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

Abstract 4498: Afatinib, an irreversible ErbB family inhibitor, demonstrates activity against HER2 mutated cervical cancer in vitro

Abstract Uterine cervical cancer (UCC) is the second leading cause of cancer deaths among women worldwide and results in 275,000 deaths annually. Functional characterization of cancer-associated genetic alterations may lead to new therapeutic approaches using molecularly targeted therapies which have the potential to improve patient outcomes. Consistent with this view, whole exome sequencing studies in a variety of human tumors including cervical cancer have recently identified somatic mutations in genes that encode for the ErbB family receptors. Importantly, some of these mutations have been shown to be “drivers” and to correlate with tumor sensitivity to the exposure to ErbB tyrosine kinase inhibitors in vitro as well as in vivo. In this study we have evaluated whether genetic alterations in the c-erbB2 gene determine the sensitivity of UCC primary cell lines to Afatinib, an EGFR, HER2, and HER4 irreversible tyrosine kinase inhibitor. Ten primary UCC cell lines, (two harboring c-erbB2 gene mutations in the extracellular HER2/neu domain [i.e., the S280F and E375D mutations-ref genome hg18] and eight harboring wild type c-erbB2), established as long term cultures in vitro, were analyzed in our study. The effect of Afatinib on cell growth, cell-cycle distribution and signaling were assessed using flow cytometry and western blot analysis following incubation of primary UCC cell lines with scalar concentrations of Afatinib for 72 hours in vitro. We found that despite similar ErbB2 mRNA expression levels by qRT-PCR in the mutated versus the wild type c-erbB2 groups, IC50 values in response to Afatinib were significantly lower in the group of mutated cell lines than in the non-mutated control UCC (MEAN±SEM = 0.55±0.11 vs. 1.64±0.09 μM, P<0.05). Furthermore, Afatinib growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells blocked in the G1 cell cycle phase as well as a dose-dependent dephosphorylation of HER2, S6, AKT and ERK in both c-erbB2 mutated and wild type UCC cell lines. Our data suggests that Afatinib, a recently FDA approved drug, is highly effective against c-erbB2 mutated UCC in vitro and could represent a valid therapeutic option for patients harboring c-erbB2 mutated advanced/recurrent cervical cancers unresponsive to radiation and/or chemotherapy. Citation Format: Salvatore Lopez, Emiliano Cocco, Bellone Stefania, Ileana Bortolomai, Elena Bonazzoli, Roberta Nicoletti, Carlton Schwab, Diana P. English, Corrado Terranova, Roberto Angioli, Alessandro D. Santin. Afatinib, an irreversible ErbB family inhibitor, demonstrates activity against HER2 mutated cervical cancer in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4498. doi:10.1158/1538-7445.AM2014-449

T‐DM1, a novel antibody–drug conjugate, is highly effective against primary HER2 overexpressing uterine serous carcinoma in vitro and in vivo

Amplification of c-erbB2 has been reported in over 30% of uterine serous carcinoma (USC) and found to confer poor survival because of high proliferation and increased resistance to therapy. In this study, we evaluated for the first time Trastuzumab emtansine (T-DM1), a novel antibody–drug conjugate, against multiple epidermal growth factor receptor-2 (HER2)-positive USC cells in vitro followed by developing a supportive in vivo model. Fifteen primary USC cell lines were assessed by immunohistochemistry (IHC) and flow cytometry for HER2 protein expression. C-erbB2 gene amplification was evaluated using fluorescent in situ hybridization. Sensitivity to T-DM1 and trastuzumab (T)-induced antibody-dependent cell-mediated cytotoxicity was evaluated in 5-h chromium release assays. T-DM1 and T cytostatic and apoptotic activities were evaluated using flow-cytometry-based proliferation assays. In vivo activity of T-DM1 versus T in USC xenografts in SCID mice was also evaluated. High levels of HER2 protein overexpression and HER2 gene amplification were detected in 33% of USC cell lines. T-DM1 was considerably more effective than trastuzumab in inhibiting cell proliferation and in causing apoptosis (P = 0.004) of USC showing HER2 overexpression. Importantly, T-DM1 was highly active at reducing tumor formation in vivo in USC xenografts overexpressing HER2 (P = 0.04) and mice treated with TDM-1 had significantly longer survival when compared to T-treated mice and control mice (P ≤ 0.0001). T-DM1 shows promising antitumor effect in HER2-positive USC cell lines and USC xenografts and its activity is significantly higher when compared to T. T-DM1 may represent a novel treatment option for HER2-positive USC patients with disease refractory to trastuzumab and traditional chemotherapy

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

Solitomab, an Epithelial Cell Adhesion Molecule/CD3 Bispecific Antibody (BiTE), Is Highly Active Against Primary Chemotherapy-Resistant Ovarian Cancer Cell Lines In Vitro and Fresh Tumor Cells Ex Vivo

BACKGROUND: Solitomab is a novel, bispecific, single-chain antibody that targets epithelial cell adhesion molecule (EpCAM) on tumor cells and also contains a cluster of differentiation 3 (CD3) (T-cell coreceptor) binding region. The authors evaluated the in vitro activity of solitomab against primary chemotherapy-resistant epithelial ovarian carcinoma cell lines as well as malignant cells in ascites. METHODS: EpCAM expression was evaluated by flow cytometry in 5 primary ovarian cancer cell lines and in 42 fresh ovarian tumor cell cultures in ascites from patients with mainly advanced or recurrent, chemotherapy-resistant disease. The potential activity of solitomab against EpCAM-positive tumor cells was evaluated by flow cytometry, proliferation, and 4-hour chromium-release, cell-mediated cytotoxicity assays. RESULTS: EpCAM expression was detected by flow cytometry in approximately 80% of the fresh ovarian tumors and primary ovarian tumor cell lines tested. EpCAM-positive, chemotherapy-resistant cell lines were identified as resistant to natural killer cell-mediated or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean +/- standard error of the mean, 3.6% 60.7% of cells killed after incubation of EpCAM-positive cell lines with control bispecific antibody). In contrast, after incubation with solitomab, EpCAM-positive, chemotherapy-resistant cells became highly sensitive to T-cell cytotoxicity (mean +/- standard error of the mean, 28.2% 62.05% of cells killed; P<.0001) after exposure to peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with EpCAM-expressing malignant cells in ascites with solitomab resulted in a significant increase in T-cell activation markers and a reduction in the number of viable ovarian tumor cells in ascites (P<.001). CONCLUSIONS: Solitomab may represent a novel, potentially effective agent for the treatment of chemotherapy-resistant ovarian cancers that overexpress EpCAM. (C) 2014 American Cancer Society

Abstract 2461: SYD985, a novel HER2-targeting antibody-drug conjugate, shows strong antitumor activity in primary USC cell lines with low (1+) and moderate (2+) HER2/Neu expression

Abstract Introduction: Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer which carries an extremely poor prognosis. Up to 35% of USC may overexpress the HER2/neu oncogene at strong (i.e., 3+) levels and and additional 45% of USC express HER2/Neu at moderate (2+) or low (1+) levels. For tumors with moderate/low HER2/neu expression, there is no sufficient targeted therapy. SYD985 (Synthon Biopharmaceuticals BV, Nijmegen, The Netherlands) is a novel HER2-targeting antibody-drug conjugate (ADC) composed of the mAb trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin). The objective of this study was to explore the anti-tumor activity of SYD985 and to compare it to trastuzumab emtansine (T-DM1) in USC in in vitro and in vivo studies. Methods: Nine primary USC cell lines were evaluated for HER2/neu surface expression by IHC and flow cytometry and gene amplification using FISH assays. The cytotoxicity of SYD985 and T-DM1 was evaluated using cell lines with differential HER2/Neu expression (i.e., 1+, 2+, and 3+). Proliferation and viability experiments were performed using propidium iodide-based, flow cytometry assays. In vivo activity of SYD985 in mouse xenograft models is ongoing. Results: SYD985 was 55 to 115 times more potent when compared to T-DM1 in primary USC cell lines with 1+ and 2+ HER2/neu expression. Specifically, in the HER2/neu 1+ the IC50's for SYD985 and T-DM1 were 0.065 μg/mL and 3.58 μg/mL, respectively (p = 0.004). In the HER2/neu 2+ cell lines the IC50's of SYD985 and T-DM1 were 0.016 μg/mL and 1.82 μg/mL, respectively (p = 0.005). In the HER2/neu 3+ cell lines a statistical trend was noted when comparing the cytotoxicity of SYD985 with T-DM1; the IC50's were 0.011 μg/mL and 0.035 μg/mL, respectively (p = 0.06). Conclusions: SYD985 is a novel ADC endowed with remarkable activity against not only USC with strong (3+) HER2/neu overexpression but also (as previously shown for SYD985 in breast cancer) versus USC with low to moderate (i.e., 1+/2+) HER2/neu expression. SYD985 is significantly more potent than T-DM1 in comparative in vitro experiments. Currently, in vivo experiments in USC xenografts not responsive to T-DM1 are ongoing. This is a significant discovery as we do not currently have an adequate targeted therapy to HER2/neu 1+ and 2+ expressing tumors. Thus, clinical studies with SYD985 in patients with biologically aggressive endometrial cancer (e.g., USC) resistant to standard salvage chemotherapy are warranted. Citation Format: Jonathan D. Black, Salvatore Lopez, Emiliano Cocco, Stefania Bellone, Elena Bonazzoli, Carlton Schwab, Diana English, Peter Goedings, Patrick Beusker, Miranda van der Lee, Marco Timmers, Wim Dokter, Thomas Rutherfor, Peter Schwartz, Alessandro Santin. SYD985, a novel HER2-targeting antibody-drug conjugate, shows strong antitumor activity in primary USC cell lines with low (1+) and moderate (2+) HER2/Neu expression. [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 2461. doi:10.1158/1538-7445.AM2015-246