Abstract: Imatinib mesylate has drastically changed Philadelphia chromosome positive (Ph+) leukemia treatment. However, remissions induced in advanced phase Ph+ leukemias tend to be short-lived even treated with imatinib. Combined therapy of imatinib with an agent which inhibits downstream signaling of BCR/ABL such as Ras is intriguing. Phase I studies of farnesyl transferase inhibitors (FTIs), which targeted farnesylation of Ras, with imatinib against Ph+ leukemias demonstrated limited combination effects. We previously reported that the third-generation bisphosphonates (BPs) zoledronate (ZOL) synergized with imatinib mesylate to inhibit Ph+ leukemia. BPs which inhibit both farnesylation and geranylgeranylation of Ras proteins may be more potent than FTIs. However, three important questions have remained unanswered to promote this attractive combination to a clinical trial. Concentrations of 20-30 μM ZOL are required in vitro to induce apoptosis in leukemic cell lines. However, peak serum concentrations after infusion of 4 mg ZOL were 1–2 μM. Therefore, it is important to investigate how ZOL achieves effective concentrations in vivo. The second is whether ZOL augments the effects of imatinib against not only cell lines but also primary cells from Ph+ leukemia patients. In addition, the safety of this combination therapy must be established, because the dose of ZOL used in our previous study was high compared with clinical doses administered for inhibition of bone lesions. To answer these questions, we herein investigated about the combination therapy of ZOL plus imatinib using NOD/SCID mice engrafted with primary Ph+ leukemic cells with or without mutations in the ATP binding site. ZOL inhibited the geranylgeranylation of Ras proteins of leukemic cells in bone marrow but not in peripheral blood, indicating that sufficient concentration of ZOL could be achieved in bone marrow. ZOL synergized with imatinib to enhance survival of mice engrafted with primary Ph+ leukemic cells without mutations and this combination was well-tolerated without any severe adverse effects. These findings suggest that ZOL inhibits leukemia growth in bone marrow and the combination of ZOL plus imatinib is a potential therapy for Ph+ leukemia patients.

Abstract: Background: Isocitrate dehydrogenase (IDH)-1 and -2 are TCA cycle-involved enzymes which convert isocitrate to alpha-ketoglutarate. Mutations that alter the enzymatic activity causes accumulation of a mal-metabolite D-2-hydroxyglutarate, which results in inhibition of DNA methylation and tumorigenesis. IDH-1 and IDH-2 mutation are present in approximately 7-10% and 10% of patients with acute myeloid leukemia (AML), respectively. Recently, whole exome sequencing has been used for the next-generation sequencing of AML, and certain gene mutations have been identified in patients with AML. The treatment strategies for leukemia have undergone drastic changes with the rapid development of new drugs. However, the proper use of newly developed agents poses a major challenge in AML treatment. Genome profiling analysis can be used to select the optimal treatment for patients with newly diagnosed AML. IDH mutant-specific inhibitors such as ivosidenib and enasidenib were already approved in the US, and combination treatment with venetoclax and Azacitidine was recently approved in Japan. Methods and Results: We lunched an actionable mutation profiling multicenter study named Hematologic Malignancies (HM)-SCREEN-Japan 01 (UMIN000035233), in which a comprehensive genomic assay was performed by Foundation One Heme (F1H) panel. The primary outcome was the frequency of each genomic alteration, as determined using F1H, which is a comprehensive genome profiling test based on next-generation sequencing, in the AML specimens. The secondary outcome was the association between each genomic alteration and the clinicopathological characteristics, prognosis, and quality of specimens used in the genetic analysis. The eligibility criteria were as follows: 1) histological diagnosis of AML through bone marrow aspiration; 2) fulfillment of either of the following conditions: i) newly diagnosed AML unfit for standard treatment (ND-unfit AML) or ii) R/R-AML; 3) sufficient sample collection via bone marrow aspiration; 4) Age of participants 20 years or above during registration; 5) provision of written informed consent by participants. Paraffin-embedded bone marrow samples were gathered from 17 Japanese faculties and the F1H reports were returned to the patients. The median turnaround time was 13 days (minimum 8 days). We found 13 patients (7.3%) with IDH1 mutation and 17 patients (9.6%) with IDH2 mutation out of 177 patients who joined this study and the F1H report was successfully returned. Only one patient had both mutations, and each mutation was mutually exclusive in all the other patients (Figure 1). The major amino acid alteration of IDH1 and IDH2 were R132C/G/H/L and R140Q/W, respectively. Frequently co-occurring mutations include FLT3 (44.8%), NPM1 (34.5%), DNMT3A (31.0%) and RUNX1 mutation (20.7%). Mutations of RAS pathway-related genes (e.g., KRAS, NRAS and PTPN11) were seen in 6 patients (20.7%). Any gene alterations didn't show statistically significant co-occurrence with IDH1 and IDH2 mutation. Serial genome profiling analyses were performed to evaluate the time-dependent changes in the genome profiles of patients administered FLT3 inhibitors, gilteritinib, and quizartinib for treating FLT3-mutated AML. Also in this cohort, we are examining the properties and distribution of IDH1/2 mutations during treatment with FLT3 inhibitors. In the several patients, expansion and persistence of IDH mutated clones seemed to be cause of resistance (Figure 2 as the representative result). The detailed clinical outcomes of AML patients with IDH1/2 mutations are under investigation. Conclusions: In our evaluation of the suitability of F1H for HM-SCREEN-Japan 01, we successfully identified IDH-1/2 mutation that can be used as therapeutic targets in AML, which have rarely been identified thus far. Figure 1 Figure 1. Disclosures Shibayama: Eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Ono: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Nippon Shinyaku: Honoraria; Daiichi Sankyo: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Chugai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Fujimoto: Honoraria; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Mundi Pharma: Honoraria; Essentia Pharma Japan: Research Funding. Yamauchi: Otsuka: Research Funding; Ono Pharmaceutical: Honoraria; Pfizer: Honoraria, Research Funding; Chugai: Honoraria; Abbie: Research Funding; Astellas: Research Funding; Daiichi Sankyo: Research Funding; Solasia Pharma: Research Funding. Kondo: Otsuka Pharmaceutical: Consultancy, Honoraria, Research Funding; Novartis Pharma KK: Honoraria; Bristol-Myers Squibb Company: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Sanwa Kagaku Kenkyusho CO.,LTD: Consultancy; Pfizer: Honoraria; Astellas Pharma Inc.: Consultancy, Honoraria; Abbvie: Honoraria. Yamamoto: AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Bristol-Myers Squibb/Celgene: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Eisai: Honoraria, Research Funding; IQIVA/Incyte: Research Funding; IQIVA/HUYA: Honoraria; HUYA: Consultancy; Janssen: Honoraria; Kyowa Kirin: Honoraria; Meiji Seika Pharma: Consultancy, Honoraria, Research Funding; MSD: Honoraria; Mundipharma: Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ono: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Sanofi: Honoraria; Solasia Pharma: Research Funding; SymBio: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Yakult: Honoraria, Research Funding; Zenyaku: Honoraria, Research Funding; Micron: Honoraria; IQIVA/Genmab: Research Funding; ADC Therapeutics: Honoraria. Kuroda: Taiho Pharmaceutical: Research Funding; Fujimoto Pharmaceutical: Current Employment, Honoraria, Research Funding; Asahi Kasei: Research Funding; Shionogi: Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Sysmex: Research Funding; Eisai: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; MSD: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Otsuka Pharmaceutical: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Research Funding; Dainippon Sumitomo Pharma: Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; Bristol-MyersSquibb: Consultancy, Honoraria, Research Funding; Janssen Pharmaceutical K.K: Consultancy. Usuki: Astellas: Research Funding, Speakers Bureau; Abbvie: Research Funding; Gilead: Research Funding; Symbio: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding, Speakers Bureau; Sumitomo Dainippon: Research Funding; Otsuka: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Brisol-Myers Squibb: Research Funding, Speakers Bureau; Ono: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Nippon Boehringer Ingelheim: Research Funding; Astellas-Amgen-Biopharma: Research Funding; Nippon shinyaku: Research Funding, Speakers Bureau; Kyowa Kirin: Research Funding, Speakers Bureau; Pfizer: Research Funding; Alexion: Speakers Bureau; Eisai: Speakers Bureau; MSD: Speakers Bureau; PharmaEssentia: Speakers Bureau; Yakult: Speakers Bureau; Mundipharma: Research Funding. Yoshimitsu: Novartis: Honoraria; Takeda: Honoraria; Sanofi: Honoraria. Ishitsuka: Asahi kasei: Research Funding; Eli Lilly: Research Funding; MSD: Research Funding; Daiichi Sankyo: Consultancy, Other: Personal fees; Kyowa Kirin: Other: Personal fees, Research Funding; Ono Pharmaceutical: Other: Personal fees, Research Funding; Celgene: Honoraria, Other: Personal fees; Chugai Pharmaceutical: Honoraria, Other: Personal fees, Research Funding; BMS: Other; Takeda: Other: Personal fees, Research Funding; Mundipharma: Other: Personal fees; Taiho Pharmaceuticals: Other: Personal fees, Research Funding; Janssen Pharmaceuticals: Other: Personal fees; Huya Japan: Other: Personal fees; Novartis: Other: Personal fees; Pfizer: Other: Personal fees; Astellas Pharma: Other: Personal fees, Research Funding; Genzyme: Other: Personal fees; Sumitomo Dainippon Pharma: Other: Personal fees, Research Funding; Eisai: Other: Personal fees, Research Funding; Mochida: Other: Personal fees, Research Funding; Shire: Other; Otsuka Pharmaceutical: Other: Personal fees; Teijin Pharma: Research Funding. Ono: Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb Company: Honoraria; Celgene: Honoraria, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K: Honoraria; Eisai Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; Takeda Pharmaceutical Company Limited.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; DAIICHI SANKYO COMPANY, LIMITED.: Honoraria; Novartis Pharma KK: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria; TAIHO PHARMACEUTICAL CO., LTD.: Research Funding; Merck Sharp & Dohme: Honoraria, Research Funding. Takahashi: Toyamakagaku: Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Research Funding; Kyowahakko-Kirin: Research Funding; Ono: Research Funding; Asahikasei: Research Funding; Eizai: Research Funding. Iyama: Alexion Pharmaceuticals: Honoraria, Research Funding; Astellas: Honoraria; CSL Behring: Honoraria; Daiichi Sankyo: Honoraria; Otsuka Pharmaceuticals Factory: Honoraria; Otsuka Pharmaceuticals Factory: Honoraria; MSD: Research Funding; Nippon Shinyaku: Honoraria; Novartis: Honoraria; Otsuka: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; SymBio Pharmaceuticals: Research Funding. Izutsu: Genmab: Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Research Funding; Fuji Film Toyama Chemical: Honoraria; Eisai: Honoraria, Research Funding; Incyte: Research Funding; Huya Biosciences: Research Funding; Chugai: Honoraria, Research Funding; Symbio: Honoraria; Solasia: Research Funding; Pfizer: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; MSD: Research Funding; Kyowa Kirin: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Beigene: Research Funding; Bayer: Research Funding; AstraZeneca: Honoraria, Research Funding; Allergan Japan: Honoraria; AbbVie: Honoraria; Takeda: Honoraria, Research Funding; Yakult: Research Funding. Minami: Novartis Pharma KK: Honoraria; Ono: Research Funding; Pfizer Japan Inc.: Honoraria; Astellas: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb Company: Honoraria; CMIC: Research Funding.

Abstract: Imatinib mesylate (also known as STI-571 and Gleevec) has drastically changed the treatment of Philadelphia chromosome positive (Ph+) leukemias. However, the resistance to imatinib has frequently been reported, particularly in patients with advanced-stage disease. A novel orally bioavailable inhibitor of the ABL tyrosine kinase (TK) named CNS-9 was developed from the 2-(phenylamino)pyrimidine class to overcome resistance mechanisms of imatinib. Inhibition of TK phosphorylation (IC50) on wild type (wt) BCR/ABL in 293T cell line by CNS-9 was 22nM, which was 2-log more potent than imatinib. Importantly, CNS-9 inhibited TK phosphorylation of E255K mutant BCR/ABL with IC50 of 98nM, while imatinib could not inhibit it with clinically relevant concentration. The T315I mutant BCR/ABL protein was resistant to CNS-9 and imatinib. CNS-9 also inhibited TK phosphorylation of platelet-derived growth factor receptor (PDGFR) or c-Kit pathways at the very similar observed IC50s when compared with imatinib, in spite of significant higher potency against ABL. The ability of CNS-9 in vitro to inhibit 101 TK molecules was assayed by KinaseProfilerTM (Upstate), showing also more specific inhibitory activity against ABL than imatinib. The growth of BCR/ABL-positive cell lines K562, KU812, BaF3 harboring wt BCR/ABL (BaF3/wt) and E255K (BaF3/E255K) was inhibited by CNS-9 with IC50 of 5, 3, 17, and 110nM, respectively (Table 1). Generally, CNS-9 was 20 to 30-fold more potent on the growth inhibition than imatinib in these same cell lines. We next investigated the in vivo effect on the leukemic growth inhibition of CNS-9. Nude mice were injected subcutaneously with 3x107 KU812 (wt BCR/ABL) on Day 0. CNS-9 or imatinib were orally administrated twice a day from Day 7 to Day 18. The dosages of CNS-9 and imatinib, which inhibited completely tumor growth were 20mg/kg/day and 200mg/kg/day, respectively, indicating that CNS-9 is 10-fold potent than imatinib in vivo. To examine the in vivo effect of CNS-9 against mutant BCR/ABL, BaF3/wt, BaF3/E255K or BaF3/T315I were engrafted to nude mice and treated with CNS-9 or imatinib. CNS-9 was also 10-fold potent than imatinib against BaF3/wt. Intriguingly, mice harboring BaF3/wt or BaF3/E255K showed significantly prolonged survival when treated with CNS-9. Consistent with in vitro assay, CNS-9 had no effect on T315I, and imatinib was not effective against both E255K and T315I. In conclusion, CNS-9 is substantially more inhibitory and more specifically than imatinib toward BCR/ABL-dependent cell growth both in vitro and in vivo Moreover, CNS-9 may be effective for leukemia patients whose leukemic cells harbor E255K mutant. The efficacy and safety of CNS-9 for Ph+ leukemias should be verified in early phase clinical trials. The IC50s values of leukemic cell lines for CNS-9 and imatinib CNS-9 (nM) imatinib (nM) K562 p210 wt BCR/ABL 5 130 KU812 p210 wt BCR/ABL 3 67 U937 BCR/ABL (−) 〉 1000 〉 1000 BaF3 p190 wt BCR/ABL 17 360 BaF3 p190 E255K BCR/ABL 110 〉 1000 BaF3 p190 T315I BCR/ABL 〉 1000 〉 1000