Abstract 3103: Cyclin E amplification predicts sensitivity of primary Uterine Serous Carcinoma (USC) cell lines to the cdk2 inhibitor CYC065

Abstract We evaluated the in vitro effectiveness of the cdk2 inhibitor CYC065 on multiple primary chemotherapy-resistant USC cell lines with or without CCNE1 amplification. CCNE1-amplified primary cell lines were significantly more sensitive than wild type USC cell lines to CYC065 in vitro (i.e., IC50: mean±STDV = 61.75±13.22 nM and 103.16± 21.9 nM for CCNE1-amplified USC-ARK-2 and USC-ARK-7 cell lines, respectively and 539.2±182.1 nM for the wild type USC-ARK-4 cell line, p = 0.0048). Consistently, low concentrations of CYC065 (i.e., 100 - 300 nM) caused a dose dependent arrest in the G1 phase of the cell cycle specifically in CCNE1-amplified primary USC cell lines. Importantly, CCNE1 knockdown in the USC-ARK-2 cell line resulted in a 9.29-fold increase in the CYC065 IC50 when compared to the MOCK-transfected USC-ARK-2 cell line (p = 0.021). Finally, when primary CCNE1-amplified USC cell lines also harboring ERBB2 amplification (50% of CCNE1-amplified USC cell lines) were incubated in vitro with the combination of CYC065 and Herceptin (a monoclonal antibody targeting the product of the ERBB2 gene, HER2/neu), an increased inhibitory effect was reported in the combination treatment when compared to Herceptin or CYC065 used as single agent (i.e.,% viable cells: mean±STDV = 71.4±0.85, 65.4±14.2, 42.2±2.1 for the Herceptin, the CYC065 and the combination treatment on USC-ARK-2, respectively; p = 0.014). Together these findings identify CYC065 as a promising drug to be considered alone or in combination in the treatment of patients harboring CCNE1-amplified USC. Citation Format: Emiliano Cocco, Stefania Bellone, Salvatore Lopez, Elena Bonazzoli, Federica Predolini, Jonathan D. Black, Alessandro D. Santin. Cyclin E amplification predicts sensitivity of primary Uterine Serous Carcinoma (USC) cell lines to the cdk2 inhibitor CYC065. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3103. doi:10.1158/1538-7445.AM2015-310

7-Hydroxymatairesinol improves body weight, fat and sugar metabolism in C57BJ/6 mice on a high-fat diet

7-Hydroxymatairesinol (7-HMR) is a plant lignan abundant in various concentrations in plant foods. The objective of this study was to test HMRLignan™, a purified form of 7-HMR, and the corresponding Picea abies extract (total extract P. abies; TEP) as dietary supplements on a background of a high-fat diet (HFD)-induced metabolic syndrome in mice and in the 3T3-L1 adipogenesis model. Mice, 3 weeks old, were fed a HFD for 60 d. Subgroups were treated with 3 mg/kg body weight 7-HMR (HMRLignan™) or 10 mg/kg body weight TEP by oral administration. 7-HMR and TEP limited the increase in body weight (-11 and -13 %) and fat mass (-11 and -18 %) in the HFD-fed mice. Epididymal adipocytes were 19 and -12 % smaller and the liver was less steatotic (-62 and -65 %). Serum lipids decreased in TEP-treated mice (-11 % cholesterol, -23 % LDL and -15 % TAG) and sugar metabolism was ameliorated by both lignan preparations, as shown by a more than 70 % decrease in insulin secretion and insulin resistance. The expression of several metabolic genes was modulated by the HFD with an effect that was reversed by lignan. In 3T3-L1 cells, the 7-HMR metabolites enterolactone (ENL) and enterodiol (END) showed a 40 % inhibition of cell differentiation accompanied by the inhibited expression of the adipogenic genes PPARγ, C/EBPα and aP2. Furthermore, END and ENL caused a 10 % reduction in TAG uptake in HEPA 1-6 hepatoma cells. In conclusion, 7-HMR and TEP reduce metabolic imbalances typical of the metabolic syndrome and obesity in male mice, whereas their metabolites inhibit adipogenesis and lipid uptake in vitro

Dual HER2/PIK3CA Targeting Overcomes Single-Agent Acquired Resistance in HER2-Amplified Uterine Serous Carcinoma Cell Lines In Vitro

HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (USC), and may represent ideal therapeutic targets against this aggressive variant of endometrial cancer. We examined the sensitivity to neratinib, taselisib and the combination of the two compounds in in vitro and in vivo experiments using PIK3CA mutated and PIK3CA-wild type HER2/neu amplified USC cell lines. Cell viability and cell cycle distribution were assessed using flow-cytometry assays. Downstream signaling was assessed by immunoblotting. Preclinical efficacy of single versus dual inhibition was evaluated in vivo using two USC-xenografts. We found both single agent neratinib and taselisib to be active but only transiently effective in controlling the in vivo growth of USC xenografts harboring HER2/neu gene amplification with or without oncogenic PIK3CA mutations. In contrast, the combination of the two inhibitors caused a stronger and long lasting growth inhibition in both USC xenografts when compared to single agent therapy. Combined targeting of HER2 and PIK3CA was associated with a significant and dose-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and a dose-dependent decline in the phosphorylation of S6. Importantly, dual inhibition therapy initiated after tumor progression in single agent-treated mice was still remarkably effective at inducing tumor regression in both large PIK3CA or pan-ErbB inhibitor-resistant USC xenografts. Dual HER2/PIK3CA blockade may represent a novel therapeutic option for USC patients harboring tumors with HER2/neu gene amplification and mutated or wild type PIK3CA resistant to chemotherapy

Dual CCNE1/PIK3CA targeting is synergistic in CCNE1-amplified/PIK3CA-mutated uterine serous carcinomas in vitro and in vivo

Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets. Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs. We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs. Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours

Supplementary Figure S3 from Dual HER2/PIK3CA Targeting Overcomes Single-Agent Acquired Resistance in HER2-Amplified Uterine Serous Carcinoma Cell Lines <i>In Vitro</i> and <i>In Vivo</i>

Cell proliferation assay of USPC-ARK-1 cells exposed for 2 weeks as described in the method section to taselisib (A, upper panel) or neratinib (B, lower panel) and treated thereafter with neratinib, taselisib and the combination of both agents at the indicated concentration for 72 hrs. Cell viability was analyzed by flow cytometry and was normalized to the mean of the control group receiving no drug, so that all data were expressed as a proportion of the control. Data are expressed as mean {plus minus} SEM from two independent experiments. As shown in the upper panel, taselisib-resistant USPC-ARK-1 cells while resistant to the PI3K inihibitor remain sensitive to single agent neratinib and highly responsive to the drug combination. In contrast, as shown in the lower panel, in vitro-neratinib-resistant cells demonstrate resistance to both single agent neratinib and taselisib but remain highly sensitive to the exposure to the drug combination.</p

Supplementary Figure S2 from Dual HER2/PIK3CA Targeting Overcomes Single-Agent Acquired Resistance in HER2-Amplified Uterine Serous Carcinoma Cell Lines <i>In Vitro</i> and <i>In Vivo</i>

Effect on pS6 in uterine serous carcinoma cells lines after exposure to neratinib, taselisib and the combination of both at the indicated concentration in a representative FISH+\PIK3CA mutated USC cell line (USPC-ARK-1) and in a representative FISH+\PIK3CA wild type USC cell line (USPC-ARK-2). A: Graph showing a statistically significant difference in the reduction of the mean fluorescence intensity (MFI) of pS6 in PIK3CA mutated/FISH+ cell line (USPC-ARK-1) after 24 hrs of treatment with taselisib, neratinib and the combination of both (*P=0.03, **P=0.03, ***P=0.0001). B: Graph showing a statistically significant difference in the reduction of the mean fluorescence intensity (MFI) of pS6 in PIK3CA wild type/FISH+ cell line (USPC-ARK-2) after 24 hrs of treatment with taselisib, neratinib and the combination of both (*P=0.01, **P=0.002).</p

Supplementary Figure S4 from Dual HER2/PIK3CA Targeting Overcomes Single-Agent Acquired Resistance in HER2-Amplified Uterine Serous Carcinoma Cell Lines <i>In Vitro</i> and <i>In Vivo</i>

Variation in body weight of the mice treated with neratinib (40 mg\kg), taselisib (10 mg\kg) and the combination of the two inhibitors. The arrows denote the time point in which we started the combination treatment in single agent resistant mice (after 31 days and after 38 days for taselisib and neratinib group respectively for USPC-ARK-1 and after 25 days and after 45 days for USPC-ARK-2)</p

Inhibition of BET Bromodomain Proteins with GS-5829 and GS-626510 in Uterine Serous Carcinoma, a Biologically Aggressive Variant of Endometrial Cancer

Uterine serous carcinoma (USC) is a rare and aggressive variant of endometrial cancer. Whole-exome sequencing (WES) studies have recently reported c-Myc gene amplification in a large number of USCs, suggesting c-Myc as a potential therapeutic target. We investigated the activity of novel BET bromodomain inhibitors (GS-5829 and GS-626510, Gilead Sciences Inc.) and JQ1 against primary USC cultures and USC xenografts. We evaluated c-Myc expression by qRT-PCR in a total of 45 USCs including fresh-frozen tumor tissues and primary USC cell lines. We also performed IHC and Western blot experiments in 8 USC tumors. USC cultures were evaluated for sensitivity to GS-5829, GS-626510, and JQ1 using proliferation, viability, and apoptosis assays. Finally, the activity of GS-5829, GS-626510, and JQ1 was studied in USC-ARK1 and USC-ARK2 mouse xenografts. Fresh-frozen USC and primary USC cell lines overexpressed c-Myc when compared with normal tissues ( = 0.0009 and 0.0083, respectively). High c-Myc expression was found in 7 of 8 of primary USC cell lines tested by qRT-PCR and 5 of 8 tested by IHC. experiments demonstrated high sensitivity of USC cell lines to the exposure to GS-5829, GS-626510, and JQ1 with BET inhibitors causing a dose-dependent decrease in the phosphorylated levels of c-Myc and a dose-dependent increase in caspase activation (apoptosis). In comparative experiments, GS-5829 and/or GS-626510 were found more effective than JQ1 at the concentrations/doses used in decreasing tumor growth in both USC-ARK1 and USC-ARK2 mouse xenograft models. GS-5829 and GS-626510 may represent novel, highly effective therapeutics agents against recurrent/chemotherapy-resistant USC-overexpressing c-Myc. Clinical studies with GS-5829 in patients with USC harboring chemotherapy-resistant disease are warranted.

Supplementary Figure S1 from Dual HER2/PIK3CA Targeting Overcomes Single-Agent Acquired Resistance in HER2-Amplified Uterine Serous Carcinoma Cell Lines <i>In Vitro</i> and <i>In Vivo</i>

Graph showing IC50 values for USPC-ARK-1 cell line after 72 hrs exposure to increasing concentration of neratinib.</p