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In mammals, either enhancer of zeste homologue 2 (EZH2) or its homolog EZH1 functions as the catalytic subunit of polycomb repressive complex 2 (PRC2), and plays an essential role for the maintenance of transcriptional repression by the trimethylation of histone H3 lysine 27 (H3K27me3). Hyperinduction of H3K27me3 mediated by EZH2 overexpression or EZH2 gain-of-function (GOF) mutation (e.g. Y641, A677 and A687) has been associated with lymphoma and myeloma progression. Many publications suggest that suppressing PRC2 activity by EZH2 inhibition is a potential therapeutic target for hematological malignancies. Up to date, there are known a few EZH2 selective inhibitors with showing anti-tumor activity in some preclinical and clinical studies. However, the inhibition of EZH2 can complementarily induce EZH1 activation, which reactivates PRC2 function in turn. Therefore, dual inhibition of EZH1 and EZH2 could give more effective than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Herein, we introduce a novel EZH1/2 dual inhibitor, HM97594, which simultaneously inhibits the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. HM97594 potently repressed trimethylation of H3K27 in lymphoma and myeloma cell lines. As a result, HM97594 showed broader and stronger activities than EZH2 selective inhibitors in in vitro studies of inhibiting the proliferation of various hematological cancer cell lines. HM97594 induced the differentiation of DLBCL to plasma cells with an increment of cell lineage specific markers (e.g. PRDM1, IRF4 and CD38) and decreased the colony forming ability. Also, HM97594 caused cell cycle G0/1 arrest and apoptosis in KARPAS-422 cells harboring EZH2 Y641 GOF mutation. In addition, HM97594 showed potent antitumor activity in KARPAS-422 lymphoma cell xenograft mouse model. Once daily orally administered HM97594 greatly inhibited tumor growth in a dose-dependent manner without body weight loss compared to the known EZH2 inhibitor. Collectively, it was demonstrated in the present studies that HM97594, EZH1/2 dual inhibitor, has promising potential as an anticancer drug for patients with subtypes of hematological malignancies. Further preclinical studies will be performed and reported soon after the establishment of a preclinical candidate. Disclosures No relevant conflicts of interest to declare.

RATIONALE HM781-36B (1-[4-[4-(3,4-dichloro-2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy]-piperidin-1-yl]prop-2-en-1-one hydrochloride) is a new anticancer drug to treat advanced solid tumors in clinical trial. In order to understand the behavior of HM781-36B in vitro and in vivo we validated an analytical method for HM781-36B and its major metabolites in plasma. METHODS In vivo and in vitro metabolism of HM781-36B was studied in dog plasma, urine and feces as well as using human and dog liver microsomes with extraction by ethyl acetate or methyl tert-butyl ether, respectively, and successfully separated by high-performance liquid chromatography diode-array detection mass spectrometry (HPLC-DAD/MS). Ten metabolites were identified by LC/ESI-ion trap mass spectrometry (MS, MS2, MS3 and MRM) and LC/Q-TOF-MS/MS for exact mass measurement. For accurate characterization of the major metabolites, authentic standards (M1, M2, M4, and M10) were synthesized. RESULTS Ten metabolites of HM781-36B in an in vitro mixture were separated and identified by LC/ESI-MSn. The MS/MS spectral patterns of the parent drug and metabolites exhibited two characteristic ions (A- and B-type ions) attributed to the cleavage of the ether bond between the piperidine ring and the quinazoline ring, providing important information on the site of chemical conversion during the metabolism. Six hydroxylated derivatives including dehalogenation and demethylation, two N-oxide forms, a demethylated form and de-acryloylpiperideine metabolites were observed. CONCLUSIONS The LC/ESI-ion trap MSn technique was effective in obtaining structural information and yielded diagnostic ions for the identification of diverse metabolites. The multiple metabolic pathways of HM781-36B were suggested in in vitro and in vivo samples and the dihydroxylation (M1) and demethylation (M2) appeared to be the major metabolites. Copyright © 2013 John Wiley & Sons, Ltd.

Introduction: Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the second most frequent cause of cancer-related death, however, treatment options are very limited. In recent studies, aberrant signaling through FGFR4 and its ligand, FGF19 have been identified as the oncogenic driver in a subset of HCCs and reported to be associated with poor prognosis. About 30% of HCC patients have altered FGF19/FGFR4 pathway signaling, therefore, the treatment with FGFR4 inhibitor may produce benefit. Materials and Method: Using the structure-based design, we have generated a novel, potent and selective FGFR4 inhibitor, HM81422 with irreversible-covalent binding mode, and evaluated its anti-tumor activity in a variety of HCC cell lines, HCC cell line xenografts and orthotopic grafts. Results: Biochemical selectivity assays demonstrated that HM81422 is highly selective towards FGFR4 compared to other FGFR isotypes as well as a panel of several kinases. The treatment of HM81422 to FGF19 amplified and overexpressed HCC cell lines led to suppression of FGF19/FGFR4 signaling pathway and concomitant reduction in cell viability in dose-dependent manner. Oral administration of HM81422 to mice bearing FGF19 altered HCC cells showed dose-dependent pharmacokinetics, pharmacodynamic modulation of FGFR4 signaling and antitumor efficacy in xenograft models. And HM81422 demonstrated inhibition of tumor growth in an orthotopic liver xenograft model of FGF19 altered HCC in nude mice. Conclusion: In conclusion, the treatment of HCC patients with a potent and selective FGFR4 inhibitor, HM81422, can be an attractive approach targeting approximately 30% of HCC patients by inhibiting altered FGF19-mediated signaling cascade. Further preclinical studies with HM81422 will be performed and reported soon. Citation Format: JaeHo Lee, Hyunjeong Kang, Kyounghwa Koo, Youngeun Ha, Sun Young Lim, Joo-Yun Byun, Hyunkyung Yu, Taehun Song, Moonsub Lee, Seung Hyun Jung, Taewoo Kim, Hyojeong Bang, Eunyoung Kim, Jahoon Kang, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. A novel, potent and selective FGFR4 inhibitor, HM81422 in hepatocellular carcinoma with FGFR4-driven pathway activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4780.

Introduction: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutations is associated with poor prognosis with a high risk of relapse after therapy and reduced overall survival. Activating mutations of internal tandem duplication (ITD) and tyrosine kinase domain point mutations (TKD) of FLT3 have been reported in approximately 30% of AML as oncogenic driver mutations. Currently, FLT3 inhibitors showed clinical benefits in the corresponding AML patients. In this study, we have characterized a novel FLT3 inhibitor, HM43239, and assessed the potential as a novel therapy for AML patients. Materials and Methods: HM43239 is a novel, highly potent FLT3 kinase inhibitor. This compound showed tight binding to FLT3 kinase in in silico docking model as a reversible Type I inhibitor. In vitro kinase assay was performed to identify kinase selectivity of HM43239. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM43239 in AML cell lines. HM43239 was evaluated in MV-4-11 and MOLM-13 xenograft mice models. Tumor sizes were measured and tumor samples were analyzed of the mechanisms of action. Results: Among 191 kinases biochemically assayed, HM43239 showed the high selectivity toward FLT3 and AML associated other kinases (e.g. SYK, JAK and TAK1). IC50s' of HM43239 against FLT3 WT, FLT3 ITD and FLT3 D835Y kinases were 1.1 nM, 1.8 nM and 1.0 nM, respectively. HM43239 potently inhibited the growth of AML cell lines harboring FLT3 ITD mutation, such as MV4-11 (IC50: 1.3 nM), MOLM-13 (5.1 nM) and MOLM-14 (2.9 nM). Furthermore, HM43239 effectively inhibited the phosphorylation levels of FLT3 and of downstream kinases related with cell proliferation. In addition, caspase 3/7-dependent apoptosis was induced by HM43239 in AML cell lines expressing FLT3 ITD mutant. And HM43239 inhibited proliferation and induced apoptosis of leukemic stem cell (LSC) marker-expressing KG1a cells (CD34+/CD38- cells) suggesting that the possibility for targeting LSC. HM43239 showed the excellent dose proportional antitumor activity in mouse models xenografted with both MV4-11 and MOLM-13 cell line without any significant toxicity. Moreover, we identified in vivo modulation of related targets (p-FLT3 & p-STAT5) in AML cell with FLT3 mutant. Conclusion: Taken together, HM43239 has demonstrated the potential therapeutic efficacy for the treatment of AML patients and implicated the mechanism of overcoming resistance and preventing relapse. Citation Format: Miyoung Lee, Young Eun Ha, Mi Jin Moon, Joo-Yun Byun, HyunKyung Yu, SeokJong Kang, JaeHo Lee, Kyuhang Lee, Eunkyung Kim, Eunyoung Kim, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. Antitumor activity of the potent and novel FLT3 inhibitor HM43239 in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 804.

HM781-36B (1-[4-[4-(3,4-dichloro-2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy]-piperidin-1-yl]prop-2-en-1-one hydrochloride) is a new anticancer drug to treat advanced solid tumors in clinical trial. In order to understand the behavior of HM781-36B in vitro and in vivo we validated an analytical method for HM781-36B and its major metabolites in plasma.In vivo and in vitro metabolism of HM781-36B was studied in dog plasma, urine and feces as well as using human and dog liver microsomes with extraction by ethyl acetate or methyl tert-butyl ether, respectively, and successfully separated by high-performance liquid chromatography diode-array detection mass spectrometry (HPLC-DAD/MS). Ten metabolites were identified by LC/ESI-ion trap mass spectrometry (MS, MS(2) , MS(3) and MRM) and LC/Q-TOF-MS/MS for exact mass measurement. For accurate characterization of the major metabolites, authentic standards (M1, M2, M4, and M10) were synthesized.Ten metabolites of HM781-36B in an in vitro mixture were separated and identified by LC/ESI-MS(n) . The MS/MS spectral patterns of the parent drug and metabolites exhibited two characteristic ions (A- and B-type ions) attributed to the cleavage of the ether bond between the piperidine ring and the quinazoline ring, providing important information on the site of chemical conversion during the metabolism. Six hydroxylated derivatives including dehalogenation and demethylation, two N-oxide forms, a demethylated form and de-acryloylpiperideine metabolites were observed.The LC/ESI-ion trap MS(n) technique was effective in obtaining structural information and yielded diagnostic ions for the identification of diverse metabolites. The multiple metabolic pathways of HM781-36B were suggested in in vitro and in vivo samples and the dihydroxylation (M1) and demethylation (M2) appeared to be the major metabolites.