Duplication Of FMS-like Tyrosine Kinase Research Articles

Abstract 429: A novel regulatory role of FLT3/ITD mutation in lipid metabolism of leukemia cells: The mechanism and therapeutic implications

Abstract Internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3/ITD), is a common type of activating mutation and frequently detected in AML. FLT3/ITD is associated with poor prognosis of AML. In this study, we investigated the regulation of FLT3/ITD mutation on sterol regulatory element-binding protein (SREBP) and the therapeutic vulnerabilities of lipid metabolism in FLT3/ITD leukemia. With the transcriptomics analysis we found that FLT3/ITD controlled lipid metabolism via the transcription factor SREBP. The analysis of TCGA_LAML dataset revealed that a gene signature involved in fatty acid and cholesterol metabolism, which is regulated by SREBP, was upregulated in FLT3/ITD patients compared with FLT3/wild-type patients. The upregulation of SREBP protein expression in FLT3/ITD leukemia was confirmed using a genetically engineered mouse model. We further demonstrated that FLT3/ITD regulates the protein degradation of SREBP through the PI3K/AKT-GSK3β axis. The metabolomics analysis further showed that FLT3/ITD primarily regulates glycerophospholipid synthesis, which contribute to the key structure lipid in the membrane, specifically, the cardiolipin. Moreover, pharmacologic inhibition of SREBP activation and its downstream enzyme fatty acid synthase (FASN) sensitized FLT3-ITD AMLs to the treatment of FLT3 inhibitor quizartinib. Collectively, our findings reveal a novel regulation of FLT3-ITD in metabolic reprogramming and indicate a targetable vulnerability in a subset of refractory leukemia. Citation Format: Yumin Hu, Feng Yin, Mingyue Zhao, Wenhua Lu, Tiantian Yu. A novel regulatory role of FLT3/ITD mutation in lipid metabolism of leukemia cells: The mechanism and therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 429.

ITDetect: a method to detect internal tandem duplication of FMS-like tyrosine kinase (FLT3) from next-generation sequencing data with high sensitivity and clinical application

Internal tandem duplication (ITD) of the FMS-like tyrosine kinase (FLT3) gene is associated with poor clinical outcomes in patients with acute myeloid leukemia. Although recent methods for detecting FLT3-ITD from next-generation sequencing (NGS) data have replaced traditional ITD detection approaches such as conventional PCR or fragment analysis, their use in the clinical field is still limited and requires further information. Here, we introduce ITDetect, an efficient FLT3-ITD detection approach that uses NGS data. Our proposed method allows for more precise detection and provides more detailed information than existing in silico methods. Further, it enables FLT3-ITD detection from exome sequencing or targeted panel sequencing data, thereby improving its clinical application. We validated the performance of ITDetect using NGS-based and experimental ITD detection methods and successfully demonstrated that ITDetect provides the highest concordance with the experimental methods. The program and data underlying this study are available in a public repository.

Mutational analysis of DNMT3A improves the prognostic stratification of patients with acute myeloid leukemia.

Nucleophosmin1 (NPM1) mutations are the most frequently detected gene mutations in acute myeloid leukemia (AML) and are considered a favorable prognostic factor. We retrospectively analyzed the prognosis of 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated AML. Although patients with NPM1-mutated AML showed a high remission rate, this was not a favorable prognostic factor for overall survival (OS); this is contrary to generally accepted guidelines. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNA methyltransferase 3A (DNMT3AR882 mutations) were a strong predicative factor indicating poor prognosis in all AML (p < 0.0001) and NPM1-mutated AML cases (p=0.0020). Furthermore, multivariate analysis of all AML cases showed that DNMT3AR882 mutations and the co-occurrence of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD), NPM1 mutations, and DNMT3AR882 mutations (triple mutations) were independent factors predicting a poor prognosis related to OS, with NPM1 mutations being an independent factor for a favorable prognosis (hazard ratios: DNMT3AR882 mutations, 1.946; triple mutations, 1.992, NPM1 mutations, 0.548). Considering the effects of DNMT3AR882 mutations and triple mutations on prognosis and according to the classification of NPM1-mutated AML into three risk groups based on DNMT3AR882 /FLT3-ITD genotypes, we achieved the improved stratification of prognosis (p < 0.0001). We showed that DNMT3AR882 mutations are an independent factor for poor prognosis; moreover, when confounding factors that include DNMT3AR882 mutations were excluded, NPM1 mutations were a favorable prognostic factor. This revealed that ethnological prognostic discrepancies in NPM1 mutations might be corrected through prognostic stratification based on the DNMT3A status.

The Effect of Sorafenib Therapy on the Outcome of Acute Myeloid Leukemia with FLT3-ITD Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Background Internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) mutation has been reported in about 25% patients with acute myeloid leukemia (AML). AML with FLT3-ITD mutations have an inferior survival, primarily due to shorter remission durations and higher relapse rates. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) improves the survival for FLT3-ITD AML, the rate of leukemia relapse remains high. Sorafenib, a multi-kinase inhibitor, has shown promising activity in AML with FLT3-ITD. A growing body of studies have suggested that sorafenib monotherapy or in combination with chemotherapy are effective in attaining complete remission (CR) for untreated and refractory relapsed FLT3-ITD AML. Sorafenib maintenance after allo-HSCT has been proved to be safe for FLT3-ITD AML, but whether it could improve survival remains uncertain. The aim of this study is to evaluate the effect of sorafenib therapy on the outcome of FLT3-ITD AML undergoing allo-HSCT.Methods A total of 118 AML with FLT3-ITD undergoing allo-HSCT were enrolled in this retrospective study between January 2012 and December 2015 at Nanfang Hospital, Peking University People's Hospital and Zhujiang Hospital. Patients with HLA-matched sibling or unrelated donors received BuCY conditioning (busulfan 3.2 mg/kg/day, on days -7 to -4; cyclophosphamide 60 mg/kg/day, on days -3, -2), and patients with HLA-haploidentical related donors received modified BuCY conditioning (cytarabine4g/m2/day, on days -10, -9; busulfan 3.2 mg/kg/day, on days -8 to -6; cyclophosphamide 1.8g/m2/day, on days -5, -4). Sorafenib therapy pre-HSCT was initiated at the time of induction, post-remission maintenance or relapse, and continued until one week prior to the start of the conditioning regimen. Sorafenib maintenance post-HSCT was started from day 30 to 180 post-transplantation. Either before or after transplantation, the initial dose of sorafenib was 400 mg twice daily and was adjusted on the basis of suspected toxicity or emerging drug resistance (dose range, 200-800 mg daily).Results Of the 118 patients, 90 were in CR, 8 in partial remission and 20 in non-remission at the time of transplantation. The 118 patients all achieved hematopoietic reconstitution and achieved CR by day +30 post-transplantation. Depending on whether patients received sorafenib pre-HSCT and sorafenib maintenance post-HSCT, the 118 patients were divided into 4 groups. Thirty-two patients only received sorafenib pre-HSCT (Group A), 29 only maintenance post-HSCT (Group B), 23 both pre-HSCT and maintenance post-HSCT (Group C), and 34 did not receive sorafenib pre-HSCT or maintenance post-HSCT (Group D). There were no significant differences in disease status pre-transplantation, donor source or HLA typing among four groups. Thirty-three patients relapsed at a median time of 137 (range, 44 to 589) days post-transplantation. Six abandoned treatment and 27 received salvage therapy of sorafenib combined with chemotherapy and DLI. Thirteen of the 27 patients achieved CR after salvage treatment. The 5-year cumulative incidence of relapse were 20.6%±9.4%, 20.7%±3.4%, 17.4%±4.3% and 50.2%±18.6% in Group A, B, C and D (P=0.006). With a median follow up of 670 (range, 63-1898) days post-transplantation, 85 patients survived and 33 died. The 5-year overall survival (OS) and disease-free survival (DFS) were 75.0%±8.8% and 70.0%±8.6%, 75.9%±8.0% and 75.9%±8.0%, 82.6%±7.9% and 78.3%±8.6%, 51.6%±9.6% and 31.2%±8.9% in Group A, B, C and D (P=0.093, P <0.001). Multivariate analysis revealed that sorafenib therapy pre-HSCT and sorafenib maintenance post-HSCT were the protective factors for lower relapse and longer OS; the nCR (not in CR) status pre-HSCT was the only risk factor for longer OS.Conclusions Sorafenib therapy pre-HSCT and sorafenib maintenance post-HSCT could reduce relapse and improve survival for AML with FLT3-ITD. DisclosuresNo relevant conflicts of interest to declare.

All-trans Retinoic Acid, Arsenic Trioxide, and Anthracycline-based Chemotherapy Improves Outcome in Newly Diagnosed Acute Promyelocytic Leukemia Regardless of FLT3-ITD Mutation Status.

All-trans retinoic acid (ATRA) and pre-upfront arsenic trioxide (ATO) have revolutionized the therapy of acute promyelocytic leukemia (APL). However, internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) mutations is associated with increased risk of relapse. The aim of this study was to analyze the prognostic impact of FLT3-ITD on APL patients who received remission induction with ATRA, idarubicin (IDA) and/or ATO, followed by ATRA plus ATO along with anthracycline, as consolidation therapy. A total of 72 patients newly diagnosed with APL were included in this study. 83.3% of the patients achieved complete remission (CR) after induction therapy. FLT3-ITD mutations were detected in 16 (22.2%) patients and closely related to bcr-3 PML-RARa transcript (P<0.001). The 5-year overall survival (OS) rate was 100% in both FLT3-ITDpositive and FLT3-ITDnegative groups, and there was no significant difference in 5-year event-free survival (EFS) between the two groups (78.3% vs. 83.3%, P=0.85). ATRA plus ATO and anthracycline-based chemotherapy achieved great outcome in newly diagnosed APL regardless of the FLT3-ITD mutation status.

Bioinformatics-based identification of SPNS3 (Spinster homolog 3) as a prognostic biomarker of apoptosis resistance in acute myeloid leukemia

ABSTRACT Spinster homolog 3 (SPNS3) belongs to the Spinster (SPNS) family which participates in sphingolipid transportation through the cell membrane. However, the functions of SPNS3 in acute myeloid leukemia (AML) are unknown. This study obtained SPNS3 from a gene set that was related to AML relapse and evaluate whether high SPNS3 expression induced apoptosis resistance in an AML cell line, which is consistent with the role of SPNS3 as a marker of poor prognosis in the clinic. Moreover, internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) mutation and the AC127521.1/ MIR-139/SPNS3 competing endogenous RNA axis were found to regulate SPNS3 expression. In addition, we noted that SPNS3 may play an important role in the Sphingosine-1-phosphate signal pathway that is involved in the maintenance of the AML microenvironment. These results highlight the anti-apoptosis effect of SPNS3 in AML, and the potential mechanism mediating this effect was explored through bioinformatics. Abbreviations: AML: acute myeloid leukemia; FLT3-ITD: internal tandem duplication of FMS-like tyrosine kinase 3; SPNS3: spinster homolog 3; SPNS1: spinster homolog 1; SPNS2: spinster homolog 2; GO: gene ontology; S1P: sphingosine-1-phosphate; ceRNA: competing endogenous RNA; dAML: acute myeloid leukemia at diagnosis; iAML: acute myeloid leukemia after induction chemotherapy; rAML: acute myeloid leukemia at relapse; DEGs: differentially expressed genes; BP: biological processes; CC: cellular components; MF: molecular functions; MRD: minimal residual disease; EFS: event-free survival; OS: overall survival; KEGG: Kyoto Encyclopedia of Genes and Genomes; SPHK: Sphingosine kinase.

Outcomes of pediatric acute myeloid leukemia patients with FLT3-ITD mutations in the pre-FLT3 inhibitor era.

BackgroundAcute myeloid leukemia (AML) with internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD) is associated with poor outcomes. This study aimed to analyze the outcomes of pediatric AML patients with FLT3-ITD mutations in the pre-FLT3 inhibitor era.MethodsWe retrospectively reviewed and identified 18 patients diagnosed with non-M3 AML with FLT3-ITD mutations at Seoul National University Children’s Hospital between May 2008 and August 2019.ResultsThe median age was 13 years (range, 6‒19 yr). The median follow-up time was 43 months (range, 6‒157 mo). Fourteen patients received BH-AC-based (N4-Behenoy1-1-b-D-arabinofuranosy1cytosine) and 4 received cytarabine-based induction chemotherapy. Complete remission (CR) was achieved in 72.2% of the patients after the first induction chemotherapy and 80% of the patients achieved CR after salvage therapy. The overall CR rate was 94% (17/18 patients). These 17 patients underwent hematopoietic stem cell transplantation (9 matched unrelated donors, 5 matched related donors, and 3 haploidentical donors). Relapse occurred in 22% of the patients. Event free survival and overall survival rates were 53.8±12.1% and 53.6±12.1%, respectively, and they were not significantly different according to the type of induction chemotherapy (P=0.690) or the type of donor (P=0.102).ConclusionThis study outlines the outcomes of pediatric AML patients with FLT3-ITD-mutations in one institution over a decade. Outcomes were significantly improved in this study compared to our previous report in 2004, where RFS and EFS were 0%. This study can provide baseline data for pediatric patients in the pre-FLT3 inhibitor era.

Natural small molecule triptonide inhibits lethal acute myeloid leukemia with FLT3-ITD mutation by targeting Hedgehog/FLT3 signaling

Acute myeloid leukemia harboring internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD AML) is a subset of highly aggressive malignancies with poor clinical outcome. Despite some advances in the development of FLT3 tyrosine kinase inhibitors (FLT3 inhibitors), most of FLT3-ITD AML patients suffer from lethal disease relapse, suggesting the requirement of novel targets and agents. Here we describe a natural small molecule, triptonide that can efficiently inhibit FLT3-ITD-driven AML in vitro and in vivo. Mechanistically, triptonide targeted Hedgehog/FLT3 signaling by inhibiting its critical effectors, which are GLI2, c-Myc and FLT3 and induced apoptosis of FLT3-ITD-driven leukemia cells. In addition, we also observed that triptonide activated tumor suppressor p53. In vivo, triptonide treatment markedly suppressed lethal FLT3-ITD-driven AML with good tolerance and prolonged survival time in orthotopic mouse model. Our studies identify Hedgehog/FLT3 axis as a novel target for treating FLT3-ITD-driven leukemia and demonstrate that triptonide is an active lead compound that can kill FLT3-ITD-driven leukemia cells.

Chlorpromazine eliminates acute myeloid leukemia cells by perturbing subcellular localization of FLT3-ITD and KIT-D816V

Mutated receptor tyrosine kinases (MT-RTKs) such as internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3 ITD) and a point mutation KIT D816V are driver mutations for acute myeloid leukemia (AML). Clathrin assembly lymphoid myeloid leukemia protein (CALM) regulates intracellular transport of RTKs, however, the precise role for MT-RTKs remains elusive. We here show that CALM knock down leads to severely impaired FLT3 ITD- or KIT D814V-dependent cell growth compared to marginal influence on wild-type FLT3- or KIT-mediated cell growth. An antipsychotic drug chlorpromazine (CPZ) suppresses the growth of primary AML samples, and human CD34+CD38- AML cells including AML initiating cells with MT-RTKs in vitro and in vivo. Mechanistically, CPZ reduces CALM protein at post transcriptional level and perturbs the intracellular localization of MT-RTKs, thereby blocking their signaling. Our study presents a therapeutic strategy for AML with MT-RTKs by altering the intracellular localization of MT-RTKs using CPZ.

Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia.

Internal tandem duplication of Fms‐like tyrosine kinase 3 (FLT3/ITD) occurs in about 30% of acute myeloid leukemia (AML) and is associated with poor response to conventional treatment and adverse outcome. Here, we reported that human FLT3/ITD expression led to axis duplication and dorsalization in about 50% of zebrafish embryos. The morphologic phenotype was accompanied by ectopic expression of a morphogen follistatin (fst) during early embryonic development. Increase in fst expression also occurred in adult FLT3/ITD‐transgenic zebrafish, Flt3/ITD knock‐in mice, and human FLT3/ITD AML cells. Overexpression of human FST317 and FST344 isoforms enhanced clonogenicity and leukemia engraftment in xenotransplantation model via RET,IL2RA, and CCL5 upregulation. Specific targeting of FST by shRNA, CRISPR/Cas9, or antisense oligo inhibited leukemic growth in vitro and in vivo. Importantly, serum FST positively correlated with leukemia engraftment in FLT3/ITD AML patient‐derived xenograft mice and leukemia blast percentage in primary AML patients. In FLT3/ITD AML patients treated with FLT3 inhibitor quizartinib, serum FST levels correlated with clinical response. These observations supported FST as a novel therapeutic target and biomarker in FLT3/ITD AML.

Targeting DNA Damage and Repair in Acute Myeloid Leukemia Carrying Internal Tandem Duplication of Fms-like Tyrosine Kinase 3 (FLT3-ITD) - a Mechanistic Study

Objective Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) is one of the most common mutations in acute myeloid leukemia (AML), occurring in nearly 30% of cases. FLT3-ITD involves in-frame duplication of 3-400 base-pairs at the juxta-membrane domain, resulting in ligand-independent activation of FLT3 signaling. Downstream effectors include activation of ERK/STAT5 via SRC kinase, activation of PI3K/AKT, phosphorylation of FOXO3A, down-regulation of equilibrative nucleoside transporter 1 (ENT1) for cytarabine, and induction of reactive oxygen species (ROS) that may lead to increased DNA damage and defective repair. The present study investigated if the latter can be effectively targeted for the treatment of this AML subtype. Methods Primary samples from patients with FLT3-ITD AML, human cell line carrying FLT3-ITD (MOLM-13) as well as mouse B-lymphoid Ba/F3 cells transduced with human FLT3-ITD were used in this study. Traffic Light Reporter (TLR) assay was used to measure fidelity of double-strand breaks (DSB) repair, either via error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Percentage of repair events by HR or NHEJ were quantified by flow cytometry. DNA DSB were examined by γ-H2AX foci staining using confocal microscopy. Single-cell DSB were quantified by neutral comet assay and analysed by OpenComet software. The tail moment was calculated as the length of comet tail multiplied by the tail DNA %. MOLM-13 was transplanted into NOD/SCID/IL2Rg-/- (NSG) mice by tail vein injection. Treatment comprised cytarabine (25mg/kg, i.p., day 1-5) and doxorubicin (1.5mg/kg, i.v., day 1-3), with or without olaparib (25mg/kg, i.p., day 1-5). Comparisons between groups of numerical data were evaluated using Student's t-test. P-values less than 0.05 were considered statistically significant. Results To investigate the link between FLT3-ITD AML and DNA damage response (DDR), expression of critical DDR genes in primary AML samples was examined by real-time quantitative PCR. The panel of genes included apical kinase ATM, ATR and DNA-PKcs; DNA damage mediators BRCA1,BRCA2 and PARP1; downstream response kinase CHEK1 and CHEK2 and effectors TP53. Among them, BRCA2 was significantly down-regulated in FLT3-ITD AML(Wild-type FLT3=18 samples; FLT3-ITD=13 samples; Average dCt 9.7 in WT vs. 10.6 in ITD; p&lt;0.05). Down-regulation of BRCA2 in FLT3-ITD AML was further validated in a microarray database GSE15434 from a multicenter study investigating gene expression profiles of normal karyotype AML( FLT3-WT=148; FL3-ITD=86; ; log2 gene expression 5.61 in WT vs 5.45 in ITD; p&lt;0.001). As BRCA2 is an important protein mediating HR, a DSB DNA repair TLR assay was performed. HR was significantly down-regulated in Ba/F3-FLT3-ITD as compared with parental Ba/F3 line by flow cytometry(1.21% HR repair in control vs. 0.44% in ITD; p&lt;0.05) while NHEJ was unaffected (1.90% NHEJ repair in control vs. 2.38% in ITD; p=0.50). Down-regulation of BRCA2 expression and defective HR in FLT3-ITD AML was reminiscent of BRCA mutant breast and ovarian cancers. Therefore, the effects of poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib were examined. Ba/F3-FLT3-ITD were more sensitive to olaparib than parental line. Olaparib inhibited base excision repair and increased DSB as indicated by increased number of γ-H2AX foci and increased tail moment in Ba/F3-FLT3-ITD cells when compared to parental line. Olaparib-induced DSB in Ba/F3-FLT3-ITD cells was mainly repaired by NHEJ as shown by colocalization of γ-H2AX foci with 53BP1. Combination of chemotherapy (cytarabine and doxorubicin) and olaparib synergistically reduced leukemic cell growth of MOLM-13 in NSG murine xenograft model. Conclusion Results from the present study supported the use of PARPi in the treatment of FLT3-ITD AML. Its therapeutic benefits in combination with chemotherapy would have to be further evaluated. Acknowledgements: Health and Medical Research Fund (Project number: 04152326) and Li Shu Fan Medical Foundation, LKS Faculty of Medicine, University of Hong Kong. Disclosures No relevant conflicts of interest to declare.

Somatic Mutations Potentiating RAS-MAPK Signaling Confer Resistant Potential Against FLT3-Inhibitors to Acute Myelogenous Leukemia

Acute myeloid leukemia (AML) is one of the most common hematologic malignancies derived from a small number of self-renewing leukemic stem cells (LSCs). AML has various genetic abnormalities such as chromosomal aberration, single nucleotide variant and insertion/deletion. In particular, internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) is the most frequently found in ~ 25 % of AML cases, moreover, AML with FLT3-ITD is classified in unfavorable. FLT3 is fundamentally essential for survival of hematopoietic stem cells (HSCs) as well as LSCs, but once ITD is acquired, FLT3-ITD promotes the proliferation of AML by activating downstream enzymes. Various FLT3 inhibitors are now available as effective and promising drugs. On the other hand, the resistance against those inhibitors has been regarded as an emerging problem in clinics. To clarify the molecular machineries how AML cells acquire the resistance against FLT3 inhibitors, we compared the mutation status of paired AML samples consisted of diagnostic and relapsed samples. LSCs, which are quiescent and highly resistant against conventional chemotherapies, are considered to play a central role in recurrence. Since we and other group previously reported that TIM-3 clearly discriminates LSCs from residual normal HSCs, we purified and evaluated the mutations of CD34+CD38-TIM-3+ LSCs or CD34+ immature AML cells. The paired diagnostic and relapsed samples obtained from the identical 5 AML patient treated with Gilteritinib were subjected to whole exome sequencing (WES) analysis. CD3+ T cells were also purified from the diagnostic samples and used as a germ line control to detect somatic mutations acquired in AML cells. Libraries for WES were prepared using Sure Select XT HS and Human All Exon V6 Capture Kit in accordance with manufacturer's instructions. Then we performed WES using NextSeq 500 with 150bp pair-end read. Surprisingly, in all 5 cases examined, AML clones lost FLT3-ITD, whereas clones harboring mutations potentiating RAS-MAPK signaling became dominant at relapse: one case acquired the activated KRAS (Q61H) mutation and the remaining four cases newly acquired the previously-unidentified mutations in RAS-MAPK inhibitory genes. Such mutations in RAS-MAPK-inhibitory genes include DIRAS3 (W173X), RASA1 (E70G), RAP1GAP2 (K18X), RIN2 (S355fs), RASA4 (C655R), RASA4B (E141G), MAP2K7 (T66P) and PTPN7 (T14M). Of note, these genes belong to RAS-MAPK-inhibitory pathways and the encoded proteins are reported to suppress the activity of RAS-MAPK pathways. These RAS-MAPK-inhibitory mutations were exclusively detected in the relapsed AML clones, and contained stop-gain and frameshift mutations. We, therefore, hypothesized that RAS/MAPK activation driven by somatic mutations contributes to the acquisition of resistance against FLT3 inhibitors in LSCs. Consistent with such hypothesis, phosflow analysis revealed that basal ERK phosphorylation was retained or rather increased in the relapse CD34+ AML cells lacking FLT3-ITD, indicating that RAS-MAPK activation was maintained independent of FLT3-ITD. Furthermore, ASP2215 and AC220 inhibited ERK phosphorylation in the diagnostic CD34+ AML cells, whereas both inhibitors failed in the inhibition of ERK activation in the relapsed AML clones in all 3 cases analyzed. Gilteritinib also failed in inhibiting ERK in other relapsed AML cases, however, the combination of ASP2215 and MEK inhibitor efficiently suppressed ERK phosphorylation. All of relapse AML cases exhibited the emergence of AML clones harboring mutations associated with RAS-MAPK pathway genes, and therefore, the clinical sequence for detecting such mutations might be useful to assess the risk of FLT3-inhibitor treatment failure in clinics. Furthermore, our study showed the efficacy of MEK inhibitors to inhibit RAS-MAPK signaling activity even in the relapsed AML cells. Thus, the combination therapy consisted of FLT3-inhibitors and MEK inhibitors might be one of the promising therapeutic strategies to overcome FLT3-inhibitor resistance in AML. In summary, our study revealed that mutations potentiating RAS-MAPK pathway should play a central role in conferring the resistance against FLT3-inhibitors to AML. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Impact of FLT3-ITD Remission Depth in Acute Myeloid Leukemia Patients Prognosis Prediction before Allogeneic Hematopoietic Cell Transplantation

Introduction: Activating internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) is one of the most frequently mutations in acute myeloid leukemia (AML) patients with a frequency of 20-30%. Although AML with FLT3-ITD mutation is not recognized as a distinct entity in the World Health Organization classification system, AML patients with FLT3-ITD usually have high white blood cell count, high risk in poor prognostic outcome and are benefited from FLT3 inhibitors. However, the outcome prediction of AML with FLT3-ITD is still not very clear in patients undergoing allogeneic hematopoietic cell transplantation (alloHCT). Recent studies showed that the depth of remission, mostly accessed by flow cytometry (FCM) based measurable residual disease (MRD) and next generation sequencing based molecular MRD, was highly predictive for relapse in AML before alloHCT. The depth of remission is considered an important criterion for evaluating AML relapse before alloHCT. Hence, FLT3-ITD remission depth showed a potential application in evaluating FLT3-ITD subtype's prognosis. Therefore, in this retrospective study we tried to evaluate prognostic impact of FLT3-ITD remission depth in AML before alloHCT. Methods: A total of 117 FLT3-ITD positive patients were obtained in our study if they were 18 years or older, had a diagnosis of AML excluding acute promyelocytic leukemia, received standard induction chemotherapy comprising anthracycline and cytoarabine, at first affiliated hospital of Soochow University between September 2011 to January 2018 . All patients were examined a panel of 51 genes mutation by next generation sequencing and FLT3-ITD allelic ratio (AR) in newly diagnosed bone marrow samples (Fig1 A). 57 patients received two courses of standard chemotherapy and immediate alloHSCT in four months. The median time from first chemotherapy to alloHSCT was 99 days. FLT3-ITD was also examined direct after secondary chemotherapy with the sensitivity level of 0.005. Distribution of OS and RFS were estimated by Kaplan-Meier method. Difference of FLT3-ITD positive ratio in FLT3-ITD subtypes was analyzed by Chi-squared test. The two-sided level of significance was set at p&amp;lt;0.05. The deadline for follow-up was May 31, 2019, with a median follow-up time of 24 (5-83) months. Results: In our cohort, the median FLT3-ITD AR of 117 patients was 0.53 (range 0.02-7.72) in newly diagnosis. Interestingly, patients with DNMT3A mutation (0.615, range 0.04-7.72) had a higher FLT3-ITD AR than those of patients with CEBPA double mutation (0.19, range 0.02-0.74), with statistical difference (p=0.007). Patients with DNMT3A mutation also had lower complete remission rate than those of patients with NPM1 mutation and DNMT3A wildtype after first chemotherapy (x2=5.987, P=0.018). In 57 patients undergoing alloHSCT, we found there were no differences in OS and RFS among patients classified by FLT3-ITD AR or concomitant NPM1 mutation. However, patients with FLT3-ITD positive had a poorer OS (p&amp;lt;0.001) and RFS (p&amp;lt;0.001) than those of patients with FLT3-ITD negative accoding to the FLT3-ITD states after secondary chemotherapy. In addition, patients with FLT3-ITD AR&amp;gt;0.01 had a poorer OS and EFS than those of patients with FLT3-ITD AR&amp;lt;0.01, indicating FLT3-ITD remission depth is helpful in prognosis prediction for patients undergoing alloHCT (Fig1 B,C). Moreover, we found patients with DNMT3A mutation had a higher FLT3-ITD positive ratio than those of patients with NPM1 mutation after secondary chemotherapy. This result was also found in patients with normal chromosome karyotype (NK). There was no FLT3-ITD positive in patients with CEBPA double mutation or t(8;21) after secondary chemotherapy in our cohort, while 4 of 8 patients with chromosome karyotype abnormalities (no favorable) had FLT3-ITD positive after secondary chemotherapy. From these results above, we finally found patients with DNMT3A mutation had a poorer OS and EFS in NK patients undergoing alloHCT(Fig1 D,E). In conclusion, our results suggested FLT3-ITD remission depth is useful in prognosis prediction for AML patients with FLT3-ITD before alloHCT. Figure Disclosures No relevant conflicts of interest to declare.

Clinical and economic analysis of patients with acute myeloid leukemia by FLT3 status and midostaurin use at a Comprehensive Cancer Center

IntroductionIdentification of cytogenetic and molecular abnormalities has become vital for the appropriate treatment of acute myeloid leukemia (AML). One of the most common molecular alterations in AML is the constitutive activation by internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3). MethodsThis observational, retrospective, cohort study at the Huntsman Cancer Institute (HCI) had two time periods: 1) a historical pre-midostaurin time period which consisted of the FLT3 mutated (FLT3m) and FLT3 wild type (FLT3wt) cohorts from January 1, 2007, to December 31, 2016, and 2) a post-midostaurin cohort which consisted of the FLT3 mutated midostaurin-user cohort (early mido) from May 01, 2017 to December 31, 2018. ResultsIn total, 39 patients were included in the FLT3m cohort, 61 in the FLT3wt cohort, and seven in the early mido cohort. FLT3m patients spent fewer days in the hospital during the first consolidation regimen and received fewer consolidation cycles compared to FLT3wt patients. Overall survival (OS) was similar between FLT3m and FLT3wt patients. For patients without hematopoietic stem cell transplant, OS was significantly shorter for FLT3m patients compared to FLT3wt patients. Mean AML related inpatient charges and physician charges for FLT3m patients were significantly higher than FLT3wt patients. ConclusionThe FLT3 mutation is historically associated with a shorter time to transplant and increased total health care charges. More information is needed to evaluate the real-world treatment strategies for FLT3-mutated patients in the presence of FLT3 inhibitors and the impact of these treatment strategies on clinical and economic outcomes.

Impact of FLT3-ITD allele ratio and ITD length on therapeutic outcome in cytogenetically normal AML patients without NPM1 mutation.

Mutations of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD) contribute to poor prognosis in cytogenetically normal acute myeloid leukemia (CN-AML). Chemotherapy has limited effect, while allogeneic hematopoietic stem cell transplantation (allo-HSCT) plus sorafenib maintenance is a promising protocol to improve their therapeutic outcome. However, the prognostic significance of FLT3-ITD mutant status remains controversial. To investigate this, we detected FLT3-ITD mutant ratio (high and low) and length (long and short) in enrolled 184 CN-AML patients without NPM1 mutation, and evaluated their impact on complete remission (CR), overall survival (OS), relapse-free survival (RFS) and relapse risk (RR) after chemotherapy or allo-HSCT plus sorafenib maintenance. Our studies showed that FLT3-ITD mutation had negative impact on chemotherapeutic response, OS and RFS in CN-AML patients. There was no significant difference in CR rate between high and low ratio, or long and short length. Increasing ITD mutant ratio and length were associated with decreasing OS, and long length had shorter RFS and higher RR than the short after chemotherapy. Allo-HSCT plus sorafenib maintenance was an effective strategy to improve RFS and decrease relapse probability in FLT3-ITD AML patients, and benefited to these regardless of mutant ratio, and those with long length instead of the short.

Homoharringtonine Combined with the Heat Shock Protein 90 Inhibitor IPI504 in the Treatment of FLT3-ITD Acute Myeloid Leukemia

As a heterogeneous group of clonal disorders, acute myeloid leukemia with internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) mutation usually shows an inferior prognosis. In the present study, we found that homoharringtonine (HHT), a protein translation inhibitor of plant alkaloid in China, exhibited potent cytotoxic effect against FLT3-ITD (+) cell lines and primary leukemia cells, and a remarkable synergistic anti-leukemia action was demonstrated in vitro and in vivo in xenograft mouse models when co-treated with the heat shock protein 90 inhibitor IPI504. Mechanistically, HHT combined with IPI504 synergistically inhibited the growth of leukemia cells by inducing apoptosis and G1 phase arrest. This synergistic action resulted in a prominent reduction of total and phosphorylated FLT3 (p-FLT3) as well as inhibition of its downstream signaling molecules such as STAT5, AKT, ERK and 4E-BP1. Furthermore, co-treatment of HHT and IPI504 led to a synergistic or additive effect on 55.56%(10/18) of acute myeloid leukemia cases tested, including three relapsed/refractory patients. In conclusion, our findings indicate that the combination of HHT and HSP90 inhibitor provides an alternative way for the treatment of FLT3-ITD positive acute myeloid leukemia, especially for relapsed/refractory AML.

Influence of FLT3-ITD Mutation and Length on the Treatment Response and Prognosis in Cytogenetically Normal AML Patients

BACKGROUND: Mutations of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD) contributed to poor prognosis in cytogenetically normal acute myeloid leukemia (CN-AML). FLT3 tyrosine kinase inhibitor sorafenib in combination with chemotherapy was applied to treat FLT3-ITD AML patients with limited efficacy. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was considered as a potent therapeutic regimen in FLT3-ITD patients, but additional sorafenib maintenance was indispensable to support their long-term survival after allo-HSCT. Studies showed that increasing ITD size may be accompanied with decreasing OS and RFS in AML patients, but it remained controversial about the prognostic implication of ITD mutant lengths, the prognostic influence was important to evaluate in determining the therapeutic strategy for AML patients with different ITD lengths.METHODS: Total 185 CN-AML patients with and without FLT3-ITD mutations were enrolled in this study. We retrospectively studied the clinical characteristic, treatment response, survival and relapse risk after chemotherapy or allo-HSCT plus sorafenib in these patients. Distribution of ITD lengths detected in AML patients suggested two groups including long (≥70bp) and short ITD length (<70bp). Influence of FLT3-ITD mutation and its length were investigated after chemotherapy or allo-HSCT plus sorafenib.RESULT:FLT3-ITD mutations were detected in 15 percentage of AML patients, and associated with leukocytosis, high blast percentage in bone morrow (BM) and increased risk of treatment failure. FLT3-ITD mutations indicated decreased complete remission (CR) rate, overall survival (OS) and relapse-free survival (RFS), and increased relapse risk (RR) in AML patients after chemotherapy plus sorafenib. Patients with long ITD length (≥70bp) had worse OS and RFS, and more relapse probability than these with short ITD length (< 0bp) or FLT3-WT, but patients with short ITD length and FLT3-WT had the similar RFS and RR after chemotherapy. Allo-HSCT plus sorafenib maintenance significantly prolonged OS and RFS, decreased RR in FLT3-ITD patients, especially in these with long ITD length instead of those with short ITD length.CONCLUSION: Our findings indicated that FLT3-ITD mutation and long ITD length had negative effect on treatment response and prognosis in CN-AML patients. Allo-HSCT plus sorafenib maintenance was an effective strategy to improve survival and decrease relapse probability, abrogated disadvantage from long ITD length in FLT3-ITD patients. DisclosuresNo relevant conflicts of interest to declare.

Concurrent FLT3 Inhibitor and PP2A Activating Drug Treatment Induces Synergistic Cytotoxicity in Acute Myeloid Leukemia Cells with FLT3 Internal Tandem Duplication through Proteasomal Degradation of Pim-1 and c-Myc

Introduction: In 30% of acute myeloid leukemia (AML) patients, internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) causes constitutive and aberrant FLT3 signaling, and these patients have short relapse-free and overall survival. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. FLT3 activation also inhibits the tumor suppressor protein phosphatase 2A (PP2A). FLT3 inhibitors and PP2A-activating drugs have been shown to induce synergistic cytotoxicity in cells with FLT3-ITD. To address mechanisms underlying this effect, we studied effects of combination therapy on the oncogenic serine/threonine kinase Pim-1 and the transcription factor c-Myc, both of which are upregulated in cells with FLT3-ITD and are also PP2A substrates.Methods: Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and/or the PP2A-activating drug fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Drug combination effects were measured by combination index determined by the Chou-Talalay method using CompuSyn software. Apoptosis was measured by Annexin V/propidium iodide staining detected by flow cytometry. c-Myc and GAPDH control mRNA was measured by real-time polymerase chain reaction. Pim-1 kinase, c-Myc, phospho-c-MycSer62, phospho-c-MycThr58, phospho-STAT5Tyr694, STAT5, phospho-PP2ATyr307, PP2A, phospho-BADSer112 and BAD levels were measured by immunoblotting. Cycloheximide treatment was used to assess protein stability. Protein expression and stability were measured with and without the proteasome inhibitor MG-132. Pim-1 kinase was inhibited with the pan-Pim inhibitor AZD1208. Ba/F3-ITD cells were infected with pMX-Flag-K67M kinase-dead (KD) Pim-1 and empty pMX retroviral vectors and with pBABE-ER-cMYC and with empty pBABE-ER retroviral vectors.Results: Concurrent treatment with 15 nM gilteritinib or 1 nM quizartinib and FTY720 2 µM in cell lines and 4 µM in patient samples decreased growth and increased apoptosis of cells with FLT3-ITD, relative to single drug treatments, and produced synergistic cytotoxicity. FLT3 inhibition was confirmed by decrease in phospho-STAT5 and PP2A activation by decreased phospho-PP2A. Concurrent treatment decreased expression of both Pim-1 and c-Myc protein, but not c-Myc mRNA, in Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD, relative to single drug treatments. Additionally, selective decrease in phospho-MycSer62, a stable c-Myc phosphoprotein that is dephosphorylated by PP2A, was seen, with persistence of phospho-c-MycThr58. FLT3 inhibitor and PP2A activator combination treatment was found to decrease stability of c-Myc and Pim-1 protein, in relation to single drugs. Moreover, pretreatment with the proteasome inhibitor MG-132 abrogated downregulation of Pim-1 and c-Myc protein expression and decrease in Pim-1 and c-Myc protein stability in Ba/F3-ITD cells treated with FLT3 inhibitor and PP2A activator. Pretreatment with the pan-Pim kinase inhibitor AZD1208, with Pim-1 inhibition confirmed by decreased phospho-BADS112 had no effect on c-Myc downregulation, and c-Myc was similarly downregulated in Pim-1 kinase-dead cells as in parental and empty-vector cells, demonstrating that combination treatment effects on c-Myc are not Pim-1 kinase-dependent. Additionally, FLT3 inhibitor and PP2A-activating drug combination induced apoptosis in 30% of cells with c-Myc overexpression, compared to 60% of parental and empty vector-infected cells. Finally, c-Myc overexpression did not abrogate Pim-1 downregulation by combination treatment.Conclusions: Concurrent FLT3 inhibitor and PP2A activating drug treatment induces synergistic cytotoxicity in AML cells with FLT3 internal tandem duplication through proteasomal degradation of Pim-1 and c-Myc, and effects on Pim-1 and c-Myc are independent. The data support in vivo testing of FLT3 inhibitor and PP2A-activating drug combinations and development of a clinical trial. DisclosuresNo relevant conflicts of interest to declare.

Superior Salvage Treatment with Regimen Containing Sorafenib for Relapse of Acute Myeloid Leukemia with FLT3-ITD after Allo-HSCT

Background Internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) mutation has been reported in about 25% of patients with acute myeloid leukemia (AML). In contrast to patients with FLT3-ITD wild-type, AML with FLT3-ITD mutations have an inferior survival, primarily due to shorter remission duration and higher relapse rate. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) improves the survival for FLT3-ITD AML, the rate of leukemia relapse remains high. Patients experiencing leukemia relapse post-transplants have a dismal prognosis. Recent studies have demonstrated that sorafenib monotherapy or in combination with other therapeutic strategies could induce sustained responses for patients with FLT3-ITD relapsed post-transplants. The aim of this study is to evaluate the efficacy of sorafenib combined with other therapeutic strategies for AML with FLT3-ITD relapsed after allo-HSCT.MethodsA total of 76 AML with FLT3-ITD relapsed after allo-HSCT from January 2012 to May 2017 were enrolled in this study. Depending on whether receiving salvage therapy containing sorafenib, patients were divided into 2 groups: sorafenib group (n=49) and non-sorafenib group (n=27). On the basis of the differences of therapeutic regimens, patients were divided into 4 subgroups, including sorafenib+ chemotherapy+donor lymphocyte infusion (DLI) (Group A, n=39), sorafenib+ chemotherapy (Group B, n=10), chemotherapy +DLI (Group C, n=15), and monochemotherapy (Group D, n=12). Outcomes of different therapeutic regimens were compared.Results Forty patients obtained complete remission (CR) and 12 partial remission after salvage therapies, with the CR and overall response (OR) rates of 52.6% and 68.4%. The CR and OR rates were 65.3% and 81.6% in the sorafenib group, compared with 29.6% and 44.4% in the non-sorafenib group (P=0.003, P=0.001). Subgroup analysis showed that the CR and OR rates in Group A were higher than that in Group D (P=0.006, P=0.001), and they were similar to that in Groups B and C (all P values >0.008). There were also no significant differences in the CR and OR rates among Groups B, C, and D (all P values >0.008). With a median follow-up of 245 (range 30-1992) days after relapse, 26 patients remained alive and 50 died. The 3-year overall survival (OS) was 33.8% (95% CI, 23.3%-44.5%). The 3-year OS in the sorafenib group was superior to that in the non-sorafenib group (42.0% vs 18.5%, P=0.002). Subgroup analysis revealed that sorafenib combined with chemotherapy followed by DLI was superior to other regimens, with 3-year OS of 47.8%, 20.0%, 20.0%, and 16.7% in Groups A, B, C, and D, respectively (P=0.007). The incidences of acute and chronic GVHD as well as the mortality of GVHD after salvage therapies were similar among the four groups (P=0.304, P=0.429, P=0.601, respectively). Multivariate analysis revealed that salvage therapy containing sorafenib was the only protective factor for OS (P=0.022, hazard ratios= 0.479).ConclusionsSalvage therapy containing sorafenib was superior to that not containing sorafenib, and sorafenib combined with chemotherapy followed by DLI revealed optimal efficacy for relapse of AML with FLT3-ITD after allo-HSCT. DisclosuresFan:National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding.

RNA-based FLT3-ITD allelic ratio is associated with outcome and ex vivo response to FLT3 inhibitors in pediatric AML

AbstractControversy exists whether internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-internal tandem duplication [ITD]) allelic ratio (AR) and/or length of the ITD should be taken into account for risk stratification of pediatric acute myeloid leukemia (AML) and whether it should be measured on RNA or DNA. Moreover, the ITD status may be of relevance for selecting patients eligible for FLT3 inhibitors. Here, we included 172 pediatric AML patients, of whom 36 (21%) harbored FLT3-ITD as determined on both RNA and DNA. Although there was a good correlation between both parameters ARspearman = 0.62 (95% confidence interval, 0.22-0.87) and ITDlengthspearman = 0.98 (95% confidence interval, 0.90-1.00), only AR ≥ 0.5 and length ≥48 base pairs (bps) based on RNA measurements were significantly associated with overall survival (AR: Plogrank = .008; ITDlength: Plogrank = .011). In large ITDs (>156 bp on DNA) a remarkable 90-bp difference exists between DNA and RNA, including intron 14, which is spliced out in RNA. Ex vivo exposure (n = 30) to FLT3 inhibitors, in particular to the FLT3-specific inhibitor gilteritinib, showed that colony-forming capacity was significantly more reduced in FLT3-ITD-AR ≥ 0.5 compared with ITD-AR-low and ITD− patient samples (P < .001). RNA-based FLT3-ITD measurements are recommended for risk stratification, and the relevance of AR regarding eligibility for FLT3-targeted therapy warrants further study.