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Mutational analysis of primary tumors and single circulating tumor cells captured by a novel dielectrophoretic microwell array system in metastatic breast cancer, oral cancer and lung cancer patients
Poster Session – Molecular targeted agents I, Wednesday 29 November 2016 54 Poster (Board P025) A novel anti-androgen candidate galeterone acts by targeting USP12, a deubiquitinating enzyme that controls prostate cancer growth and survival U. McClurg1 , D. Dransfield2 , N. Namdev2 , D. Jacoby3 , N. Chit1 , S. Nakjang1 , J. Edwards4 , S. McCracken1 , C. Robson1 . 1 Newcastle University, Northern Institute for Cancer Research, Newcastle upon Tyne, United Kingdom; 2 Tokai Pharmaceuticals, Translational Medicine, Boston, USA; 3 Tokai Pharmaceuticals, Research, Boston, USA; 4 University of Glasgow, Institute of Cancer, Glasgow, United Kingdom Background: Galeterone is a highly selective, orally bioavailable small molecule drug candidate that disrupts androgen receptor (AR) signalling through AR degradation. Galeterone is also a potent CYP17 inhibitor and AR antagonist. We previously demonstrated that USP12 functions as an AR co-activator by deubiquitinating the AR and stabilising its protein levels. USP12 protein targets the PHLPP phosphatases and thereby reduces the level of activated AKT (pAKT) and as such indirectly stabilises the AR. Galeterone has previously been reported to induce pAKT and proteasomal inhibition abrogates galeterone’s ability to induce AR degradation consistent with the involvement of the ubiquitination pathway. Our project focused on determining the main enzymes involved in galeterone-driven AR degradation. Materials and Methods: We utilised a series of biochemical, biophysical and cell-based in vitro methodologies to elucidate the enzymes responsible for the proteasomal-dependent mechanism of galeterone-induced AR degradation. We screened a panel of 22 deubiquitinating enzymes (DUBs) in vitro to test whether galeterone inhibited enzymatic activity. Surface plasmon resonance (SPR) was applied to confirm direct binding to DUBs which were inhibited by galeterone. We then used next-generation RNA sequencing and immunohistochemistry to determine the role of USP12 in prostate cancer (PCa). Results: We discovered that galeterone acts on AR by selectively inhibiting USP12, a DUB that relies on UAF1, for its enzymatic activity. Galeterone dose-dependently bound to USP12, alone or when pre-complexed with UAF1. Global transcriptome profiling identified that in addition to regulating the AR signalling cascade, USP12 controls the p53 signalling pathway. USP12 directly targets the E3 MDM2, stabilising MDM2 protein and consequently controlling p53 protein levels. The clinical importance of USP12 was confirmed in PCa samples where increased USP12 was found to be a marker of poor prognosis that correlated with shorter relapse-free survival and reduced overall survival. Additionally, USP12 may play a role in the development of castration-resistant PCa (CRPC) as USP12 protein levels were significantly increased in CRPC patients from two independent clinical cohorts. Conclusions: We report that galeterone, a candidate small molecule drug currently in a phase 3 registration trial in metastatic CRPC inhibits AR activity in prostate cancer by inhibiting a DUB that stabilises the AR, namely USP12. We determined that USP12 additionally controls the p53 pathway with USP12 protein levels elevated in PCa and associated with decreased relapse-free survival and overall survival. Our results indicate that galeterone could offer a therapeutic alternative in PCa treatment as no other anti-androgens to date have been shown to inhibit the DUBs that regulate AR levels in PCa. No conflict of interest. 55 Poster (Board P026) Lactate and choline metabolites are potential biomarkers for monitoring response to mTOR pathway inhibitors in combination with the ALK inhibitor crizotinib in ALK-mutated neuroblastoma N.M.S. Al-Saffar1 , L.E. Jackson1 , J. Sidhu1 , L. Chesler2 , M.O. Leach1 . 1 The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, Cancer Research UK Cancer Imaging Centre, Division of Radiotherapy and Imaging, London, United Kingdom; 2 The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, Paediatric Solid Tumour Biology and Therapeutics Team, Division of Cancer Therapeutics, London, United Kingdom Background: Clinical outcome remains poor in patients with high-risk neuroblastoma (NB). Novel treatments are required. One promising avenue for targeted therapy in NB focuses on ALK. However, the ALK inhibitor crizotinib (Criz) has shown limited activity against the various ALK mutations identified in NB patients. Therefore, some criz resistant mutants will require a combinational therapeutic approach to achieve tumour response. Berry T et al 2012 showed that combining criz with the mTOR inhibitor Torin2 (Tor2) potentiates response in ALKF1174L/MYCN-positive models compared to single agent treatment. This combination is now being pursued clinically in paediatrics. Our aim is to assess whether this
Poster abstracts
S25
combination results in metabolic changes that are detectable by NMR providing noninvasive biomarkers of pathway inhibition and potentially of tumour response to combination therapy during early stage clinical trials. Material and Methods: The human NB Kelly cells (ALKF1174L mutant) were treated for 24 h with criz, Tor2 (both at 2xGI50 ), or the combination. Treatment effects were studied at molecular and cellular levels. Metabolic changes were evaluated by 1 H NMR. Results: Table 1 summarizes the cellular and metabolic effects of treatment with Criz, Tor2 vs. the combination. Table 1. Effects/Treatments
Criz
Tor2
Criz+Tor2
cell count Lac PC
↓74±12* ↓50±21* 86±27
↓61±13* ↓58±17* 99±24
↓↓53±6* ↓↓33±5* ↓↓↓66±7*
Data are expressed as %T/C and presented as mean±SD, n 3. Two-tailed unpaired t test (*P < 0.05). We used immunoblotting to explore potential mechanisms underlying the detected metabolic changes. A decrease in the protein levels of the facilitative glucose transporter GLUT1 and the glycolytic enzymes HK2 and LDHA was detected following single and the combination treatments, suggesting mechanisms for the depletion of lactate (Lac). Furthermore, single and combination treatments resulted in a decrease in the protein levels of CHKA, the enzyme responsible for choline phosphorylation into PC. However, the decrease in CHKA was only low enough to reduce the level of PC when both compounds were combined. The changes in the assessed metabolic enzymes followed similar patterns to those of molecular markers downstream of ALK and mTOR pathways, including the decrease in pAKT (Ser473) and pRPS6 (Ser240/244). This suggests that the metabolic changes are a consequence of inhibition of ALK and mTOR signalling pathways. Conclusions: Our results show that by using NMR we can detect metabolic changes following ALK and mTOR inhibition, including the decrease in Lac following single agents and the combination, and a decrease in PC which was unique to the combination treatment. This is the first study reporting that Lac and choline metabolites could provide potential noninvasive biomarkers for monitoring response to combination of ALK and mTOR inhibitors during early clinical trials in children with NB. No conflict of interest. 56 Poster (Board P027) Mutational analysis of primary tumors and single circulating tumor cells captured by a novel dielectrophoretic microwell array system in metastatic breast cancer, oral cancer and lung cancer patients T. Sawada1 , A. Morimoto2 , Y. Akiyama2 , T. Suzuki3 , Y. Hosomi4 , Y. Okuma4 , H. Horio5 , M. Harada5 , S. Oyama6 , C. Ogawa6 , T. Yamashita7 , Y. Maeda1 , T. Shimoyama1 , T. Hishima8 , F. Koizumi3 . 1 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Medical Oncology, Tokyo, Japan; 2 TOSOH Corporation, Research and Development, Tokyo, Japan; 3 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Clinical Research Support, Tokyo, Japan; 4 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Thoracic Oncology and Internal Medicine, Tokyo, Japan; 5 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Thoracic Surgery, Tokyo, Japan; 6 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Oral Surgery, Tokyo, Japan; 7 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Breast Surgery, Tokyo, Japan; 8 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Pathology, Tokyo, Japan Background: Analysis of circulating tumor cells (CTCs) in a single-cell level potentially allows us to understand cancer biology in metastasis, however it remains technically challenging. We have developed a capture system for detection and molecular characterization of single CTCs based on a high-density dielectrophoretic microwell array technology. Material and Methods: CTC enrichment was performed by density gradient centrifugation with RosetteSep® . After CTC enrichment, samples were loaded into the cell entrapment chamber, followed by application of 20 Vp−p AC voltage (1 MHz for 3 minutes), so as to entrap cells in the microwells using the dielectrophoretic force. Then samples were fixed and labeled with DAPI, FITC-anti-cytokeratin (CK), PE-anti-CD45 antibodies, and detected by image-based analysis using a fluorescence microscope. The results of the CTC enumeration were compared with those by CellSearch® system. In this study, the DAPI+, CK slightly+, CD45− cells with tumor cell morphology were picked up by micropipette, and isolated
S26
Poster abstracts
Poster Session – Molecular targeted agents I, Wednesday 29 November 2016
single cells were subjected to whole genome amplification followed by sequencing in 50 cancer-related genes on the Ion Torrent PGM platform. CTCs were isolated from 9 metastatic lung cancer, 4 metastatic oral cancer and 4 metastatic breast cancer patients. Among them, primary tumor tissue from 8 metastatic lung cancer, 3 metastatic oral cancer and 1 metastatic breast cancer patients were obtained. Comparison mutational analysis between primary tumor tissue and CTCs were performed. Results: The number of CTCs detected by our system ranged from 1 to 70 CTCs (median: 11 / 3 mL), and 100% (21/21) of the samples were above the threshold level (1 / 3 mL). On the other hand, with CellSearch® system, 17.6% of the samples had a 2 / 7.5 mL threshold level (median: 0 / 7.5 mL). Single CTC sequencing revealed that CTCs have various cancer related gene mutations. From the results of comparison mutational analysis between primary tumor tissue and CTCs, the same mutations in three particular genes were detected among CTCs from multiple patients regardless of cancer types. Discussion: We established a platform enabling us to capture and characterize CTCs in single cell level. The results of the present study show that single cells isolated by our system are useful for further genomic analysis. In addition, the result of comparison mutational analysis between primary tumor tissue and CTCs suggested that specific mutations might allow tumor cells to become CTCs. Further investigation is essential to confirm these facts. Conflict of interest: Other Substantive Relationships: Mr. Morimoto and Mr. Akiyama are salaried employees of TOSOH Corporation. 57 Poster (Board P028) TPC-107, a next generation, HER2 selective covalent inhibitor demonstrates potent inhibition for activated mutations of HER2 while sparing EGFR inhibition K. Oguchi1 , H. Irie1 , K. Ito1 , Y. Fujioka1 , Y. Yamada1 , T. Shimamura1 , Y. Kawai1 , Y. Shibata1 , H. Araki1 , T. Haruma1 , A. Fujioka1 , T. Sagara1 , K. Matsuo1 , T. Utsugi1 , Y. Iwasawa1 . 1 Taiho Pharmaceutical Co., Ltd., Discovery And Preclinical Research Division Background: Amplified HER2 is a well-established therapeutic target in breast and gastric cancer. In addition, non-amplified somatic mutations in HER2, an alternate HER2 activation mechanism, have been found in various cancers. HER2-directed antibodies appear to be ineffective in HER2 non-amplified mutation cancers, while small molecule HER2 inhibitors might be expected to be active for the indications. Although there are several pan-ERBB covalent inhibitors in development, dose-limiting diarrhea related to EGFR inhibition is a common adverse event. Here we report a novel, HER2 selective, covalent inhibitor, TPC-107, which displays high potency for HER2 active mutations while sparing wild type EGFR (wtEGFR). In this study, we evaluate potency of TPC-107 against cancer cell lines harboring HER2 active mutations both in vitro and in vivo. Materials and Methods: To evaluate the target engagement of TPC-107, the cellular phosphorylation of HER2 (Y1196) was examined by Western blot analysis. NIH/3T3 cell lines harboring wtHER2 or mutations were established using Piggyback transposon vector system. Anchorage-independent growth of NIH/3T3 cells was evaluated in the three-dimensional culture using FCeM® medium. For evaluation of pharmacodynamics (PD), and antitumor effect of TPC-107 in vivo, we dosed TPC-107 orally in mouse models. Results: In HEK293 cells, transient expression of HER2 mutations (S310F, V777L, and V842I) strongly increased the phosphorylation of HER2, HER3 and downstream signaling to a greater extent than over-expressed wtHER2. TPC-107 inhibited proliferation of HER2 mutations as well as over-expressed wtHER2 at 100 nM while sparing activity against wtEGFR. In NIH/3T3 cell lines, constitutive expression of HER2 mutations (S310F, V777L, and V842I) conferred anchorage-independent growth in vitro, and tumorigenicity in vivo with an accompanying significantly increased phosphorylation level of HER2. TPC-107 inhibited cell growth and colony formation of those NIH/3T3 cell lines at 1 mM. Also, TPC-107 exhibited a robust inhibition of pHER2 in NIH/3T3 tumor expressing HER2 S310F mutation in a mouse model. Consistent with the results in cell-based assays, TPC-107 displayed significant, dose-dependent antitumor effect in the mouse model at PD effective dose levels of 30 and 60 mg/kg/day without significant body weight changes. Conclusion: TPC-107 is highly selective covalent HER2 inhibitor that can potently inhibit HER2 active mutations while sparing wtEGFR. TPC-107 demonstrated substantial antitumor activity in HER2 mutated tumors in vivo, with an accompanying robust inhibition of pHER2 in the tumors. These data suggest TPC-107 may provide a promising therapeutic option for HER2 mutated tumors with a large therapeutic window. No conflict of interest.
58 Poster (Board P029) TPC-107, a next generation, HER2 selective covalent inhibitor demonstrates potent and sustained inhibition against the HER2– HER3 signaling while sparing EGFR activity, leading to a large therapeutic window H. Irie1 , K. Ito1 , Y. Kataoka1 , Y. Fujioka1 , K. Oguchi1 , T. Shimamura1 , Y. Kawai1 , T. Sagara1 , Y. Shibata1 , H. Araki1 , T. Haruma1 , A. Hashimoto1 , K. Matsuo1 , T. Utsugi1 , Y. Iwasawa1 . 1 Taiho Pharmaceutical Co., Ltd., Discovery And Preclinical Research Division Background: HER2 (human epidermal growth receptor 2) is a member of ERBB family kinases. The amplification of HER2 is a well-established therapeutic target in breast and gastric cancer. Although there are several reports of pan-ERBB covalent inhibitors in development, a selective covalent HER2 inhibitor is yet to be reported. Diarrhea is one of the most common adverse events for EGFR or pan-ERBB TKIs and is most often the dose limiting toxicity for these inhibitors. However, due to the high structural similarity between EGFR and HER2, the development of a selective HER2 inhibitor remains very difficult. Here we report on a highly selective, covalent HER2 inhibitor that blocks HER2 and the downstream substrate HER3, with reduced activity against wild type EGFR. Materials and Methods: Selectivity for kinases by TPC-107 was determined in a panel of 386 kinases. For growth inhibition assays, various cell lines were treated with TPC-107 for 3 days, and the number of living cells was determined. To evaluate the effect of TPC-107 and lapatinib (a reversible HER2/EGFR dual inhibitor) on HER2-HER3 signaling, the phosphorylation level of HER2, HER3 and the downstream pathway molecules in HER2 positive breast cancer SKBR3 cells were examined after 3 or 48 hours treatment by Western blot analysis. For evaluation of efficacy of TPC-107 and lapatinib in vivo, we dosed the compounds orally in a N87 (HER2 positive gastric cancer cell line) peritoneal dissemination xenograft model. Results: TPC-107 was a potent inhibitor of HER2 with an IC50 value of 13 nM, and demonstrated little activity against the majority of other kinases including EGFR. TPC-107 exhibited antiproliferative activity only against HER2 activated cells across various cell lines. In SKBR3 cells, although lapatinib initially inhibited pHER2, pHER3, and the downstream pathway at 100 nM after 3 hours incubation, reactivation of those molecules was observed after 48 hours of continuous exposure. In contrast, TPC-107 exhibited a sustained and robust inhibition of pHER2, pHER3, and the downstream signaling pathway at 100 nM during 48 hours treatment. Consistent with these results, TPC-107 showed superior efficacy compared to lapatinib in N87 peritoneal dissemination xenograft model without any evident toxicities including diarrhea. Conclusion: TPC-107 is a highly potent, selective inhibitor of HER2. TPC-107 demonstrated sustained and robust inhibition of HER2-HER3 in vitro and a survival benefit in a HER2-positive xenograft model without diarrhea. These data suggest that TPC-107 may be a promising therapeutic option for HER2 amplified cancer and would be expected to have an improved therapeutic window compared to current HER2 inhibitors. No conflict of interest. 59 Poster (Board P030) Combination treatment with novel GLUT1 inhibitors and sorafenib in hepatocellular carcinoma S.H.H. Low1 , G.H. Tan2 , M.J. Han1 , B. Tang1 , S.C. Chang1 , A. Wise3 , R. Soong1 , B. Bhattacharya1 . 1 National University Of Singapore, Cancer Science Institute, Singapore, Singapore; 2 National University Health System, National Cancer Science Institute Of Singapore, Singapore, Singapore; 3 Iomet Pharma, Merck And Company, Edinburgh, United Kingdom Background: Hepatocellular carcinomas (HCC) are characterised by having increased glycolysis and reduced oxidative phosphorylation than normal hepatocytes, supporting that a therapeutic index in HCC could be obtained through inhibition of glucose transport. This study aimed to characterise the effects of combining novel GLUT1 inhibitors with sorafenib, the standard of care drug in HCC. Materials and Methods: The IC50 values and combination indices of combination treatment of 6 HCC cell lines with sorafenib and four GLUT1 inhibitors (IOM1−4) were assessed by MTS assay and the method of Chou and Talalay, where CI = 1 denotes additivity, >1 antagonism and <1 synergy. Phenotypic analysis was carried out using standard techniques. Results: Combination of GLUT1 inhibitors and sorafenib led to at least additivity or synergy (Table 1). At least 2-fold reduction in the IC50s of both GLUT1 inhibitors and sorafenib was noted when combined together. However, the combination of IOM4 and sorafenib was selected for further analysis as a result of desirable pharmacokinetic properties of IOM4 for future in vivo evaluation. Glucose consumption was decreased at 3 h
Poster abstracts
S25
combination results in metabolic changes that are detectable by NMR providing noninvasive biomarkers of pathway inhibition and potentially of tumour response to combination therapy during early stage clinical trials. Material and Methods: The human NB Kelly cells (ALKF1174L mutant) were treated for 24 h with criz, Tor2 (both at 2xGI50 ), or the combination. Treatment effects were studied at molecular and cellular levels. Metabolic changes were evaluated by 1 H NMR. Results: Table 1 summarizes the cellular and metabolic effects of treatment with Criz, Tor2 vs. the combination. Table 1. Effects/Treatments
Criz
Tor2
Criz+Tor2
cell count Lac PC
↓74±12* ↓50±21* 86±27
↓61±13* ↓58±17* 99±24
↓↓53±6* ↓↓33±5* ↓↓↓66±7*
Data are expressed as %T/C and presented as mean±SD, n 3. Two-tailed unpaired t test (*P < 0.05). We used immunoblotting to explore potential mechanisms underlying the detected metabolic changes. A decrease in the protein levels of the facilitative glucose transporter GLUT1 and the glycolytic enzymes HK2 and LDHA was detected following single and the combination treatments, suggesting mechanisms for the depletion of lactate (Lac). Furthermore, single and combination treatments resulted in a decrease in the protein levels of CHKA, the enzyme responsible for choline phosphorylation into PC. However, the decrease in CHKA was only low enough to reduce the level of PC when both compounds were combined. The changes in the assessed metabolic enzymes followed similar patterns to those of molecular markers downstream of ALK and mTOR pathways, including the decrease in pAKT (Ser473) and pRPS6 (Ser240/244). This suggests that the metabolic changes are a consequence of inhibition of ALK and mTOR signalling pathways. Conclusions: Our results show that by using NMR we can detect metabolic changes following ALK and mTOR inhibition, including the decrease in Lac following single agents and the combination, and a decrease in PC which was unique to the combination treatment. This is the first study reporting that Lac and choline metabolites could provide potential noninvasive biomarkers for monitoring response to combination of ALK and mTOR inhibitors during early clinical trials in children with NB. No conflict of interest. 56 Poster (Board P027) Mutational analysis of primary tumors and single circulating tumor cells captured by a novel dielectrophoretic microwell array system in metastatic breast cancer, oral cancer and lung cancer patients T. Sawada1 , A. Morimoto2 , Y. Akiyama2 , T. Suzuki3 , Y. Hosomi4 , Y. Okuma4 , H. Horio5 , M. Harada5 , S. Oyama6 , C. Ogawa6 , T. Yamashita7 , Y. Maeda1 , T. Shimoyama1 , T. Hishima8 , F. Koizumi3 . 1 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Medical Oncology, Tokyo, Japan; 2 TOSOH Corporation, Research and Development, Tokyo, Japan; 3 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Clinical Research Support, Tokyo, Japan; 4 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Thoracic Oncology and Internal Medicine, Tokyo, Japan; 5 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Thoracic Surgery, Tokyo, Japan; 6 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Oral Surgery, Tokyo, Japan; 7 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Breast Surgery, Tokyo, Japan; 8 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Pathology, Tokyo, Japan Background: Analysis of circulating tumor cells (CTCs) in a single-cell level potentially allows us to understand cancer biology in metastasis, however it remains technically challenging. We have developed a capture system for detection and molecular characterization of single CTCs based on a high-density dielectrophoretic microwell array technology. Material and Methods: CTC enrichment was performed by density gradient centrifugation with RosetteSep® . After CTC enrichment, samples were loaded into the cell entrapment chamber, followed by application of 20 Vp−p AC voltage (1 MHz for 3 minutes), so as to entrap cells in the microwells using the dielectrophoretic force. Then samples were fixed and labeled with DAPI, FITC-anti-cytokeratin (CK), PE-anti-CD45 antibodies, and detected by image-based analysis using a fluorescence microscope. The results of the CTC enumeration were compared with those by CellSearch® system. In this study, the DAPI+, CK slightly+, CD45− cells with tumor cell morphology were picked up by micropipette, and isolated
S26
Poster abstracts
Poster Session – Molecular targeted agents I, Wednesday 29 November 2016
single cells were subjected to whole genome amplification followed by sequencing in 50 cancer-related genes on the Ion Torrent PGM platform. CTCs were isolated from 9 metastatic lung cancer, 4 metastatic oral cancer and 4 metastatic breast cancer patients. Among them, primary tumor tissue from 8 metastatic lung cancer, 3 metastatic oral cancer and 1 metastatic breast cancer patients were obtained. Comparison mutational analysis between primary tumor tissue and CTCs were performed. Results: The number of CTCs detected by our system ranged from 1 to 70 CTCs (median: 11 / 3 mL), and 100% (21/21) of the samples were above the threshold level (1 / 3 mL). On the other hand, with CellSearch® system, 17.6% of the samples had a 2 / 7.5 mL threshold level (median: 0 / 7.5 mL). Single CTC sequencing revealed that CTCs have various cancer related gene mutations. From the results of comparison mutational analysis between primary tumor tissue and CTCs, the same mutations in three particular genes were detected among CTCs from multiple patients regardless of cancer types. Discussion: We established a platform enabling us to capture and characterize CTCs in single cell level. The results of the present study show that single cells isolated by our system are useful for further genomic analysis. In addition, the result of comparison mutational analysis between primary tumor tissue and CTCs suggested that specific mutations might allow tumor cells to become CTCs. Further investigation is essential to confirm these facts. Conflict of interest: Other Substantive Relationships: Mr. Morimoto and Mr. Akiyama are salaried employees of TOSOH Corporation. 57 Poster (Board P028) TPC-107, a next generation, HER2 selective covalent inhibitor demonstrates potent inhibition for activated mutations of HER2 while sparing EGFR inhibition K. Oguchi1 , H. Irie1 , K. Ito1 , Y. Fujioka1 , Y. Yamada1 , T. Shimamura1 , Y. Kawai1 , Y. Shibata1 , H. Araki1 , T. Haruma1 , A. Fujioka1 , T. Sagara1 , K. Matsuo1 , T. Utsugi1 , Y. Iwasawa1 . 1 Taiho Pharmaceutical Co., Ltd., Discovery And Preclinical Research Division Background: Amplified HER2 is a well-established therapeutic target in breast and gastric cancer. In addition, non-amplified somatic mutations in HER2, an alternate HER2 activation mechanism, have been found in various cancers. HER2-directed antibodies appear to be ineffective in HER2 non-amplified mutation cancers, while small molecule HER2 inhibitors might be expected to be active for the indications. Although there are several pan-ERBB covalent inhibitors in development, dose-limiting diarrhea related to EGFR inhibition is a common adverse event. Here we report a novel, HER2 selective, covalent inhibitor, TPC-107, which displays high potency for HER2 active mutations while sparing wild type EGFR (wtEGFR). In this study, we evaluate potency of TPC-107 against cancer cell lines harboring HER2 active mutations both in vitro and in vivo. Materials and Methods: To evaluate the target engagement of TPC-107, the cellular phosphorylation of HER2 (Y1196) was examined by Western blot analysis. NIH/3T3 cell lines harboring wtHER2 or mutations were established using Piggyback transposon vector system. Anchorage-independent growth of NIH/3T3 cells was evaluated in the three-dimensional culture using FCeM® medium. For evaluation of pharmacodynamics (PD), and antitumor effect of TPC-107 in vivo, we dosed TPC-107 orally in mouse models. Results: In HEK293 cells, transient expression of HER2 mutations (S310F, V777L, and V842I) strongly increased the phosphorylation of HER2, HER3 and downstream signaling to a greater extent than over-expressed wtHER2. TPC-107 inhibited proliferation of HER2 mutations as well as over-expressed wtHER2 at 100 nM while sparing activity against wtEGFR. In NIH/3T3 cell lines, constitutive expression of HER2 mutations (S310F, V777L, and V842I) conferred anchorage-independent growth in vitro, and tumorigenicity in vivo with an accompanying significantly increased phosphorylation level of HER2. TPC-107 inhibited cell growth and colony formation of those NIH/3T3 cell lines at 1 mM. Also, TPC-107 exhibited a robust inhibition of pHER2 in NIH/3T3 tumor expressing HER2 S310F mutation in a mouse model. Consistent with the results in cell-based assays, TPC-107 displayed significant, dose-dependent antitumor effect in the mouse model at PD effective dose levels of 30 and 60 mg/kg/day without significant body weight changes. Conclusion: TPC-107 is highly selective covalent HER2 inhibitor that can potently inhibit HER2 active mutations while sparing wtEGFR. TPC-107 demonstrated substantial antitumor activity in HER2 mutated tumors in vivo, with an accompanying robust inhibition of pHER2 in the tumors. These data suggest TPC-107 may provide a promising therapeutic option for HER2 mutated tumors with a large therapeutic window. No conflict of interest.
58 Poster (Board P029) TPC-107, a next generation, HER2 selective covalent inhibitor demonstrates potent and sustained inhibition against the HER2– HER3 signaling while sparing EGFR activity, leading to a large therapeutic window H. Irie1 , K. Ito1 , Y. Kataoka1 , Y. Fujioka1 , K. Oguchi1 , T. Shimamura1 , Y. Kawai1 , T. Sagara1 , Y. Shibata1 , H. Araki1 , T. Haruma1 , A. Hashimoto1 , K. Matsuo1 , T. Utsugi1 , Y. Iwasawa1 . 1 Taiho Pharmaceutical Co., Ltd., Discovery And Preclinical Research Division Background: HER2 (human epidermal growth receptor 2) is a member of ERBB family kinases. The amplification of HER2 is a well-established therapeutic target in breast and gastric cancer. Although there are several reports of pan-ERBB covalent inhibitors in development, a selective covalent HER2 inhibitor is yet to be reported. Diarrhea is one of the most common adverse events for EGFR or pan-ERBB TKIs and is most often the dose limiting toxicity for these inhibitors. However, due to the high structural similarity between EGFR and HER2, the development of a selective HER2 inhibitor remains very difficult. Here we report on a highly selective, covalent HER2 inhibitor that blocks HER2 and the downstream substrate HER3, with reduced activity against wild type EGFR. Materials and Methods: Selectivity for kinases by TPC-107 was determined in a panel of 386 kinases. For growth inhibition assays, various cell lines were treated with TPC-107 for 3 days, and the number of living cells was determined. To evaluate the effect of TPC-107 and lapatinib (a reversible HER2/EGFR dual inhibitor) on HER2-HER3 signaling, the phosphorylation level of HER2, HER3 and the downstream pathway molecules in HER2 positive breast cancer SKBR3 cells were examined after 3 or 48 hours treatment by Western blot analysis. For evaluation of efficacy of TPC-107 and lapatinib in vivo, we dosed the compounds orally in a N87 (HER2 positive gastric cancer cell line) peritoneal dissemination xenograft model. Results: TPC-107 was a potent inhibitor of HER2 with an IC50 value of 13 nM, and demonstrated little activity against the majority of other kinases including EGFR. TPC-107 exhibited antiproliferative activity only against HER2 activated cells across various cell lines. In SKBR3 cells, although lapatinib initially inhibited pHER2, pHER3, and the downstream pathway at 100 nM after 3 hours incubation, reactivation of those molecules was observed after 48 hours of continuous exposure. In contrast, TPC-107 exhibited a sustained and robust inhibition of pHER2, pHER3, and the downstream signaling pathway at 100 nM during 48 hours treatment. Consistent with these results, TPC-107 showed superior efficacy compared to lapatinib in N87 peritoneal dissemination xenograft model without any evident toxicities including diarrhea. Conclusion: TPC-107 is a highly potent, selective inhibitor of HER2. TPC-107 demonstrated sustained and robust inhibition of HER2-HER3 in vitro and a survival benefit in a HER2-positive xenograft model without diarrhea. These data suggest that TPC-107 may be a promising therapeutic option for HER2 amplified cancer and would be expected to have an improved therapeutic window compared to current HER2 inhibitors. No conflict of interest. 59 Poster (Board P030) Combination treatment with novel GLUT1 inhibitors and sorafenib in hepatocellular carcinoma S.H.H. Low1 , G.H. Tan2 , M.J. Han1 , B. Tang1 , S.C. Chang1 , A. Wise3 , R. Soong1 , B. Bhattacharya1 . 1 National University Of Singapore, Cancer Science Institute, Singapore, Singapore; 2 National University Health System, National Cancer Science Institute Of Singapore, Singapore, Singapore; 3 Iomet Pharma, Merck And Company, Edinburgh, United Kingdom Background: Hepatocellular carcinomas (HCC) are characterised by having increased glycolysis and reduced oxidative phosphorylation than normal hepatocytes, supporting that a therapeutic index in HCC could be obtained through inhibition of glucose transport. This study aimed to characterise the effects of combining novel GLUT1 inhibitors with sorafenib, the standard of care drug in HCC. Materials and Methods: The IC50 values and combination indices of combination treatment of 6 HCC cell lines with sorafenib and four GLUT1 inhibitors (IOM1−4) were assessed by MTS assay and the method of Chou and Talalay, where CI = 1 denotes additivity, >1 antagonism and <1 synergy. Phenotypic analysis was carried out using standard techniques. Results: Combination of GLUT1 inhibitors and sorafenib led to at least additivity or synergy (Table 1). At least 2-fold reduction in the IC50s of both GLUT1 inhibitors and sorafenib was noted when combined together. However, the combination of IOM4 and sorafenib was selected for further analysis as a result of desirable pharmacokinetic properties of IOM4 for future in vivo evaluation. Glucose consumption was decreased at 3 h