Quizartinib Treatment Research Articles

Dobutamine Enhances the Targeted Inhibitory Effect of Quizartinib on FLT3-ITD Mutant Acute Myeloid Leukemia

To observe the inhibitory effect of dobutamine on proliferation of FLT3-ITD mutated acute myeloid leukemia (AML) cells and explore the feasibility of dobutamine as a monotherapy or in combination with quizartinib for the treatment of this type of AML. FLT3-ITD mutant cell lines MOLM13 and MV4-11 were cultured in vitro and divided into control group, dobutamine treatment group, quizartinib treatment group, and dobutamine combined with quizartinib treatment group. Cell viability, ROS levels, and apoptosis rate were detected by CCK-8, Flow cytometry, respectively, as well as the expression of YAP1 protein by Western blot. Both dobutamine and quizartinib inhibited the proliferation of FLT3-ITD mutant AML cell lines. Compared with the control group, the dobutamine group exhibited a significant increase in ROS levels (P < 0.01), an increase in apoptosis rates (P < 0.05), and a decrease in YAP1 protein expression (P < 0.01), and decreased YAP1 expression (P < 0.05). Dobutamine as a monotherapy can inhibit theproliferation of FLT3-ITD mutated AML cells, inducing apoptosis. Additionally, the combination of quizartinib enhances the targeted inhibitory effect on FLT3-ITD mutated AML. The mechanism may involve the inhibition of YAP1 protein expression in AML cells of this type, leading to an increase in ROS levels and exerting its anti-tumor effects.

Key role of glutamine metabolism in persistence of leukemic cells upon exposition to FLT3 tyrosine kinase inhibitors

Acute myeloid leukemias are a group of hematological malignancies characterized by a poor prognosis for survival. The discovery of oncogenic mutations in the FMS-like tyrosine kinase 3 (FLT3) gene has led to the development of tyrosine kinase inhibitors such as quizartinib. However, achieving complete remission in patients remains challenging because these new tyrosine kinase inhibitors (TKIs) are unable to completely eradicate all leukemic cells. Residual leukemic cells persist during quizartinib treatment, leading to the rapid emergence of drug-resistant leukemia. Given that mitochondrial oxidative metabolism promotes the survival of leukemic cells after exposure to multiple anticancer drugs, we characterized the metabolism of leukemic cells that persisted during quizartinib treatment and developed metabolic strategies to eradicate them. In our study, employing biochemical and metabolomics approaches, we confirmed that the survival of leukemic cells treated with FLT3 inhibitors critically depends on maintaining mitochondrial metabolism, specifically through glutamine oxidation. We uncovered a synergistic interaction between the FLT3 inhibitor quizartinib and L-asparaginase, operating through antimetabolic mechanisms. Utilizing various models of persistent leukemia, we demonstrated that leukemic cells resistant to quizartinib are susceptible to L-asparaginase. This combined therapeutic strategy shows promise in reducing the development of resistance to FLT3 inhibitors, offering a potential strategy to enhance treatment outcomes.

The Sensitization of Murine Hematopoietic Progenitors and Stem Cells to DNA Damaging Agents By Quizartinib Is Associated with the Transcriptional Downregulation of DNA Damage Repair Genes

Background: The FLT3 inhibitor quizartinib induces quiescence of murine hematopoietic progenitors, and this affords their protection from S phase specific chemotherapies. In this study we investigated whether quizartinib can also protect hematopoietic progenitors from the cytotoxic effects of DNA damaging chemotherapies and radiation. Methods: C57BL/6 mice were primed with 30 mg/kg of quizartinib or vehicle (5% cyclodextrin) by oral gavage, and treated with DNA damaging chemotherapies, either cyclophosphamide, carboplatin, cisplatin, temozolomide or mitoxantrone 6 hours later, or 5.5Gy total body irradiation (TBI) after 9 hours. This TBI dose represents half the 11 Gy split dose used for bone marrow conditioning of C57BL/6 mice prior to HSC transplantation. Bone marrow cells were analyzed by flow cytometry after 3 to 6 days to identify progenitor and stem cell populations. Results: In contrast to quizartinib's protection against the cytotoxic effects of 5-FU and gemcitabine, all 5 DNA-damaging chemotherapies caused an increased loss of total bone marrow cellularity when mice were primed with quizartinib compared to vehicle primed controls. Flow cytometry analysis revealed that the numbers of myeloid and multipotent progenitors, and short-term and long-term hematopoietic stem cells (HSCs), were markedly lower in the quizartinib-treated mice. DNA damage and the resultant cell death caused by ionizing radiation is cell cycle specific, particularly for cells traversing from G 1 to S phases of the cell cycle. The ability of quizartinib to induce quiescence in myeloid and multipotent progenitors suggested that quizartinib may protect these cells from radiation. To test this, mice were dosed with vehicle or 30 mg/kg of quizartinib and 9 hours later exposed mice to 5.5 Gy of TBI. Analysis of bone marrow cells at day 6 revealed that total bone marrow cellularity and the numbers of progenitor and stem cells were markedly reduced in quizartinib-dosed mice compared to vehicle controls. For subsequent experiments mice were treated with 3 Gy TBI because the combination of quizartinib and 5.5 Gy caused severe myeloablation. As part of a series of experiments examining the kinetics of quizartinib's sensitization of bone marrow cells we tested whether the sensitization was maintained when mice were dosed with quizartinib 24 hours prior to 3 Gy TBI. Analysis of bone marrow cells 6 days later revealed that quizartinib dosed mice showed a significantly greater loss of total bone marrow cells, progenitor cells and stem cells compared to vehicle dosed mice (Figure 1). To identify possible mechanisms by which quizartinib sensitizes hematopoietic progenitors to DNA damaging agents we investigated quantitative gene expression profiling of the HPC7 hematopoietic progenitor cell line. These cells provide a suitable in vitro model since quizartinib induces their quiescence and provides protection against 5-FU. HPC7 cells were treated with 100 µM quizartinib, or vehicle, for 18 hr before RNA was harvested and sent for Next-generation sequencing. We found that quiescence induction in HPC7 cells by quizartinib correlated with the transcriptional downregulation of a wide range of genes involved in DNA damage repair pathways (including Brca1/2, Rad51, Palb2, Chek1 and Nbn), thus providing a possible mechanism for the enhanced cytotoxic effects of DNA damaging agents. Conclusions: Quizartinib sensitizes hematopoietic progenitors and stem cells to DNA-damaging agents, an effect that correlates with the transcriptional downregulation of many DNA damage repair genes. The enhanced depletion of hematopoietic progenitors and stem cells by DNA damaging agents in the presence of quizartinib has important clinical implications. Administering concurrent DNA damaging chemotherapy or radiotherapy with quizartinib treatment should be avoided. However, this interaction could facilitate a reduction in radiation/chemotherapy doses administered for bone marrow conditioning prior to HSC transplantation. By reducing these doses, but not compromising the conditioning process, non-hematopoietic side effects could be minimized and may allow currently excluded patients to be considered for HSC transplantation. Disclosure: This study was partly funded by Daiichi Sankyo.

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.

FLT3-ITD Activation Mediates Resistance to the BCL-2 Selective Antagonist, Venetoclax, in FLT3-ITD Mutant AML Models

FLT3 internal tandem duplication (FLT3-ITD) mutations account for ~20-25% of adult AML cases and are associated with worse prognosis. FLT3-ITD is a constitutively activated kinase with differential signaling compared to wild type (WT) FLT3. Notably, FLT3-ITD activates STAT5, which can regulate the pro-survival proteins BCL-XL and MCL-1. Venetoclax (ABT-199/GDC-0199), a potent, selective inhibitor of the pro-survival protein BCL-2, recently demonstrated monotherapy activity in relapsed/refractory AML (ORR 19%); however, no activity was seen in FLT3 mutant cases. BCL-XL and MCL-1 are known venetoclax resistance factors; therefore targeting their regulation in combination with venetoclax may enhance cell death. Based on this hypothesis, we interrogated the combination of venetoclax and quizartinib (AC220), a potent FLT3 inhibitor, in FLT3-ITD+ AML models.Sensitivity to venetoclax was assessed in FLT3-ITD+ (Molm13 and MV4;11) and FLT3 WT (HL60 and OCI-AML3) cells in vitro . Dose dependent growth inhibition and induction of apoptosis was seen in MV4;11, Molm13 and HL60 cells following 48hr venetoclax treatment, with the MV4;11 cells being the most sensitive to venetoclax. Twenty-four hour quizartinib treatment reduced expression of BCL-XL and MCL-1 protein, but not BCL-2, in FLT3-ITD+ cells, while quizartinib had no effect on BCL-XL, MCL-1 or BCL-2 expression in FLT3 WT cells, confirming FLT3-ITD mediated regulation of BCL-XL and MCL-1. To interrogate combined FLT3-ITD and BCL-2 inhibition, cells were treated for 48hrs with venetoclax, quizartinib or the combination. Combination treatment led to significant reduction in growth and increased apoptosis in FLT3-ITD+ cells compared to either single agent. FLT3 WT cells were not sensitive to quizartinib alone or in combination with venetoclax. To determine the dependence of FLT3-ITD+ cells on BCL-XL and MCL-1, cells were treated with selective BCL-XL (A1331852) or MCL-1 (A1210477) inhibitors in combination with venetoclax. Treatment with either of the selective inhibitors in combination with venetoclax reduced the growth of cells compared to single agents alone, suggesting that all three pro-survival proteins may contribute to the survival of FLT3-ITD+ cells. Consistent with FLT3-ITD regulating BCL-XL and MCL-1, co-treatment of quizartinib with either BCL-XL or MCL-1 inhibitor had no combination effect in FLT3-ITD+ cells. To elucidate the signaling pathway(s) regulated by FLT3-ITD that confer resistance to venetoclax, cells were treated with PI3K, MEK or JAK2 kinase selective inhibitors alone or in combination with venetoclax. Each pathway inhibitor combined with venetoclax to reduce cell growth when compared to each agent alone, indicating that multiple signaling pathways can contribute to venetoclax resistance rather than a single pathway dependence for driving the pro-survival effects of FLT3-ITD. This data underscores the reliance of these models on FLT3-ITD for survival and regulation of multiple survival pathways that confer resistance to venetoclax, suggesting FLT3-ITD itself could be a predictive biomarker of venetoclax resistance.Further, the combination of quizartinib and venetoclax was assessed in vivo utilizing MV4;11 and Molm13 xenograft models grown orthotopically. Mice were treated with venetoclax (100 mg/kg), quizartinib (5 mg/kg) or the combination for 21 continuous days. Quizartinib treatment significantly increased survival compared to vehicle in both models [56 days vs. 26 days (p<0.0001) and 32 days vs. 14 days (p<0.0001) for MV4;11 and Molm13, respectively]. However, venetoclax alone only marginally improved survival [34 days vs. 26 days (p<0.0001)] in the MV4;11 model and was not efficacious in the Molm13 model. Despite lack of single agent activity, addition of venetoclax to quizartinib led to a further improvement in survival when compared to quizartinib alone [91 days vs. 56 days (p<0.0001) and 40 days vs. 32 days (p<0.0001) in MV4;11 and Molm13, respectively]. Monotherapy and combination treatments were tolerated based on minimal changes in animal body weight throughout the duration of the study.Taken together, our data demonstrate that FLT3-ITD inhibition combines with venetoclax in vitro and in vivo in FLT3-ITD+ AML models. These results provide a strong mechanistic rationale to support clinical investigation of FLT3 inhibitors in combination with venetoclax to treat FLT3-ITD+ AML. [Display omitted] DisclosuresMali:Genentech: Employment. Lasater:Genentech: Employment. Doyle:Abbvie: Employment. Malla:Genentech: Employment. Boghaert:Abbvie: Employment. Souers:Abbvie: Employment.

Dysregulation of Follistatin Promotes Leukemogenesis in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an abnormal increase of myeloblasts in bone marrow (BM) and peripheral blood (PB). Chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However, these approaches have reached an impasse and despite an initial remission, leukemia relapses are frequent, with an overall cure rate of only 30-40% (Rowe and Tallman 2010). Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene is the commonest mutation in AML, occurring in nearly 30% cases with inferior disease-free and overall survivals (Boissel et al. 2002; Meshinchi et al. 2001). FLT3-ITD as a therapeutic target has been tested in phase I/II clinical trials (Man et al. 2012; Fischer et al. 2010; Levis et al. 2011; Cortes et al. 2011). In most circumstances, there was an initial response to FLT3 inhibitors with reduction or clearance of myeloblasts in BM and PB. However, resistance invariably developed despite continuous treatment (Kindler, Lipka, and Fischer 2010). Therefore, the identification of novel therapeutic targets for FLT3-ITD+ AML becomes urgent.In our previous study (He et al. 2014), we showed that over-expression of human leukemic gene FLT3-ITD resulted in significant myeloid cells expansion which could be effectively rescued by FLT3-ITD inhibitor AC220 treatment. Interestingly, a proportion of zebrafish embryos expressing FLT3-ITD showed double-headed and axis-duplication phenotype as shown by the whole mount in situ hybridization (WISH) of a notochord-specific marker col9a2 . Mechanistically, FLT3-ITD-induced axis-duplication was correlated with the expansion of Spemann's Organizer as shown by the ectopic expression of Spemann's Organizer-specific marker goosecoid (gsc) by WISH as well as by the -2,067gsc-GFP reporter assay. It has been reported that some key signaling pathways are conserved between embryogenesis and tumorigenesis. We hypothesized that certain Spemann's Organizers were dysregulated in FLT3-ITD+ AML.To test this hypothesis, we performed an in silico analysis to quantify the gene expression of Spemann's Organizers in normal HSC and AML by the public database BloodSpot. Intriguingly, the Activin antagonist Follistatin (FST) was significantly increased in hematological malignancies, comparing to the normal HSC. In human malignancies, FST has been shown to express in prostate (Sepporta et al. 2013; Tumminello et al. 2010), ovarian (Ren et al. 2012; Karve et al. 2012; Di Simone et al. 1996), and liver cancers (Patella et al. 2006; Rossmanith et al. 2002) promote cancer cell growth (Gao et al. 2010; Rossmanith et al. 2002). However, the role of FST in human AML and other hematological malignancies is currently unknown. Firstly, we confirmed the upregulation of FST in AML by real-time quantitative PCR, Western Blotting, and immunofluorescence imaging. Mechanistically, FLT3-ITD up-regulated FST via the phosphorylation of CREB as demonstrated by ChIP-PCR. Moreover, the upregulation of FST by FLT3-ITD was effectively blocked by the pharmacological inhibition of CREB in a dosage-dependent manner. FST gene knock-down (by shRNA) or knock-out (by CRISPR/Cas9) in FLT3-ITD+ MOLM-13 AML cell line significantly reduced leukemic growth in vitro, reduced leukemic burden, and prolonged survival of mice in the xenotransplantation experiment. Over-expression of FST in MOLM-13 and ML-2 AML cell lines promoted leukemic growth in vitro and in vivo xenograft mouse model via AKT/p70S6K/mTOR signaling. Furthermore, proteins (RPS19, RPS6, RPS13, RPL6, RPS21, RPSA, RPS18, RPLP2, RPL10A, WIBG, and PPP2R1A) associated with nonsense-mediated decay of mRNA were decreased after FST over-expression in ML-2 AML cells, while the chemoresistant and anti-apoptotic protein CD44 was consistently increased by a label-free proteomics analysis. Notably, the serum FST levels were significantly increased in a patient-derived xenograft AML mouse model. Consistently, plasma FST levels from FLT3-ITD+ AML patients were significantly reduced after an FLT3-ITD inhibitor quizartinib treatment in a phase I/II clinical trial.In conclusion, the CREB targeted gene FST promotes AML cell growth via AKT/p70S6K/mTOR signaling and CD44-mediated anti-apoptosis. Therefore, FST may be a promising therapeutic target for AML treatment. DisclosuresNo relevant conflicts of interest to declare.

Clinical Outcomes in Patients with FLT3-ITD-Mutated Relapsed/Refractory Acute Myelogenous Leukemia Undergoing Hematopoietic Stem Cell Transplantation after Quizartinib or Salvage Chemotherapy in the QuANTUM-R Trial

Despite the substantial clinical activity of fms-related tyrosine kinase 3 (FLT3) inhibitors in relapsed or refractory (R/R) FLT3-ITD‒positive acute myelogenous leukemia (AML), durable remissions and prolonged survival in this population require allogeneic hematopoietic stem cell transplantation (allo-HSCT). Quizartinib, a once-daily oral, highly potent, and selective FLT3 inhibitor, significantly prolonged overall survival (OS) and improved clinical benefit compared with salvage chemotherapy (median OS, 6.2 months versus 4.7 months; hazard ratio [HR], .76; 95% confidence interval [CI], .58 to .98; P=.018; composite complete remission [CRc] rate, 48% versus 27%; median duration of CRc, 2.8 months versus 1.2 months; mortality rate, .8% versus 14% by day 30, 7% versus 24% by day 60) in patients with R/R FLT3-ITD AML in the phase 3 QuANTUM-R trial. In this post hoc analysis, we described the characteristics of and clinical outcomes in patients who underwent on-study HSCT in QuANTUM-R at the investigator's discretion and institutional practices. Of 367 randomized patients, 78 (32%) in the quizartinib arm and 14 (11%) in the salvage chemotherapy arm underwent on-study allo-HSCT without any intervening therapy for AML after quizartinib or study-specified salvage chemotherapy. Pooled data of patients from both treatment arms showed a longer median overall survival (OS) in transplant recipients versus those treated without allo-HSCT (12.2 months versus 4.4 months; HR, .315; 95% CI, .233 to .427). Pooled data also showed a longer median OS in patients with a last recorded response of CRc before allo-HSCT versus patients without a CRc (20.1 months versus 8.8 months; HR, .506; 95% CI, .296 to .864). By treatment arm, the median OS was 25.1 months with quizartinib and 20.1 months with salvage chemotherapy in patients with a last recorded response of CRc before allo-HSCT. Forty-eight patients in the quizartinib arm continued quizartinib treatment after allo-HSCT. In the 31 patients with a last recorded response of CRc before allo-HSCT who continued quizartinib after allo-HSCT, the median OS was 27.1 months. Continuation of quizartinib after allo-HSCT was tolerable, and no new safety signals were identified. These results suggest that post-transplantation survival following salvage chemotherapy and quizartinib treatment are similar. However, quizartinib response occurs more frequently than with salvage chemotherapy, potentially allowing more patients to undergo transplantation and achieve durable clinical benefit. In addition, post-transplant quizartinib was found to be tolerable and may be associated with prolonged survival in some patients, highlighting its potential value in the management of patients with FLT3-ITD R/R AML.

Antimetabolic Cooperativity with l-Asparaginase and Tyrosine Kinase Inhibitor Quizartinib to Eradicate Persistant FLT3-ITD Leukemic Cells

Introduction Acute myeloid leukemias are a group of malignant hemopathies characterized by a poor prognosis for survival. The discovery of oncogenic mutations in the FLT3 gene (eq FLT3-ITD) has led to the development of new tyrosine kinase inhibitors such as quizartinib. But complete remissions of patients remains difficult because these new TKIs are not able to completely eradicate all leukemia cells. Residual leukemia cells persist during treatment with quizartinib and lead to the rapid emergence of drug-resistant leukemia. Since mitochondrial oxidative metabolism supports the survival of leukemia cells after exposure to several anticancer drugs, we characterized the metabolism of leukemia cells that persisted within quizartinib treatment and developed metabolic strategies to eradicate them. Results First, we evaluated glycolysis activity in FLT3-ITD leukemia cell lines (MOLM13 / MOLM14 / MV4-11) under quizartinib treatment (5-10nM). Quizartinib reduced extracellular acidification rate ECAR, but this glycolytic activity is not fully inhibited (50% of untreated condition). These results obtained using the XFe24 Seahorse were in agreement with the metabolomic analysis carried out in a medium containing isotopic U-13C6 glucose. Next we evaluated mitochondrial oxidative phosphorylation in FLT3-ITD leukemia cell lines. After treatment with quizartinib, the basal and maximal oxygen consumption (OCR) of leukemia cells decreased. Metabolomic analysis using isotopic glucose U-13C6 or glutamine U-13C5 have shown that pyruvate derived from glucose was weakly oxidized in the mitochondria of untreated or quizartinib-treated cells. In contrast, a large amount of glutamine was oxidized by the tricarboxylic acid (TCA) cycle in untreated FLT3-ITD cells. Quizartinib reduced but did not abolish the complete oxidation of glutamine in leukemia cells. This result showed that even in the presence of quizartinib, FLT3-ITD cells maintained partially oxygen consumption trough glutamine oxidation. L-asparaginases (Kidrolase, Erwinase) are enzymes capable of hydrolyzing amino acids such as asparagine and glutamine. These clinical drugs have been approved for the treatment of chronic lymphocytic leukemia (CLL) and pediatric acute myeloid leukemia. We have shown that L-asparaginases weakly induced cell death in FLT3-ITD leukemia cells. Interestingly, our isobologram analysis showed that L-asparaginase acted synergistically with quizartinib to induce apoptosis. To determine whether glutamine metabolism also promoted the persistence of AML under treatment with quizartinib, we treated MOLM13 with quizartinib for several days. After long-term treatment, the percentage of surviving cells (annexin-V negative) was less than 5%. These persistent cells were characterized by an increased mitochondrial membrane potential (Δψm) and mitochondrial ROS. After treatment with the combination of L-asparaginase and quizartinib, the percentage of persistent cells decreased drastically. The combination of L-asparaginase and quizartinib was also more effective than quizartinib alone in reducing the size and number of colonies of MOLM13 in a model based on the formation of leukemia colonies growing in methylcellulose. Conclusion Persistent leukemia cells that survive after exposure to FLT3 inhibitor quizartinib can be targeted by the clinical drug L-asparaginases. This metabolic strategy could reduce the emergence of leukemic cells resistant to quizartinib. Disclosures Kluza: Daiichi-Sankyo: Research Funding.

FF-10101 Retains Potent Inhibitory Activities Against Resistant Mutations to FLT3 Inhibitors, Newly Identified in Random Mutagenesis Screens

Background: FLT3-internal tandem duplication (FLT3-ITD) mutation is a poor prognostic factor for acute myeloid leukemia (AML). Several second-generation FLT3-targeted tyrosine kinase inhibitors with high selectivity and potency have been developed to date. Recently, gilteritinib was approved for the FLT3 mutation-positive relapsed or refractory AML patients. However, acquired mutations at the F691 residue in FLT3 kinase domain were identified in the patients who had disease progression after the treatment with gilteritinib, as with quizartinib treatment that caused resistant mutations at F691 and D835 residues. Therefore, it is very important to clarify the potencies of each FLT3 inhibitors against acquired FLT3 mutations for clinically selecting an appropriate FLT3 inhibitor according to the mutation type. In this study, we explored the resistant mutations against FLT3 inhibitors by random mutagenesis analysis and evaluated the cross-reactivity of FLT3 inhibitors against each resistant mutation. Methods: For random mutagenesis assay, 32D cells were infected with retroviruses encoding randomly mutagenized human FLT3-ITD. FLT3-ITD dependent 32D cells were established without an addition of IL-3, and then treated with FLT3 inhibitors, gilteritinib, FF-10101 and quizartinib, at concentrations of GI95 and 3 x GI95. Two weeks after treatment, full length FLT3-ITD sequences of viable clones were analyzed. The identified mutated FLT3-ITDs were introduced into 32D cells to confirm the resistance to FLT3 inhibitors. For cell growth assay, 32D transfectants were incubated with 5 FLT3 inhibitors (gilteritinib, FF-10101, quizartinib, crenolanib and midostaurin) for 3 days followed by determination of cell viability. Results: We identified the gilteritinib-resistant mutation (FLT3-ITD+D698N), quizartinib-resitant mutation (FLT3-ITD+N676T), and FF-10101-resistant mutation (FLT3-ITD+C695W) (Table). Inhibitory activity of gilteritinib against FLT3-ITD+D698N-expressing 32D cells (GI50, 27 nM) was decreased by 12-fold as compared with that against original FLT3-ITD-expressing 32D cells (GI50, 2.3nM). FF-10101 (GI50, 0.73 nM), quizartinib (GI50, 0.99 nM) and crenolanib (GI50, 19 nM) retained potency against FLT3-ITD+D698N, while midostaurin (GI50, 47 nM) did not have a potency. In FLT3-ITD+N676T-expressing 32D cells, inhibitory activities of quizartinib and midostaurin were decreased by 11 and 15-fold (GI50, 6.6 nM and 83 nM, respectively), while FF-10101 (GI50, 0.73 nM), gilteritinib (GI50, 6.6 nM) and crenolanib (GI50, 19 nM) retained potency. In FF-10101-resistant mutation (FLT3-ITD+C695W)-expressing 32D cells, the other inhibitors retained growth inhibitory activity. Conclusions: Resistant mutations to gilteritinib and quizartinib were newly identified in FLT3 kinase domain by random mutagenesis analysis. FF-10101 retained potent inhibitory activities against FLT3-ITD+N676T conferring resistance to quizartinib and midostaurin, and FLT3-ITD+D698N resistant to gilteritinib and midostaurin, although FF-10101 was vulnerable to FLT3-ITD+C695W substituted for C695 residue which forms covalent bond with FF-10101. These results indicated that FF-10101 was a promising agent for the treatment of patients with AML with FLT3 inhibitor-resistant mutations newly identified in this study. Disclosures Ishikawa: Bristol-Myers Squibb: Honoraria; Abbvie GK.: Honoraria; Celgene Co., Ltd.: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria. Saito:FUJIFILM Corporation: Employment. Morimoto:FUJIFILM Corporation: Employment. Murao:FUJIFILM Corporation: Employment. Terada:FUJIFILM Corporation: Employment. Yamaura:FUJIFILM Corporation: Employment. Hagiwara:FUJIFILM Coporation: Employment. Kiyoi:Daiichi Sankyo Co., Ltd: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Research Funding; Perseus Proteomics Inc.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding.

Individualized Mitochondrial Functional Approach to Combination of BCL-2 and MCL-1 Antagonism in Acute Myeloid Leukemia

BCL-2 antagonist venetoclax combined with hypomethylating agents or low-dose cytarabine is a new standard of care for treatment-naive elderly or unfit AML patients. Despite the striking overall response of 70%, only 30% achieved MRD negative status with a short remission duration of 11.3 months. This highlights the need for new agents that can overcome venetoclax resistance. We exploited mitochondrial functional assay called BH3 profiling to identify predictors of clinical responses and to discover combination partners of venetoclax. BH3 profiling measures apoptotic priming of a cell (proximity to the apoptotic threshold) by measuring mitochondrial response to a standardized panel of pro-apoptotic BH3 oligopeptides. By using pretreatment myeloblasts of patients (N=16) from clinical trial of venetoclax and azacitidine, we found that response to HRK + MS1 peptides (infers BCL-XL and MCL-1 dependency, respectively) inversely correlates with the achievement of remission. The serial sampling on treatment revealed the emergence of differential patterns of anti-apoptotic dependency that correlated with patient response. Of note, mitochondrial sensitivity to the MS-1 peptide, an indication of MCL-1 dependence, increased from 0% at diagnosis to 40% at relapse, suggesting an opportunity for MCL-1 antagonism in the relapsed setting. First, we exposed 6 AML cell lines with a BCL-2 (venetoclax) and MCL-1 antagonist (S63845). As previously reported, combination treatment resulted in synergistic cell line killing. Venetoclax treatment (within an hour) led to a dynamic increase in mitochondrial sensitivity to the MCL-1 selective MS-1 peptide, while S63845 caused increased mitochondrial susceptibility to the BCL-2 and BCL-XL selective BAD peptide. There was a correlation between % of priming caused by MS-1 and BAD peptide with Loewe synergy score (Spearman r=0.79, p&lt;0.05). This suggests that BH3 profiling can provide a rapid pharmacodynamic marker of the activity of BH3 mimetics. We next investigated combination MCL-1 and BCL-2 antagonism in vivo in venetoclax resistant AML PDXs. We transplanted NSG mice with human AML cells and started them on venetoclax (100mg/kg, PO) until leukemia relapsed (N=6 models). We subjected resistant myeloblasts to BH3 profiling and identified dependency on BCL-2 and MCL-1 to guide therapeutic choices. As a single agent, S63845 showed limited activity in venetoclax resistance models (p&lt;0.01 vs. vehicle; N=2/4 models). We next tested combinations at 3 different dosing schedules where BH3 mimetics were given either in a simultaneous week, alternative weeks or switched based on leukemia burden. Mice receiving venetoclax and S63845 in the same week showed the greatest anti-leukemia effects (p&lt;0.01 to p&lt;0.0005; N=4/4 models). We found superior benefit of BCL-2 + MCL-1 combination antagonism over MCL-1 alone in venetoclax resistance settings, implies that resistant myeloblasts escape apoptosis by switching their anti-apoptotic dependencies. Next, we applied the dynamic BH3 profiling technique that measures drug-induced changes in mitochondrial priming to discover additional agents that may overcome venetoclax resistance. We compared mitochondrial priming signals of 40 targeted agents in isolated parental and resistant cells. Although venetoclax resistant cells gained cross resistance to most of the agents, inhibitors of the FLT-3 pathway, including quizartinib, crenolanib, and gilteritinib retained similar priming responses as in parental cells. To validate this, we re-transplanted venetoclax resistant cells and found that quizartinib treatment maintained anti-leukemia efficacy compared to venetoclax or vehicle-treated mice (p&lt;0.0001). In an unbiased whole transcriptome analysis, venetoclax resistant PDXs showed enrichment for JAK/STAT, RAS/MAPK, and PI3K/AKT/mTOR pathways, and corresponding upregulation in protein levels of p-FLT3, p-STAT5, p-MAPK, and p-AKT. In addition we identified that FLT3-ITD mutation measured via capillary electrophoresis was maintained in resistant clones while point mutation in BCL2 (BCL2G101V) was undetectable. In conclusion, our study illustrates the power of mitochondrial measurements as a predictive biomarker for BH3 mimetic based therapy. We provide an evidence for the persistent activity of FLT-3 inhibitors in venetoclax resistance settings, a discovery made possible by dynamic BH3 profiling. Disclosures Ryan: Vivid Biosciences: Consultancy. Erick:Novartis: Employment. Weinstock:Celgene: Research Funding. Garcia:Abbvie: Research Funding; Genentech: Research Funding. Letai:Novartis: Research Funding; Flash Therapeutics: Equity Ownership; Abbvie: Consultancy, Research Funding; Vivid Bioscience: Equity Ownership; Astrazeneca: Research Funding.

Effect of Co-Mutations and FLT3-ITD Variant Allele Frequency (VAF) on Response to Quizartinib or Salvage Chemotherapy (SC) in Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML)

Effect of Co-Mutations and FLT3-ITD Variant Allele Frequency (VAF) on Response to Quizartinib or Salvage Chemotherapy (SC) in Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML)

Abstract B052: FLT3-ITD activation mediates resistance to the BCL-2 selective antagonist, venetoclax, in FLT3-ITD mutant AML models

Abstract 20-25% of acute myeloid leukemia (AML) patients harbor FLT3 internal tandem duplication (ITD) mutations, which confer poorer prognosis. FLT3-ITD is a constitutively activated kinase with differential signaling compared to wild type (WT) FLT3. Notably, FLT3-ITD activates STAT5, which can regulate the prosurvival proteins BCL-XL and MCL-1. Venetoclax, a potent, selective inhibitor of the prosurvival protein BCL-2, demonstrated monotherapy activity in relapsed/refractory AML (ORR 19%); however, no activity was seen in FLT3 mutant cases. BCL-XL and MCL-1 are known venetoclax resistance factors and targeting their regulation in combination with venetoclax may enhance cell death. Based on this hypothesis, we interrogated the combination of venetoclax and quizartinib, a potent FLT3 inhibitor, in FLT3-ITD+ AML models. In vitro, quizartinib reduced expression of BCL-XL and MCL-1, but not BCL-2, in FLT3-ITD+ cells (Molm13 and MV4;11). However, quizartinib had no effect on BCL-XL or MCL-1 expression in FLT3 WT cells (HL60 and OCI-AML3). Combination treatment with venetoclax led to significant reduction in growth and increased apoptosis in FLT3-ITD+ cells compared to single agents while FLT3 WT cells were not sensitive to quizartinib alone or in combination with venetoclax. Consistent with FLT3-ITD regulating BCL-XL and MCL-1, cotreatment of quizartinib with either BCL-XL or MCL-1 specific inhibitors had no combination effect in FLT3-ITD+ cells. Additionally, quizartinib and venetoclax were assessed in vivo utilizing MV4;11 and Molm13 orthotopic xenograft models. Mice were treated with venetoclax (100 mg/kg), quizartinib (5 mg/kg), or the combination of both drugs for 21 continuous days. Quizartinib treatment significantly increased survival compared to the vehicle group in both xenograft models (56 days vs. 26 days and 32 days vs. 14 days for MV4;11 and Molm13 xenografts, respectively). However, venetoclax only marginally improved survival (34 days vs. 26 days) in the MV4;11 model and was not efficacious in the Molm13 model. Despite lack of single-agent activity, addition of venetoclax to quizartinib led to a further improvement in survival when compared to quizartinib (91 days vs. 56 days and 40 days vs. 32 days in MV4;11 and Molm13 xenografts, respectively). These data underscore the dependency of these models on FLT3-ITD for survival and its regulation of survival pathways that confer resistance to venetoclax, suggesting FLT3-ITD could be a predictive biomarker of venetoclax resistance. To determine mechanisms of venetoclax resistance mediated by FLT3-ITD, mutant cells were treated with PI3K, MEK or JAK2 kinase selective inhibitors alone or in combination with venetoclax. Each pathway inhibitor combined with venetoclax to reduce cell growth compared to single agents. This observation suggests that multiple pathways may contribute to venetoclax resistance rather than a single pathway dependency to confer the prosurvival effects of FLT3-ITD. Additional mechanistic studies are under way to define the role of each pathway in regulating survival. Together, our data demonstrate that FLT3-ITD inhibition combines with venetoclax in vitro and in vivo in FLT3-ITD+ AML models. These results provide a strong mechanistic rationale to support clinical investigation of FLT3 inhibitors in combination with venetoclax to treat FLT3-ITD+ AML. Citation Format: Raghuveer Mali, Elisabeth A. Lasater, Kelly Doyle, Ritu Malla, Erwin Boghaert, Andrew Souers, Joel Leverson, Deepak Sampath. FLT3-ITD activation mediates resistance to the BCL-2 selective antagonist, venetoclax, in FLT3-ITD mutant AML models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B052.

FGF2 from Bone Marrow Stroma Protects Acute Myeloid Leukemia Cells from the FLT3 Inhibitor Quizartinib and Facilitates Acquisition of Resistance Mutations

Background: Acute myeloid leukemia (AML) patients with internal tandem duplication of FLT3 (FLT3-ITD AML) routinely develop resistance to FLT3 inhibitor monotherapy. Resistance to inhibitors such as quizartinib can develop through mutation of the FLT3 kinase domain, but not all resistant patients have mutations indicating that additional mechanisms of resistance are important. Although quizartinib induces a rapid reduction of circulating leukemia blasts, residual blasts in the marrow persist, suggesting that the microenvironment protects blasts from quizartinib and may serve as a reservoir for development of resistance.Results: Using a screen of molecules from the microenvironment, we found that FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) were the two most protective proteins of FLT3-ITD MOLM14 cells when treated with 10 nM quizartinib in vitro. FL protection has been previously described by Mark Levis' group, so we focused on the mechanism of FGF2 protection. FGF2 bound FGFR1, activating the MAPK pathway to mediate resistance. FGF2-mediated resistance could be overcome by concomitant inhibition of the FGFR receptor using the inhibitor PD173074 or by siRNA knock-down of FGFR1. FGFR1 is highly expressed in primary AML cells, and exogenous FGF2 protected primary FLT3-ITD AML cells from quizartinib ex vivo in a dose-dependent fashion (p=0.0007 at 100 ng/ml).MOLM-14 cells were cultured continuously with quizartinib in media alone, or media supplemented with 10 ng/ml FGF2 or FL to mimic the effects of the microenvironment. MOLM14 cells supplemented with FL or FGF2 resumed growth after 6-8 weeks, significantly faster than cells cultured in media alone, of which only 2/4 developed resistance after 12 weeks. To test the dependence of resistance cultures on exogenous ligand, we removed ligand from the FGF2- and FL-dependent cultures after 4 months and continued quizartinib treatment. After a temporary pause in growth, the cells regained exponential growth within 1 month. We assayed FLT3 resistance mutations by Sanger sequencing during this time, and again at 8 months with a targeted next-generation sequencing panel of commonly mutated genes in AML. Deep sequencing revealed multiple FLT3 mutations along with recurrent mutations of KRAS and NRAS, indicating the importance of the FLT3 and MAPK signaling pathway in resistance (Figure 1). Multiple FLT3 mutations were identified strongly suggesting that FLT3 mutations were not pre-existing but developed during culture with quizartinib (Figure 1). In contrast, the frequency of the KRAS G13D mutation suggested this mutation was present at a low frequency in MOLM14 cells.FGF2 expression was evaluated by immunohistochemistry in serial bone marrow core biopsies from 10 FLT3-ITD AML patients on the phase II quizartinib trial. Stromal FGF2 expression increased significantly with quizartinib therapy (p<0.01) and remained elevated until patients acquired FLT3 resistance mutations or other intrinsic mechanisms of resistance. Once stromal FGF2 became redundant for survival, expression decreased again. FGF2 protected cells in a paracrine fashion since FGF2 immunofluorescence did not overlap with CD45 (hematopoietic marker) in core biopsies of quizartinib-treated patients.To test the effect of combined FLT3 and FGFR inhibition in a more complex in vitro model of the microenvironment, MOLM14 cells were co-cultured in transwells over HS-5 (FGF2 high expression) or HS-27 stromal cell lines (FGF2 low). HS-5 stromal cells were highly protective of MOLM14 cells treated with quizartinib alone, but this protection was attenuated by combined FGFR and FLT3 inhibition (p<0.01). In contrast, protection of MOLM14 cells in HS-27 co-culture was minimal, and unaffected by combined FLT3 and FGFR inhibition.Conclusions: Our data supports a two-step model of resistance to quizartinib with initial resistance mediated by microenvironmental proteins such as FGF2 and/or FL, followed by kinase domain mutation of FLT3 and/or activating mutations of the RAS pathway. Early resistance mediated by FGF2-expressing stroma can be overcome by concomitant inhibition of FLT3 and FGFR suggesting that pre-emptively targeting extrinsic resistance pathways, in combination with newer FLT3 inhibitors that have activity against kinase domain mutations, will improve the durability of response to FLT3 inhibitors. [Display omitted] DisclosuresAgarwal:CTI BioPharma Corp: Research Funding. Kovacsovics:Seattle Genetics: Research Funding. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.

Quizartinib Significantly Improves Overall Survival in FLT3-ITD Positive AML Patients Relapsed after Stem Cell Transplantation or after Failure of Salvage Chemotherapy: A Comparison with Historical AML Database (UK NCRI data)

Introduction: The prognosis of patients with a FLT3-ITD mutation in acute myeloid leukemia (AML) is worse than FLT3 wild type patients, with increased early relapse including after allogeneic stem cell transplantation (SCT) and with a poor response to salvage. FLT3-ITD mutant patients who relapse after SCT or fail salvage chemotherapy have a dismal prognosis with no approved treatment options. Quizartinib (AC220), an oral FLT3 receptor tyrosine kinase inhibitor, has shown a remission rate of 44%, and median overall survival of 23 weeks in FLT3-ITD mutant patients who were relapsed or refractory to 2nd line treatment, or relapsed following SCT in a non-randomised phase 2 study [1]. While these outcomes compare favourably to published data, which give a median survival of 1.5 months in all comers [2], and median survival in FLT3-ITD mutant patients of 13 weeks following first relapse [3], there is an overall paucity of data available for the FLT3-ITD+ subpopulation examined in the quizartinib studies . The UK NCRI database is the largest dataset for FLT3-ITD+ AML patients, containing 6872 patients with ITD status (1388 mutant) and systematic collection of data from diagnosis with follow-up for at least 5 years or to death.Methods: 97 FLT3-ITD+ patients from the AC220-002 study who had received intensive chemotherapy, and had either relapsed following SCT (n=39); or were relapsed (n=53) or refractory (n=5) following salvage therapy prior to enrolment were identified. The equivalent eligibility criteria were applied to the UK MRC/NCRI database of AML trial patients treated intensively from 1988-2013 who had confirmed FLT3-ITD mutation: 183 patients matching the same criteria were identified (post-SCT n= 65; relapsed n=99; refractory n=19). Patients in this group received only recognised intensive chemotherapy regimens prior to fulfilling the eligibility criteria. To reflect the screening period in AC220-002, patients in the MRC/NCRI cohort entered the analysis 14 days following being identified as relapsed/refractory. The primary endpoint was overall survival. Multivariable Cox/logistic regression was performed stratified for known prognostic factors, including route to eligibility (post SCT/relapsed/refractory). Sensitivity analyses were performed excluding deaths within 14 and 28 days from eligibility from the MRC/NCRI cohort only to allow for patients who might not have been considered for quizartinib treatment on grounds of fitness.Results: There was no significant difference in the ages of the two cohorts (median age AC220-002 47y vs. MRC 48y). Outcomes in the MRC/NCRI cohort did not improve over time from 1988-2013 (p=0.7). Analyses were performed adjusted for route to eligibility, ITD allelic ratio, duration of remission (in relapsed patients), and cytogenetics. Overall survival was improved in the Quizartinib patients (adjusted hazard ratio (HR) 0.40 (0.29-0.55) p<.0001), with no interaction with route to eligibility (p=0.4). Median survival was 231 vs 60 d for relapse post SCT, 128 vs 53 d in relapsed patients, and 146 vs 61 d in refractory patients. Response rates (CRc) overall were 43% vs 11% (adjusted OR 0.24 (0.10-0.61) p=0.002). In sensitivity analyses excluding deaths within 14 days (HR 0.50 (0.36-0.69), p<.0001) and 28 days (HR 0.5 (0.38-0.76) p=0.0004) within the MRC/NCRI cohorts quizartinib retained superiority.Discussion: Quizartinib has previously shown activity in relapsed/refractory patients in a large Phase 2 single arm study, and is currently being studied in a randomized Phase 3 study vs. standard salvage chemotherapy. This analysis examines a patient population that has failed salvage therapy or SCT and for whom there is no standard therapy. It confirms the prolonged overall survival seen in the Phase 2 study when compared to an equivalent large historical cohort of previously intensively treated patients from the UK MRC/NCRI trials, with median survival in quizartinib patients was 159 days compared to 56 days in the historical cohort. Even if deaths within 28 days are excluded entirely from the MRC/NCRI group, there is a highly significant survival benefit for quizartinib. Overall, the activity seen in the AC220-002 Phase 2 study is greater than that seen in comparable patients in a large historical cohort.[1] Levis M et al. ASH meeting Abstr 615, 2014. [2] Giles F et al. Cancer 104 (3), p547, 2005. [3] Ravandi F et al. Leukemia Research 34, p752, 2010. [Display omitted] DisclosuresOff Label Use: Quizartinib for AML. Gammon:Ambit: Employment. Trone:Ambit: Employment.

FLT3-Kinase Inhibitors Quizartinib and Midostaurin Do Not Impair T-Cell Reactivity and Activation

AbstractAbstract 1045In patients with FLT3-ITD mutated AML, FLT3-inhibitors have been used successfully as a ‘bridging therapy' before allogeneic transplantation. Inhibitors of other kinases (such as imatinib for BCR-ABL positive CML) have previously been used successfully after allogeneic transplantation – even before discontinuation of immunosuppressive medication. However, it is known that some BCR-ABL inhibitors such as dasatinib exert strong inhibitory effects on primary T-cells through inhibition of Src-kinases relevant for T-cell receptor signaling. Even imatinib and nilotinib - although not affecting Src kinase activity – showed decreased T-cell activation and reactivity to some extent. Thus, the influence of FLT3-kinase inhibitors on T-cell function may be critical in the context of allogeneic bone marrow transplantation for FLT3-ITD-positive AML. Besides inhibition of FLT3-kinase, midostaurin (PKC412) exerts activity against PDGFR, VEGFR or c-KIT. In contrast, second generation inhibitors such as quizartinib (AC220) act in a far more FLT3-specific manner.Therefore, we aimed to investigate the effects of both clinically relevant FLT3-inhibitors on T-cell receptor signaling in comparison to the well characterized and potent BCR-ABL inhibitor dasatinib. Investigating primary T-cells derived from healthy donors, we applied a dose range of 10–50 nM dasatinib, 5–50nM midostaurin and 10–50 nM quizartinib. These dose ranges have been previously described to be achievable as trough levels during inhibitor therapy in early clinical trials. Upon incubation with dasatinib (10nM and 50nM), we found overall reduction in global tyrosine phosphorylation as detected by Western-blotting using the 4G10 antibody. In contrast, treatment with midostaurin left the activation of T-cell receptor signaling pathways unaffected. Comparable to DMSO control, overall phosphorylation was induced almost immediately after stimulation. Western-blotting of LCK and Plcg1 showed similar time dependent activation compared to total phosphorylation. Likewise, quizartinib did not reduce overall tyrosine phosphorylation level and left activation of downstream kinases (ZAP70, MAPK, LCK, Plcg1) largely unaffected.As activation of primary T-cells is a critical step in immune responses against viral and tumor antigens we aimed to investigate the influence of FLT3-kinase inhibitors quizartinib and midostaurin on activation of CD8+ T-cells. T-cells from healthy donors were stimulated using either PHA 0.5% or CD3/CD28 beads to ensure a more T-cell receptor specific stimulation. Using CD3/CD28 stimulation, CD69 expression was almost abrogated following dasatinib treatment. Applying clinically relevant doses of midostaurin or quizartinib to isolated T-cells did not influence CD69 expression. Expression levels upon PHA or CD3/CD28 stimulation were comparable to DMSO-control - even in the presence of 50nM midostaurin or quizartinib. Proliferation of T-cells upon CD3/CD28 stimulation was impaired by dasatinib treatment, while midostaurin and quizartinib left T-cell proliferation largely unaffected – as determined by CSFE staining. In order to investigate the T cell allo-reactivity, mixed lymphocyte culture was performed, where human pan-T-cells are co-cultured with allogeneic antigen presenting cells. T-cell proliferation – as measured by 3H-thymidine incorporation – was significantly impaired by dasatanib but neither midostaurin nor quizartinib treatment. Investigation of leukemia- and virus-antigen-specific T-cell responses are currently under way to gain deeper insight regarding this clinically relevant scenario.Overall, we found FLT3-kinase inhibitors midostaurin and quizartinib to leave T-cell activation, proliferation and function unaffected in-vitro. This information may be useful for the design of up-coming clinical trials testing the safety and efficacy of FLT3-kinase inhibitors in combination with allogeneic stem-cell transplantation. Disclosures:Lipka:Novartis Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heidel:Novartis Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.