions (DMRs) being called with methylation differences ≥25% and false discovery rates (FDR) < 0.1. Notably, paired analysis of pre- and post-treatment samples revealed very little impact of quizartinib on the DNA methylation landscape of patients with cytotoxic responses, with only 21 DMRs detected. Similarly, those who exhibited a differentiation response, showed moderate methylation changes with 84 DMRs enriched at introns compared to whole coverage of mERRBS (Background [BG] 32% vs DMRs 56%; exact binomial test [EBT] p-value 5.053e-06). Thus, FLT3 inhibition did not appear to modify DNA methylation in these patients. However, and importantly, a direct comparison at baseline, prior to the administration of quizartinib, between patients who developed either differentiation or cytotoxic response, revealed robust epigenetic differences between these two groups of patients, with 580 DMRs. The majority of these (414 DMRs, 71%) were hypermethylated in patients with cytotoxic response. These DMRs were strongly depleted from promoter regions (BG 24% vs DMRs 9%; EBT p-value 2.2x10E-16) while they were enriched at intergenic regions (BG 35% vs DMRs 49%; EBT p-value = 1.064e-10). Given the preferential location of DMRs at these distal intergenic regions, we analyzed their relationship to enhancer elements. A total of 46.2% of DMRs (n=268) overlapped with ENCODE-defined enhancers (EBT p-value 3.57x10E-13 compared to 31.8% of BG). Pathway annotation of DMRs to the nearest genes revealed that they were associated with ion transmembrane transporter activity and extracellular matrix binding. In addition, amongst the DMRs were several annotated to HOX genes, including HOXB3 , HOXA7 and HOXD13 genes, which were hypomethylated in patients with a differentiation response. In summary, our analyses reveal that pre-existing epigenetic programs are encoded in FLT3 -mutant AML cells that correlate with the type of response they will undergo upon quizartinib treatment. The precise consequences of these epigenetic differences at the transcriptional level are still being explored and will be discussed at the meeting. We hypothesize that the pathologic role of FLT3 in AML may be modified by the epigenetic state of the transformed cells. DisclosuresPerl:Arog Pharmaceuticals: Consultancy; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Pfizer: Other: Advisory Board; Astellas: Consultancy; Asana Biosciences: Other: Scientific advisory board; Daiichi Sankyo: Consultancy; Novartis: Other: Advisory Board; Seattle Genetics: Other: Advisory board. Carroll:Incyte Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding.

Cost-Effectiveness of Midostaurin in the Treatment of Newly Diagnosed FLT3-Mutated Acute Myeloid Leukemia in the United States

IntroductionAcute myeloid leukemia (AML) is the most common form of acute leukemia in adults and is fatal if left untreated. Patients with FLT3-mutated AML account for approximately one-third of all AML patients and have a poorer prognosis than patients with FLT3 wild-type AML. Midostaurin has been shown to effectively improve overall survival (OS) and event-free survival (EFS) compared to existing regimens among newly diagnosed FLT3-mutated AML patients in the Phase III randomized controlled RATIFY trial. The current study aimed to assess the cost-effectiveness of midostaurin + cytarabine + daunorubicin (midostaurin arm) versus placebo + cytarabine + daunorubicin (placebo arm), considering direct medical costs and treatment benefits, in the treatment of adult patients newly diagnosed with FLT3-mutated AML who are eligible for standard cytarabine + daunorubicin chemotherapy from a US third-party payer perspective.MethodsCosts and benefits associated with the midostaurin arm and the placebo arm were estimated based on a partitioned survival model with four health states (active disease, complete remission [CR], relapse, and death) over a lifetime. Efficacy inputs (time to CR or death, time to relapse or death, and OS) for the two treatment arms were estimated using the RATIFY trial data. OS rates were adjusted using natural US mortality rates. Treatment costs (including drug and drug administration costs and hospitalization costs for induction and consolidation therapies), drug monitoring costs, stem cell transplant (SCT) procedure costs, adverse event (AE) costs, and medical costs associated with health states were obtained from publicly available databases, literature, and real-world database analysis. All costs were inflated to 2016 USD. Health state utilities were estimated based on a de novo utility study and expert opinions. Incremental costs per quality-adjusted life year (QALY) and life year (LY) gained were estimated comparing the midostaurin versus placebo arm. Deterministic sensitivity analysis (DSA) and probabilistic sensitivity analysis (PSA) were performed to assess the robustness of the model.ResultsCompared to the placebo arm, patients in the midostaurin arm incurred higher total direct costs ($4,043,470 vs. $3,959,741) over a lifetime. Specifically, the midostaurin arm was associated with higher treatment and monitoring costs ($251,883 vs. $187,282), higher medical costs associated with health states (due to longer OS; $3,523,343 vs. $3,500,095), lower SCT procedure costs (due to lower cumulative SCT rate in the midostaurin arm vs. the placebo arm in the RATIFY trial; $155,794 vs. $157,423), lower terminal care costs ($57,239 vs. $61,389), and higher AE costs ($55,210 vs. $53,551). Modeling efficacy based on the RATIFY trial, the midostaurin arm was associated with more LYs (11.44 vs. 9.85), and more QALYs (7.30 vs. 5.94) over a lifetime. In the model, when cost and effectiveness outcomes were combined, the midostaurin arm led to an incremental cost per LY gained of $52,596, and an incremental cost per QALY gained of $61,167 compared to the placebo arm over a lifetime. DSA results showed that the estimated incremental cost-effectiveness ratio (ICER) was generally consistent with the base case result. The model was most sensitive to the methods used to estimate efficacy outcomes, but not sensitive to the variations in midostaurin drug costs, other treatment costs, medical costs, and utility values. Results from the PSA based on the base case scenario showed that the probability of the midostaurin arm to be cost-effective compared to the placebo arm was 69.0% at a willingness to pay (WTP) of $175,000. In scenario analyses where cost effectiveness was modeled under the assumption that midostaurin was not used in the post-consolidation setting, midostaurin continued to be cost-effective compared to the placebo arm. In fact, it was shown to be the dominant strategy (i.e., the midostaurin arm was associated with lower costs and better effectiveness).ConclusionsCompared to the placebo arm, the midostaurin arm has a relatively low ICER over a lifetime, and thus should be considered as a cost-effective strategy in the treatment of adult patients with newly diagnosed FLT3-mutated AML from a US third-party payer perspective. DisclosuresStein:Pfizer: Consultancy, Other: Travel expenses; GSK: Other: Advisory Board, Research Funding; Seattle Genetics: Research Funding; Novartis: Consultancy, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Constellation Pharma: Research Funding. Xie:Analysis Group, Inc.: Employment; Celgene Corporation: Other: I am an employee of Analysis Group, which received consulting fees from Celgene for this study. Duchesneau:Analysis Group: Employment, Other: Author is an employee of Analysis Group, which has received consultancy fees from Novartis for this study. Bhattacharyya:Novartis Healthcare Pvt. Ltd.: Employment. Ndife:Novartis Pharmaceuticals Corporation: Employment. Bonifacio:Novartis Pharmaceuticals Corporation: Employment. Joseph:Novartis Pharmaceuticals Corporation: Employment; Pfizer: Other: stock/stock options; Amgen: Other: stock/stock options; Express Scripts: Other: stock/stock options.

Pre-Clinical Activity of SN37169, a Novel Hypoxia-Activated FLT3 Inhibitor Prodrug in FLT3-Mutated AML

Activating mutations in the Fms-like tyrosine kinase 3 gene (FLT3) are the most frequently observed molecular abnormalities in AML, which lead to overexpression or constitutive activation of the tyrosine kinase. While several mechanisms of resistance to FLT3 inhibitors have been identified, commonly failure is associated with lack of complete and sustained inhibition of FLT3 tyrosine kinase activity, with dose escalation limited by toxicities. Widespread hypoxia has been shown by us and others to be prevalent within the confines of the leukemic BM space, and to limit the efficacy of standard chemotherapy (Benito et al., PLoS One 6(8):e23108, 2011). With the goal of exploiting hypoxia as a physiological target and increasing selectivity to FLT3-mutant AML blasts, we have developed SN37169, a hypoxia-activated FLT3 inhibitor pro-drug based on an analogue of the known clinical compound Quizartinib (AC220). Bioreductive trigger conjugation was performed at the final step of the synthesis to provide SN37169 as previously described (Lu et al., Tetrahedron, 69, p9130-9138, 2013). Under hypoxia, SN37169 can undergo an enzymatic one-electron reduction and then fragment selectively to release the cell permeable SN37168 effector, generating higher local concentrations of active FLT3 inhibitor SN37168 and limiting normal tissue exposures.Pulse radiolysis was first used to determine the electron affinity (E(1)) and rate of fragmentation (kfrag) of SN37169 following one-electron reduction under hypoxia. SN37169 demonstrated E (1) and kfrag parameters ideal for hypoxia-selective cellular metabolism (-449 mV, 62 s-1, respectively).We have previously reported SN37169 to selectively inhibit FLT3 auto phosphorylation in MV4-11 cell line under hypoxic but not oxic conditions, with similar potency to the parental inhibitor (Quizartinib) under normal oxygen culture conditions (Cavazos et al. ASH 2016). We then examined the hypoxia-dependent anti-leukemia activity of SN37169 in 3 primary FLT3-mutated AML samples. SN37169 exhibited hypoxia enhanced apoptosis and growth arrest, with Hypoxia Cytotoxicity Ratios (Oxic/Hypoxic IC50 ratio) of 10.2, 9.9 and 2.8 (Figure1A).To test the efficacy of SN37169 in vivo, we injected NSGS mice with genetically engineered MOLM14/luc/GFP cells. Mice were randomized to treatment groups on day 7 when engraftment was documented by bioluminescent imaging (BLI), and treated daily with either vehicle or SN37169 at 42.3mg/kg/d IP for 4 weeks. BLI demonstrated 88-99% leukemia reduction during the course of SN37169 treatment compared to vehiclel (Figure 1B and 1C). Overall survival rate in each group was estimated by the Kaplan-Meier method. The median survival for vehicle group was 25 days while for SN37169 group was 56 days (p= 0.0043) (Figure 1D).To compare the efficacy of SN37169 with that of Quizartinib in vivo, we established an AML FLT3-ITD Patient Derived Xenograft (PDX) in NSG mice. A dose of 2.5 mg/kg/day Quizartinib in mice was estimated to provide a plasma PK profile comparable to 30 mg/day Quizartinib in human subjects based on calculated total AUC exposure across species (Zarrinkar et al., Blood 114:2984-2992, 2009). Weekly human CD45 engraftment analysis in peripheral blood using flow cytometry and daily body weight change measurements has demonstrated a significantly reduced leukemia burden in SN37169 treated group compared to vehicle (p < 0.0001) and Quizartinib (p < 0.0001) treatment groups with no indication of dose related toxicity (Figure 1E and 1F). The efficacy/PD/PK study is ongoing and median survival for all groups is yet to be determined.In summary, our data indicate hypoxia-enhanced inhibition of mutant FLT3 and its selected downstream signaling, hypoxia-dependent growth arrest and apoptosis in FLT3-mutant AML cells and notable in vivo activity of a novel hypoxia-activated FLT3 inhibitor SN37169. These findings support the functional role of hypoxia in the leukemia microenvironment and implicate potential clinical utility of hypoxia-activated kinase inhibitors with the goal of increasing efficacy and reducing toxicity. [Display omitted] DisclosuresDaver:Pfizer Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Jazz: Consultancy; Incyte Corporation: Honoraria, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Immunogen: Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Karyopharm: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding; Kiromic: Research Funding.

NPM1c Alters FLT3-D835Y Localization and Signaling Thereby Inducing a Fulminant Myeloproliferative Disorder in Mice

Activating mutations in FLT3 and NPM1 are the most common alterations in AML and frequently coincidental. The NPM1 mutation (NPM1c) dislocates the protein to the cytoplasm due to a disruption of the nuclear localization signal. In FLT3, two types of mutations are present: Tandem duplications of the juxtamembraneous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). In murine models, both NPM1c and FLT3-ITD induce a myeloproliferative disease (MPN), while FLT3-TKD expression alone is not sufficient to cause a myeloid disease in mice. Co-expression of NPM1c and FLT3-ITD rapidly induces an AML in C57Bl/6 mice. In the present study, we investigated the impact of NPM1c co-expression with the TKD mutation FLT3-D835Y and the molecular mechanism underlying the functional dependency of those two proteins.For this purpose, we used a heterozygous conditional Npm1c knockin mouse model, where bone marrow cells were harvested, retrovirally infected with MiG- Flt3-D835Y and injected into lethally irradiated C57Bl/6 wt mice. Lymphatic/leukemic organs of moribund mice were analyzed for their immune phenotype by flow cytometry. To investigate the molecular function and mechanism leading to disease induction by these oncogenes, intracellular flow cytometry as well as immunofluorescence imaging of primary murine cells were performed to analyze signaling and localization differences. In order to examine the clinical relevance, AML patient samples harboring either NPM1c and FLT3-TKD mutations or FLT3-TKD mutation in combination with wildtype NPM1 were analyzed by immunohistochemistry and flow cytometry for intracellular signaling mediators.Flt3-D835Y+ Npm1c+ mice rapidly developed an MPN with a median latency of 33 days, whereas Flt3-D835Y+ Npm1 wt mice did not develop a leukemic disease. To investigate the underlying mechanism leading to MPN induction in Flt3-D835Y+ Npm1c+ mice, we analyzed the activation of signaling transducer and activator of transcription (STAT) pathways in primary splenocytes of moribund mice. Strikingly, we were able to detect a profound activation of STAT5 in murine Flt3-D835Y+ Npm1c+ cells, which could not be seen in the Flt3-D835Y+ Npm1 wt cells. Next we asked for the clinical significance of these differential FLT3-TKD signaling capacities and performed immunohistochemistry stainings as well as intracellular flow cytometry of primary AML patient BM at initial diagnosis. In line with the findings in the murine model, immunohistochemic analyses showed significantly increased pSTAT5 signal in AML samples harboring both NPM1c and FLT3-TKD mutations, when compared to FLT3-TKD+ samples without NPM1 alteration. These results could be confirmed by intracellular pSTAT5 staining of primary human AML blasts, where STAT5 activation was significantly elevated in FLT3-TKD patients harboring an NPM1c mutation.To investigate the molecular mechanism underlying these differential signaling capacities we performed immunofluorescence and flow cytometry analyses of murine leukemic cells and could show a profound localization shift of FLT3-TKD from the cell surface to the endoplasmic reticulum only in cells harboring the NPM1c mutation. In NPM1 wt cells FLT3-TKD could exclusively be detected at the cell surface. These results could be confirmed by immunoblotting analyses, where we detect an underglycosylated, intracelullar FLT3-TKD 130 kDa-form in NPM1c+ cells while it is found as a highly glycosylated, cell-surface bound 150 kDa form in cells lacking NPM1c.In the present study, we could demonstrate that co-expression of Npm1c and Flt3-D835Y induces a rapid-onset MPN in C57Bl/6 mice. Interestingly, the presence of NPM1c also alters the cellular localization of FLT3-TKD from the cell surface to the endoplasmic reticulum, and thereby leads to STAT5 activation by FLT3-D835Y. We could further show that STAT5 activation not only occurs in murine cells but also in AML patients with FLT3-TKD in combination with NPM1c mutation. Thus, our data indicate that the NPM1c mutation shifts the signaling properties of FLT3-TKD towards those of the FLT3-ITD mutation, which may explain its occurrence in myeloid disorders and suggest that STAT5 might be a potential unique therapeutic target in FLT3-mutated AML. DisclosuresNo relevant conflicts of interest to declare.

Comparison of Induction Chemotherapy in NSG and NOD- Rag1 null IL2rg null Mouse Models of FLT3 Mutant AML

Background and objectives: Induction chemotherapy that combines Ara-C (cytarabine, 7 days) with an anthracycline such as daunorubicin or doxorubicin (Adriamycin, 3 days) (DA) is a standard induction therapy in AML. Immunodeficient NSG or NSGS mice are extremely useful for preclinical drug testing and are promiscuous recipients of primary AML engraftment. However, these models have limitations for use of genotoxic chemotherapy that often induces toxicity and mortality to the recipient mice, particularly in AML patient-derived xenograft (PDX) models when irradiation is required. The sensitivity of NSG or NSGS mice to genotoxic drugs and irradiation are due to SCID mutation in the mice. In this study we have compared tolerability and efficacy of DA chemotherapy in NSG and NRG (NOD- Rag1 null IL2rg null) AML-bearing mice. We used a FLT3 mutant AML Molm13 cell line to generate AML xenograft in NRG and NSG mice and then tested the response to chemotherapy using combined Ara-C and doxorubicin chemotherapy with or without sub-lethal irradiation. Testing of chemotherapy regimen in human AML PDX models is ongoing and will be presented.Methods and results: The AML mouse models were established by i.v. injection of GFP and firefly luciferase transduced Molm13 cells (0.6 x 106 cells per mouse) into NSG and NRG mice (n=5 per group, F, 8 weeks old). In experiments testing sub-lethal radiation, NSG mice were irradiated with 200 cGy and NRG mice with 400 cGy before cell injection. AML cell engraftment and growth in vivo was monitored by bioluminescent imaging (BLI). Engraftment was confirmed on day-3 (Fig. 1A-B) and chemotherapy started on day-4 using combined Ara-C (50 mg/kg, i.v. 5 days) and doxorubicin (1.5 mg/kg, i.v., 3 days) (DA 5+3) or PBS. NSG and NRG mice (n=3 each) that served as naïve controls without bearing Molm13 cells also received the chemotherapy. Mouse health conditions and body weights were monitored daily, and mice were followed for survival.As shown by BLI in Figure 1 A-B, DA 5+3 significantly reduced leukemic burden in both NRG and NSG mice compared to PBS treated mice (p<0.001 on day 13 or 14). The chemotherapy also induced transient toxicity in all treated mice with significant body weight loss in the sub-lethal irradiated AML NSG mice (Figure 1C, 30% loss on day 10, p=0.016 vs PBS treated mice), resulting in lethality earlier than PBS treated mice (Figure 1D, p=0.0019). Complete blood cell counts showed pancytopenia with anemia of the moribund treated Molm13 NSG mice. Overall, chemotherapy induced more body weight loss in NSG than that in NRG mice (a transient 25% body weight loss in naive NSG and AML NSG mice compared to 10% loss in NRG mice on day 10), indicating that NSG mice are more susceptible to chemotherapy-induced toxicity. All AML NRG mice and NSG mice without prior sub-lethal irradiation survived the chemotherapy and had significantly prolonged survival.We further challenged the NRG mice with a more intensive chemotherapy regimen by administrating Ara-C for 7 days (DA 7+3) mimicking the one used in AML patients. BLI confirmed more effective leukemia control by this regimen compared to DA 5+3 and significantly extended survival.Conclusion: The results demonstrate that Molm13 bearing NSG and NRG mice have identical latency, leukemia growth kinetics, and survival, suggesting that like NSG mice, NRG mice are suitable recipients for AML xenograft studies. Both Molm13 NSG mice and NRG mice showed strong efficacy in response to chemotherapy using Ara-C (cytarabine, 50 mg/kg) and doxorubicin (1.5 mg/kg) with the regimen of DA 5+3. However, this regimen was not tolerable when mice required prior sublethal irradiation, with excessive bodyweight loss and mortality in NSG but not in NRG mice. In contrast, Molm13 NRG mice can tolerate a more intensive combination chemotherapy regimen of AraC administered for 7 days (DA 7+3). In summary, NRG mouse model showed better tolerance to chemotherapy and irradiation than the NSG model, thus representing a superior in vivo AML model for pre-clinical studies with genotoxic agents. [Display omitted] DisclosuresCarter:Daiichi Sankyo: Research Funding; novartis: Research Funding.

Phase II Trial of Vosaroxin and Infusional Cytarabine for Frontline Treatment of Acute Myeloid Leukemia (VITAL) Patients Eligible for Intensive Chemotherapy: A Planned Interim Analysis

Phase II Trial of Vosaroxin and Infusional Cytarabine for Frontline Treatment of Acute Myeloid Leukemia (VITAL) Patients Eligible for Intensive Chemotherapy: A Planned Interim Analysis

Phase 1 first-in-human study of irreversible FLT3 inhibitor FF-10101-01 in relapsed or refractory acute myeloid leukemia.

7008 Background: FF-10101-01 is a selective and irreversible FMS-like tyrosine kinase 3 (FLT3) inhibitor with potent in vitro activity against FLT3-mutated AML. FF-10101-01 is highly active against FLT3 internal tandem duplication (ITD) mutations associated with high relapse and low survival/remission rates, as well as resistance-conferring D835 and F691 tyrosine kinase domain (TKD) and non-canonical FLT3 activating mutations. Here we report on a Phase 1 dose escalation trial examining the safety, efficacy, pharmacokinetics, and pharmacodynamics of FF-10101-01 in patients (pts) with relapsed/refractory primary or secondary AML. Methods: To determine the recommended Phase 2 dose, pts with or without a FLT3 mutation received FF-10101-01 orally once (QD) or twice (BID) daily until unacceptable toxicity was observed or pts had no further clinical benefit (1 cycle = 28 days). Composite complete remission (CRc) and partial remission (PR) rates were assessed. Inhibition of FLT3 phosphorylation was evaluated using a plasma inhibitory activity assay and was correlated with associated FF-10101-01 exposure. Results: Fifty-two pts [median age 61 (range, 21-84); 52% female; FLT3: ITD [22 (42%)], TKD [5 (10%)], ITD+TKD [1 (2%)], Wt [24 (46%)] received continuous dosing of FF-10101-01 at 10 - 225 mg QD or 50 - 100 mg BID. Median number of prior therapies was 3 (range, 0-6) and the majority [23/28 (82%)] of pts with known FLT3 mutations had received prior FLT3 inhibitors. The median duration on study was 5.7 (range, 0.1-36) weeks. FF-10101-01 was generally well-tolerated up to total daily doses of 150 mg. The most common treatment related adverse events included nausea [n = 18 (35%)] diarrhea [14 (27%), 2 Grade (Gr) 3/4], elevations in creatine kinase [CK; 14 (27%), 4 Gr 3/4], vomiting [10 (19%)] and increased AST [10 (19%), 2 Gr 3]. Grade 3/4 differentiation syndrome (n = 4, 8%) was observed at 75 - 150 mg/day. Dose-limiting cardiac toxicity (heart failure with reduced ejection fraction; Gr 3 increased troponin/CK) was observed at total daily doses ≥200 mg. The CRc rate was 13% (4/30 pts evaluable for response): 1 CR at 75 mg BID (FLT3-ITD); 1 CRp at 100 mg BID (Wt-FLT3); and 2 CRi’s at 50 mg BID, one that previously progressed on gilteritinib. The median time to overall response was ̃13.3 weeks. Four pts achieved a PR (≥50% decrease in BM blasts to 5 - 25% abnormal cells) at total daily doses of 50 - 150 mg; 2 had ITD mutations, and all had received prior FLT3 kinase inhibitors. At ≥75 mg BID, trough plasma concentrations were &gt; 90 ng/ml and associated with &gt; 90% p-FLT3 inhibition maintained over the dosing interval. Conclusions: The FF-10101-01 FLT3 inhibitor has shown activity in pts with refractory/relapsed AML, including those with activating FLT3-ITD mutations resistant to gilteritinib and other FLT3 kinase inhibitors. Doses of 50-75 mg BID were well tolerated and resulted in sustained FLT3 inhibition. Clinical trial information: NCT03194685.

58 - The Role of Cytogenetic Risk Stratification in FLT3 Mutated NPM1 Negative AML Patients Undergoing Allogeneic Stem Cell Transplantation (alloSCT) in Remission: A Study on Behalf of the ALWP of the EBMT

58 - The Role of Cytogenetic Risk Stratification in FLT3 Mutated NPM1 Negative AML Patients Undergoing Allogeneic Stem Cell Transplantation (alloSCT) in Remission: A Study on Behalf of the ALWP of the EBMT