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Preclinical validation of a novel compound targeting p70S6 kinase in breast cancer
S36
Poster abstracts
Poster Session – Molecular targeted agents I, Wednesday 29 November 2016
tumor malignancy. Therefore, ROS1 and NTRKs may be promising therapeutic targets. The MET/ALK1/ROS1 inhibitor crizotinib has shown responses in patients with ROS1 fusions, however, acquired resistance to crizotinib has been a concern and potential resistance mechanisms including ROS1 kinase mutations are implicated. DS-6051b is a novel, orally available, small molecule tyrosine kinase inhibitor of ROS1 and NTRKs, and is currently being investigated in phase I clinical trials. In this study, we examined in vitro and in vivo activities of DS-6051b against ROS1 and NTRKs, including ROS1 mutations to characterize the compound. Material and Methods: In vitro inhibitory activity against ROS1 and NTRKs was tested in biochemical and cell-based assays. Antitumor efficacy was examined in a mouse subcutaneous xenograft model using cells expressing ROS1 or NTRK fusion gene. In addition, the activity against crizotinibresistant ROS1 mutant was evaluated using Ba/F3 cells expressing ROS1 fusion with gatekeeper mutation (L2026M). Results: Potent in vitro activity of DS-6051b was shown against ROS1, NTRK1, NTRK2, and NTRK3 kinases with IC50s of 0.2 nM to 2.3 nM. When the compound was administered orally to xenografted mice, the growth of KM12 cells harboring TPM3-NTRK1 fusion and U-118MG cells harboring FIG-ROS1 fusion were significantly inhibited at 25 mg/kg and above. In Ba/F3-ROS1 allograft mice, tumor regression with inhibition of phosphoROS1 in the tumor was observed even against the tumor with a ROS1 gatekeeper mutation, in which crizotinib has no significant effect. Conclusions: These results indicated potent in vitro and in vivo activities of DS-6051b against ROS1 and NTRKs, suggesting the potential of the compound for the targeted therapy against cancers with ROS1 or NTRKs gene rearrangements. Moreover, the potential for the effectiveness against acquired ROS1 resistant tumors is also demonstrated. DS-6051b is currently being evaluated in phase I clinical trials. Conflict of interest: Other Substantive Relationships: All authors are employees at Daiichi Sankyo Co., Ltd. 88 Poster (Board P059) Influence of N-acetyltransferase 2 (NAT2) gene polymorphisms on the in vitro metabolism of the epidermal growth factor receptor inhibitor rociletinib J. Ramirez1 , L. House1 , M.J. Ratain1 . 1 University of Chicago, Medicine, Chicago, USA Background: Rociletinib (ROCI) has shown evidence of activity in T790Mpositive non-small cell lung cancer patients. The drug undergoes biotransformation by amide hydrolysis to form M502, followed by N-acetylation to M544 or amide hydrolysis to M460. Treatment of cancer patients with ROCI has been associated with hyperglycemia and corrected QT (QTc) prolongation that are caused by M502 and M460, respectively. Our aim was to elucidate the enzymes responsible for the metabolism of ROCI and metabolites, and investigate the relationship between M544 formation and N-acetyltransferase 2 (NAT2) polymorphisms. Material and Methods: Human hepatocytes from fast (NAT2*6/*12A) and slow (NAT2*5B/*5B) acetylators (one each) were treated with ROCI and metabolites (10 mM; 24 hrs). Also, experiments were performed for 1 hr with recombinant CES1b, CES1c, CES2, NAT1, NAT2, pooled human liver microsomes (HLM) and cytosols (HLC), with and without inhibitors. M502 and M460 incubations with HLC and NAT enzymes included acetyl coenzyme A. Cytosols (n = 110) were genotyped for NAT2 polymorphisms (rs1041983 and rs1801280) using TaqMan® assays and incubated with M502. Metabolite formation was measured by HPLC. Results: Hepatocytes treated with ROCI formed M502, M544 and M460 by primary and sequential metabolism. The fast acetylator had 5.1-fold higher production of M544 than the slow acetylator. M502 incubations also showed higher N-acetylation (6.4-fold) by the fast acetylator, while formation of M460 was 2.1-fold higher in the slow acetylator. M502 could also be produced by deacetylation of M544, independent of acetylator status. M460 incubations showed formation of an N-acetylated metabolite (N-acetylM460), and higher N-acetyl-M460 formation (3.4-fold) was observed in incubations with cells from the fast acetylator. Incubations with expressed enzymes showed that (1) M502 was formed from ROCI and M544 by CES2, (2) M544 and N-acetyl-M460 were formed by NAT2, and (3) M460 was not formed by CES enzymes. Additionally, (1) M502 formation by HLM was inhibited by 10 mM BNPP (CES inhibitor) and 10 mM eserine (CES2 and AADAC inhibitor); (2) M544 formation in HLC was inhibited by 100 mM quercetin (NAT inhibitor) and was associated with NAT2 genotype (p < 0.0001; additive model); (3) M460 formation in HLM was inhibited by eserine; and (4) M460 was metabolized by N-acetylation in HLC. Conclusions: Our results strongly suggest that extensive metabolism of ROCI by polymorphic NAT2 and CES2 could alter drug exposure in patients. As M502 and M460 are eliminated by NAT2, we expect slow acetylators to have higher exposure to these metabolites and consequently, to be at increased risk of experiencing hyperglycemia and QTc prolongation. No conflict of interest.
89 Poster (Board P060) Preclinical validation of a novel compound targeting p70S6 kinase in breast cancer I. Segatto1 , S. Massarut2 , R. Boyle3 , G. Baldassarre1 , D. Walker3 , B. Belletti1 . 1 CRO Aviano, National Cancer Institute, Division of Experimental Oncology 2, Aviano, Italy; 2 CRO Aviano, National Cancer Institute, Breast Surgery Unit, Aviano, Italy; 3 Sentinel Oncology, Chemistry, Cambridge, United Kingdom Background: Triple Negative Breast Cancer (TNBC) is an aggressive malignancy with dismal prognosis owing to high levels of recurrence. It has been shown that p70S6 kinase (p70S6K) is activated via post-surgical inflammation and can mediate recurrence in a mouse model of TNBC. The purpose of the current study is to validate whether a novel p70S6K1 inhibitor, FS115, can control the formation of recurrence and metastasis in pre-clinical models of TNBC. Material and Methods: FS115 was synthesized by Sentinel Oncology. IC50s were determined via radiometric kinase assays. Cell colony assay: MDA-MB-231 cells (basal, TNBC) were treated with FS115 or vehicle then seeded onto plates and incubated for 14 d prior to colony counting. The pharmacokinetic (PK) profile was determined in two separate cohorts of CD-1 mice (dosed PO or IV with FS115). Pharmacodynamics (PD) were determined as follows: nude mice bearing MDA-MB-231 tumours were dosed with FS115 (125 mg/kg BID ×3 PO) or vehicle, then 12 h after the last dose mice were sacrificed and tumour ELISA carried out to measure levels of p-S6 240/244, t-S6, p-AKT 473 and t-AKT. Local recurrence was modelled in vivo: MDA-MB-231 orthotopic primary tumors were first grown in nude mice. The mice were treated with FS115 or vehicle in a peri-operative schedule (day −1, day 0, day +1; surgery on day 0) then monitored for recurrence for 56 d. Metastasis was modelled in vivo as follows: On day −1, nude mice were assigned to treatment groups. On day 0, mice were given an intracardiac injection of MDA-MB-231-luc cells (luciferase-expressing). Mice were dosed for a further 68 days and were then imaged for total photon flux to show metastatic spread. Results: In vitro, FS115 potently inhibits p70S6K1 (IC50 0.035 uM) with high selectivity over PI3K pathway kinases (AKT2 IC50 23.8 uM). In MDAMB-231, a TNBC cell line, FS115 inhibits p70S6K activity in a dosedependent manner (optimal effect at 10 uM) and suppresses colony growth in the range 1−10 uM. The PK profile of FS115 in mice shows high oral bioavailability (>95%) and favorable brain penetration (4:1 B:P). PD studies reveal that FS115 inhibits phosphorylation of p70S6K substrate S6 in tumour by 55% without concomitant AKT activation. In mouse models of TNBC, FS115 was found to inhibit multiple facets of the disease including (a) tumor take rate and growth, (b) local recurrence and (c) metastasis, including brain metastasis. Conclusions: Here, we show that a small molecule oral inhibitor of p70S6K1, FS115, dosed to mice in a peri-operative schedule was effective in decreasing local recurrence of breast cancer and in long-term treatment schedule was well-tolerated and efficiently suppressed distant metastasis formation. Altogether, these findings suggest that an inhibitor of p70S6K1 could provide a targeted treatment option for TNBC patients at high risk of recurrence. Conflict of interest: Other Substantive Relationships: Robert Boyle and David Walker are both employees of Sentinel Oncology Ltd. 90 Poster (Board P061) Antitumor effects of a novel diaminothiazole inhibitor of GSK3/CDK9 C. Smith1 , R. Schrecengost1 , L. Maines1 , Y. Zhuang1 , S. Keller1 , R. Smith1 , C. Green1 . 1 Apogee Biotechnology Corporation, Research, Hummelstown, USA Background: Diaminothiazoles (DATs) that inhibit various cyclindependent kinases (cdks) or that inhibit microtubule assembly have been previously described as potential anticancer agents. In characterizing a new series of DATs, we focused on defining the mechanism of action and the antitumor activity of a novel compound (ABC1183). Materials and Methods: A library of DATs was synthesized by reaction of substituted phenylisothiocyanates, cyanamide and substituted phenylacylbromides, and tested for cytotoxicity against several tumor cell lines. These compounds were further assessed for their mechanism(s) of action using a variety of cellular and biochemical assays, and ABC1183 was tested in vivo for toxicity and antitumor activity in syngeneic xenograft models using PAN02 (pancreatic), B16 (melanoma) or TRAMP (prostate) tumor cells. Results: A panel of more than 70 DATs was produced, and these compounds demonstrated IC50s for cytotoxicity against PAN02 cells ranging from 0.04 to >100 mM. Several of these compounds depolymerized microtubules in cells, consistent with previous reports; however, several compounds with sub-mM potency did not have this activity. One such compound, ABC1183, was selected for detailed characterization because
Poster abstracts
Poster Session – Molecular targeted agents I, Wednesday 29 November 2016
tumor malignancy. Therefore, ROS1 and NTRKs may be promising therapeutic targets. The MET/ALK1/ROS1 inhibitor crizotinib has shown responses in patients with ROS1 fusions, however, acquired resistance to crizotinib has been a concern and potential resistance mechanisms including ROS1 kinase mutations are implicated. DS-6051b is a novel, orally available, small molecule tyrosine kinase inhibitor of ROS1 and NTRKs, and is currently being investigated in phase I clinical trials. In this study, we examined in vitro and in vivo activities of DS-6051b against ROS1 and NTRKs, including ROS1 mutations to characterize the compound. Material and Methods: In vitro inhibitory activity against ROS1 and NTRKs was tested in biochemical and cell-based assays. Antitumor efficacy was examined in a mouse subcutaneous xenograft model using cells expressing ROS1 or NTRK fusion gene. In addition, the activity against crizotinibresistant ROS1 mutant was evaluated using Ba/F3 cells expressing ROS1 fusion with gatekeeper mutation (L2026M). Results: Potent in vitro activity of DS-6051b was shown against ROS1, NTRK1, NTRK2, and NTRK3 kinases with IC50s of 0.2 nM to 2.3 nM. When the compound was administered orally to xenografted mice, the growth of KM12 cells harboring TPM3-NTRK1 fusion and U-118MG cells harboring FIG-ROS1 fusion were significantly inhibited at 25 mg/kg and above. In Ba/F3-ROS1 allograft mice, tumor regression with inhibition of phosphoROS1 in the tumor was observed even against the tumor with a ROS1 gatekeeper mutation, in which crizotinib has no significant effect. Conclusions: These results indicated potent in vitro and in vivo activities of DS-6051b against ROS1 and NTRKs, suggesting the potential of the compound for the targeted therapy against cancers with ROS1 or NTRKs gene rearrangements. Moreover, the potential for the effectiveness against acquired ROS1 resistant tumors is also demonstrated. DS-6051b is currently being evaluated in phase I clinical trials. Conflict of interest: Other Substantive Relationships: All authors are employees at Daiichi Sankyo Co., Ltd. 88 Poster (Board P059) Influence of N-acetyltransferase 2 (NAT2) gene polymorphisms on the in vitro metabolism of the epidermal growth factor receptor inhibitor rociletinib J. Ramirez1 , L. House1 , M.J. Ratain1 . 1 University of Chicago, Medicine, Chicago, USA Background: Rociletinib (ROCI) has shown evidence of activity in T790Mpositive non-small cell lung cancer patients. The drug undergoes biotransformation by amide hydrolysis to form M502, followed by N-acetylation to M544 or amide hydrolysis to M460. Treatment of cancer patients with ROCI has been associated with hyperglycemia and corrected QT (QTc) prolongation that are caused by M502 and M460, respectively. Our aim was to elucidate the enzymes responsible for the metabolism of ROCI and metabolites, and investigate the relationship between M544 formation and N-acetyltransferase 2 (NAT2) polymorphisms. Material and Methods: Human hepatocytes from fast (NAT2*6/*12A) and slow (NAT2*5B/*5B) acetylators (one each) were treated with ROCI and metabolites (10 mM; 24 hrs). Also, experiments were performed for 1 hr with recombinant CES1b, CES1c, CES2, NAT1, NAT2, pooled human liver microsomes (HLM) and cytosols (HLC), with and without inhibitors. M502 and M460 incubations with HLC and NAT enzymes included acetyl coenzyme A. Cytosols (n = 110) were genotyped for NAT2 polymorphisms (rs1041983 and rs1801280) using TaqMan® assays and incubated with M502. Metabolite formation was measured by HPLC. Results: Hepatocytes treated with ROCI formed M502, M544 and M460 by primary and sequential metabolism. The fast acetylator had 5.1-fold higher production of M544 than the slow acetylator. M502 incubations also showed higher N-acetylation (6.4-fold) by the fast acetylator, while formation of M460 was 2.1-fold higher in the slow acetylator. M502 could also be produced by deacetylation of M544, independent of acetylator status. M460 incubations showed formation of an N-acetylated metabolite (N-acetylM460), and higher N-acetyl-M460 formation (3.4-fold) was observed in incubations with cells from the fast acetylator. Incubations with expressed enzymes showed that (1) M502 was formed from ROCI and M544 by CES2, (2) M544 and N-acetyl-M460 were formed by NAT2, and (3) M460 was not formed by CES enzymes. Additionally, (1) M502 formation by HLM was inhibited by 10 mM BNPP (CES inhibitor) and 10 mM eserine (CES2 and AADAC inhibitor); (2) M544 formation in HLC was inhibited by 100 mM quercetin (NAT inhibitor) and was associated with NAT2 genotype (p < 0.0001; additive model); (3) M460 formation in HLM was inhibited by eserine; and (4) M460 was metabolized by N-acetylation in HLC. Conclusions: Our results strongly suggest that extensive metabolism of ROCI by polymorphic NAT2 and CES2 could alter drug exposure in patients. As M502 and M460 are eliminated by NAT2, we expect slow acetylators to have higher exposure to these metabolites and consequently, to be at increased risk of experiencing hyperglycemia and QTc prolongation. No conflict of interest.
89 Poster (Board P060) Preclinical validation of a novel compound targeting p70S6 kinase in breast cancer I. Segatto1 , S. Massarut2 , R. Boyle3 , G. Baldassarre1 , D. Walker3 , B. Belletti1 . 1 CRO Aviano, National Cancer Institute, Division of Experimental Oncology 2, Aviano, Italy; 2 CRO Aviano, National Cancer Institute, Breast Surgery Unit, Aviano, Italy; 3 Sentinel Oncology, Chemistry, Cambridge, United Kingdom Background: Triple Negative Breast Cancer (TNBC) is an aggressive malignancy with dismal prognosis owing to high levels of recurrence. It has been shown that p70S6 kinase (p70S6K) is activated via post-surgical inflammation and can mediate recurrence in a mouse model of TNBC. The purpose of the current study is to validate whether a novel p70S6K1 inhibitor, FS115, can control the formation of recurrence and metastasis in pre-clinical models of TNBC. Material and Methods: FS115 was synthesized by Sentinel Oncology. IC50s were determined via radiometric kinase assays. Cell colony assay: MDA-MB-231 cells (basal, TNBC) were treated with FS115 or vehicle then seeded onto plates and incubated for 14 d prior to colony counting. The pharmacokinetic (PK) profile was determined in two separate cohorts of CD-1 mice (dosed PO or IV with FS115). Pharmacodynamics (PD) were determined as follows: nude mice bearing MDA-MB-231 tumours were dosed with FS115 (125 mg/kg BID ×3 PO) or vehicle, then 12 h after the last dose mice were sacrificed and tumour ELISA carried out to measure levels of p-S6 240/244, t-S6, p-AKT 473 and t-AKT. Local recurrence was modelled in vivo: MDA-MB-231 orthotopic primary tumors were first grown in nude mice. The mice were treated with FS115 or vehicle in a peri-operative schedule (day −1, day 0, day +1; surgery on day 0) then monitored for recurrence for 56 d. Metastasis was modelled in vivo as follows: On day −1, nude mice were assigned to treatment groups. On day 0, mice were given an intracardiac injection of MDA-MB-231-luc cells (luciferase-expressing). Mice were dosed for a further 68 days and were then imaged for total photon flux to show metastatic spread. Results: In vitro, FS115 potently inhibits p70S6K1 (IC50 0.035 uM) with high selectivity over PI3K pathway kinases (AKT2 IC50 23.8 uM). In MDAMB-231, a TNBC cell line, FS115 inhibits p70S6K activity in a dosedependent manner (optimal effect at 10 uM) and suppresses colony growth in the range 1−10 uM. The PK profile of FS115 in mice shows high oral bioavailability (>95%) and favorable brain penetration (4:1 B:P). PD studies reveal that FS115 inhibits phosphorylation of p70S6K substrate S6 in tumour by 55% without concomitant AKT activation. In mouse models of TNBC, FS115 was found to inhibit multiple facets of the disease including (a) tumor take rate and growth, (b) local recurrence and (c) metastasis, including brain metastasis. Conclusions: Here, we show that a small molecule oral inhibitor of p70S6K1, FS115, dosed to mice in a peri-operative schedule was effective in decreasing local recurrence of breast cancer and in long-term treatment schedule was well-tolerated and efficiently suppressed distant metastasis formation. Altogether, these findings suggest that an inhibitor of p70S6K1 could provide a targeted treatment option for TNBC patients at high risk of recurrence. Conflict of interest: Other Substantive Relationships: Robert Boyle and David Walker are both employees of Sentinel Oncology Ltd. 90 Poster (Board P061) Antitumor effects of a novel diaminothiazole inhibitor of GSK3/CDK9 C. Smith1 , R. Schrecengost1 , L. Maines1 , Y. Zhuang1 , S. Keller1 , R. Smith1 , C. Green1 . 1 Apogee Biotechnology Corporation, Research, Hummelstown, USA Background: Diaminothiazoles (DATs) that inhibit various cyclindependent kinases (cdks) or that inhibit microtubule assembly have been previously described as potential anticancer agents. In characterizing a new series of DATs, we focused on defining the mechanism of action and the antitumor activity of a novel compound (ABC1183). Materials and Methods: A library of DATs was synthesized by reaction of substituted phenylisothiocyanates, cyanamide and substituted phenylacylbromides, and tested for cytotoxicity against several tumor cell lines. These compounds were further assessed for their mechanism(s) of action using a variety of cellular and biochemical assays, and ABC1183 was tested in vivo for toxicity and antitumor activity in syngeneic xenograft models using PAN02 (pancreatic), B16 (melanoma) or TRAMP (prostate) tumor cells. Results: A panel of more than 70 DATs was produced, and these compounds demonstrated IC50s for cytotoxicity against PAN02 cells ranging from 0.04 to >100 mM. Several of these compounds depolymerized microtubules in cells, consistent with previous reports; however, several compounds with sub-mM potency did not have this activity. One such compound, ABC1183, was selected for detailed characterization because