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

FLT3 mutation is found in about 30% of acute myeloid leukemia (AML) patients and is associated with a poor prognosis. Several FLT3 inhibitors are undergoing investigation, while their clinical efficacies were lower than expected and several resistant mechanisms to FLT3 inhibitors have been demonstrated. Although most AML cells harboring FLT3 mutation co-express wild-type (Wt)-FLT3, it is not fully understood how Wt-FLT3 expression is associated with the resistance to FLT3 inhibitors. In this study, we elucidated a resistant mechanism by which FL-dependent Wt-FLT3 activation reduced inhibitory effects of FLT3 inhibitors. We demonstrated that FL-stimulation much more strongly reduced growth inhibitory effects of FLT3 inhibitors on Wt- and mutant-FLT3 co-expressing cells than sole mutant-FLT3 expressing cells both in vitro and in vivo. It was also confirmed that FL impaired the anti-leukemia effects of FLT3 inhibitors on primary AML cells. We elucidated that FL impeded the inhibitory effects of FLT3 inhibitors mainly through the activation of Wt-FLT3, but not mutated FLT3, in the Wt- and ITD-FLT3 co-expressing cells. Furthermore, FL-induced activation of Wt-FLT3-MAPK axis was the dominant pathway for the resistance, and the glycosylation of Wt-FLT3 was also vital for FL-dependent kinase activation and following resistance to FLT3 inhibitors. Thus, we clarified the importance of co-expressing Wt-FLT3 in resistance to FLT3 inhibitors. These findings provide us with important implications for clinical application and new strategies to improve clinical outcomes of FLT3 inhibitors.

No abstract

Genetic alterations in myelodysplastic syndromes (MDS) are critical for pathogenesis. We previously showed that peripheral blood cell‐free DNA (PBcfDNA) may be more sensitive for genetic/epigenetic analyses than whole bone marrow (BM) cells and mononuclear cells in peripheral blood (PB). Here we analyzed the detailed features of PBcfDNA and its utility in genetic analyses in MDS. The plasma‐PBcfDNA concentration in MDS and related diseases (N = 33) was significantly higher than that in healthy donors (N = 14; P = 0.041) and in International Prognostic Scoring System higher‐risk groups than that in lower‐risk groups (P = 0.034). The concentration of plasma‐/serum‐PBcfDNA was significantly correlated with the serum lactate dehydrogenase level (both P < 0.0001) and the blast cell count in PB (P = 0.034 and 0.025, respectively). One nanogram of PBcfDNA was sufficient for one assay of Sanger sequencing using optimized primer sets to amplify approximately 160‐bp PCR products. PBcfDNA (approximately 50 ng) can also be utilized for targeted sequencing. Almost all mutations detected in BM‐DNA were also detected using corresponding PBcfDNA. Analyses using serially harvested PBcfDNA from an RAEB‐2 patient showed that the somatic mutations and a single nucleotide polymorphism that were detected before allogeneic transplantation were undetectable after transplantation, indicating that PBcfDNA likely comes from MDS clones that reflect the disease status. PBcfDNA may be a safer and easier alternative to obtain tumor DNA in MDS.

No abstract

Patient-derived xenografts (PDX) are widely used as human cancer models. Previous studies demonstrated clonal discordance between PDX and primary cells. However, in acute myeloid leukemia (AML)-PDX models, the significance of the clonal dynamics occurring in PDX remains unclear. By evaluating changes in the variant allele frequencies (VAF) of somatic mutations in serial samples of paired primary AML and their PDX bone marrow cells, we identify the skewing engraftment of relapsed or refractory (R/R) AML clones in 57% of PDX models generated from multiclonal AML cells at diagnosis, even if R/R clones are minor at <5% of VAF in patients. The event-free survival rate of patients whose AML cells successfully engraft in PDX models is consistently lower than that of patients with engraftment failure. We herein demonstrate that primary AML cells including potentially chemotherapy-resistant clones dominantly engraft in AML-PDX models and they enrich pre-existing treatment-resistant subclones.

All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are essential for acute promyelocytic leukemia (APL) treatment. It has been reported that mutations in PML-RARA confer resistance to ATRA and ATO, and are associated with poor prognosis. Although most PML-RARA mutations were point mutations, we identified a novel seven amino acid deletion mutation (p.K227_T233del) in the RARA region of PML-RARA in a refractory APL patient. Here, we analyzed the evolution of the mutated clone and demonstrated the resistance of the mutated clone to retinoic acid (RA). Mutation analysis of PML-RARA was performed using samples from a chemotherapy- and ATRA-resistant APL patient, and the frequencies of mutated PML-RARA transcript were analyzed by targeted deep sequencing. To clarify the biological significance of the identified PML-RARA mutations, we analyzed the ATRA-induced differentiation and PML nuclear body formation in mutant PML-RARA-transduced HL-60 cells. At molecular relapse, the p.K227_T233del deletion and the p.R217S point-mutation in the RARA region of PML-RARA were identified, and their frequencies increased after re-induction therapy with another type of retinoiec acid (RA), tamibarotene. In deletion PML-RARA-transduced cells, the CD11b expression levels and NBT reducing ability were significantly decreased compared with control cells and the formation of PML nuclear bodies was rarely observed after RA treatment. These results indicate that this deletion mutation was closely associated with the disease progression during RA treatment.

Background: FLT3 is one of the most frequently mutated genes in acute myeloid leukemia (AML). Internal tandem duplication (ITD) of juxtamembrane domain sequence and missense point mutations at D835 residue within kinase domain are major mutations of FLT3 in AML. These mutations induce constitutive activation of FLT3 and its downstream pathway, resulting in aberrant cell proliferation of AML cells. FLT3 is, therefore, believed to be an attractive drug target for AML. Several FLT3 inhibitors were evaluated in clinical trials and they demonstrated clinical efficacy; however, drug-resistant secondary mutations such as F691L and D835 mutations with FLT3-ITD were often appeared. Therefore development of novel FLT3 inhibitors is required to overcome resistance to current FLT3 inhibitors. Here we report that a novel irreversible FLT3 inhibitor, FF-10101 is a promising agent for AML therapy. Methods: Bone marrow samples from patients with AML were subjected to Ficoll-Hypaque density gradient centrifugation. Informed consent was obtained from all patients, and approval was obtained from the ethics committee of our institute. In vitro growth inhibitory assay was performed with leukemia cell lines and 32D transfectants in liquid culture and primary AML cells in semisolid culture. Cell viability was determined by MTS assay or ATP quantification assay. For in vivo efficacy study, leukemia xenograft mouse model was prepared by tale vein injection of MOLM-13 cells or primary AML cells. Efficacies of tested compounds were evaluated by detection of human CD45-positive cells in bone marrow cells obtained from femurs at the end of studies. Results: Kinase profiling assay with 216 human recombinant kinases revealed that FF-10101 selectively and potently inhibited kinase activities of wtFLT3 and FLT3 D835Y with IC50 values of 0.20 nM and 0.16 nM, respectively. In MV4-11 cells, FF-10101 treatment decreased phosphorylation of FLT3 and its downstream molecules in a dose-dependent manner. FF-10101 treatment for 2 days demonstrated growth inhibitory effect on FLT3-dependent human cell lines, MV4-11, MOLM-13, MOLM-14 and 32D transfectants expressing FLT3-ITD with equal to or greater potency than Quizartinib, a highly potent FLT3 inhibitor currently under clinical development for AML (FF-10101 IC50=0.83 nM-2.4 nM, Quizartinib IC50=0.95 nM-4.5 nM). Cell cycle arrest was observed followed by increased sub-G1 population in MV4-11 cells treated with 1 nM FF-10101. Importantly, FF-10101 retained growth inhibitory activities against 32D transfectants expressing drug resistance mutations such as FLT3-ITD/D835Y, FLT3-ITD/Y842C or FLT3-ITD/Y842H with IC50 values of 0.66-3.1 nM, although Quizartinib demonstrated weak inhibitory effects with IC50 values of 85-150nM. In mice xenografted with MOLM-13, oral administration of 5 mg/kg FF-10101 once daily for 8 days significantly decreased MOLM-13 cells in bone marrow as compared to vehicle administration (p<0.001). Next, anti-leukemic effect of FF-10101 was assessed by using primary AML cells. In vitro cell growth assay, 1 week treatment of FF-10101 significantly reduced primary AML cells harboring FLT3-ITD. Growth inhibitory effect was also observed in primary AML cells harboring FLT3 D835H mutation, although Quizartinib had little effect. When 10 mg/kg FF-10101 was orally administrated twice daily to mice xenografted with primary AML cells with FLT3-ITD, AML cells in bone marrow were significantly reduced with comparable efficacy of Quizartinib. Furthermore, FF-10101 demonstrated more potent efficacy than Quizartinib in mice xenografted with primary AML cells harboring FLT3 D835H mutation. FF-10101 also retained its efficacy against mice xenografted with residual AML cells in Quizartinib-treated mice inoculated with primary AML cells harboring FLT3 D835H. Conclusions: We have developed a novel irreversible FLT3 inhibitor, FF-10101. FF-10101 showed potent anti-leukemic effect on cell lines and primary AML cells by selective inhibition of FLT3 both in vitro and in vivo. Notably, FF-10101 also has potency against drug resistance mutations. These results strongly indicate that FF-10101 is a promising agent for AML patients with FLT3 mutations. Phase I study of FF-10101 for AML patients is planned for 2016. Disclosures Nakatani: FUJIFILM Corporation: Employment. Uda:FUJIFILM Corporation: Employment. Yamaura:FUJIFILM Corporation: Employment. Takasaki:FUJIFILM Corporation: Employment. Ishikawa:GlaxoSmithKline K.K.: Research Funding. Hagiwara:FUJIFILM Corporation: Employment. Kiyoi:Eisai Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; Toyama Chemical CO., LTD.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding.