Gert Jan Meersma

Drug-induced caspase 8 upregulation sensitises cisplatin-resistant ovarian carcinoma cells to rhTRAIL-induced apoptosis

Background:Drug resistance is a major problem in ovarian cancer. Triggering apoptosis using death ligands such as tumour necrosis factor-related apoptosis inducing ligand (TRAIL) might overcome chemoresistance.Methods:We investigated whether acquired cisplatin resistance affects sensitivity to recombinant human (rh) TRAIL alone or in combination with cisplatin in an ovarian cancer cell line model consisting of A2780 and its cisplatin-resistant subline CP70.Results:Combining cisplatin and rhTRAIL strongly enhanced apoptosis in both cell lines. CP70 expressed less caspase 8 protein, whereas mRNA levels were similar compared with A2780. Pre-exposure of particularly CP70 to cisplatin resulted in strongly elevated caspase 8 protein and mRNA levels. Caspase 8 mRNA turnover and protein stability in the presence or absence of cisplatin did not differ between both cell lines. Cisplatin-induced caspase 8 protein levels were essential for the rhTRAIL-sensitising effect as demonstrated using caspase 8 small-interfering RNA (siRNA) and caspase-8 overexpressing constructs. Cellular FLICE-inhibitory protein (c-FLIP) and p53 siRNA experiments showed that neither an altered caspase 8/c-FLIP ratio nor a p53-dependent increase in DR5 membrane expression following cisplatin were involved in rhTRAIL sensitisation.Conclusion:Cisplatin enhances rhTRAIL-induced apoptosis in cisplatin-resistant ovarian cancer cells, and induction of caspase 8 protein expression is the key factor of rhTRAIL sensitisation.

Human papilloma virus 16 E6 RNA interference enhances cisplatin and death receptor-mediated apoptosis in human cervical carcinoma cells.

In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small interfering RNA (siRNA) restores p53 functionality and sensitizes the HPV16-positive cervical cancer cell line SiHa to apoptosis by cisplatin, irradiation, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), or agonistic anti-Fas antibody. E6 siRNA resulted in decreased E6 mRNA levels and enhanced p53 and p21 expression, demonstrating the restoration of p53 functionality in SiHa cells, without inducing high levels of apoptosis (<10%). Cell surface expression of the proapoptotic death receptors (DRs) DR4, DR5, and Fas was not affected by E6 suppression. E6 suppression conferred susceptibility to cisplatin-induced apoptosis but not to irradiation-, rhTRAIL-, or anti-Fas antibody-induced apoptosis. Combining cisplatin with rhTRAIL or anti-Fas antibody induced even higher apoptosis levels in E6-suppressed cells. At the molecular level, cisplatin treatment resulted in elevated p53 levels, enhanced caspase-3 activation, and reduced p21 levels in E6-suppressed cells. Cisplatin in combination with death receptor ligands enhanced caspase-8 and caspase-3 activation and reduced X-linked inhibitor-of-apoptosis protein (XIAP) levels in these cells. We showed using siRNA that the enhanced apoptosis in E6-supressed cells was related to reduced XIAP levels and not due to reduced p21 levels. In conclusion, targeting E6 or XIAP in combination with cisplatin can efficiently potentiate rhTRAIL-induced apoptosis in HPV-positive cervical cancer cells.

Biobanking of patient and patient-derived xenograft ovarian tumour tissue: efficient preservation with low and high fetal calf serum based methods.

Using patient-derived xenografts (PDXs) for preclinical cancer research demands proper storage of tumour material to facilitate logistics and to reduce the number of animals needed. We successfully established 45 subcutaneous ovarian cancer PDXs, reflecting all histological subtypes, with an overall take rate of 68%. Corresponding cells from mouse replaced human tumour stromal and endothelial cells in second generation PDXs as demonstrated with mouse-specific vimentin and CD31 immunohistochemical staining. For biobanking purposes two cryopreservation methods, a fetal calf serum (FCS)-based (95%v/v) “FCS/DMSO” protocol and a low serum-based (10%v/v) “vitrification” protocol were tested. After primary cryopreservation, tumour take rates were 38% and 67% using either the vitrification or FCS/DMSO-based cryopreservation protocol, respectively. Cryopreserved tumour tissue of established PDXs achieved take rates of 67% and 94%, respectively compared to 91% using fresh PDX tumour tissue. Genotyping analysis showed that no changes in copy number alterations were introduced by any of the biobanking methods. Our results indicate that both protocols can be used for biobanking of ovarian tumour and PDX tissues. However, FCS/DMSO-based cryopreservation is more successful. Moreover, primary engraftment of fresh patient-derived tumours in mice followed by freezing tissue of successfully established PDXs is the preferred way of efficient ovarian cancer PDX biobanking.

Abundant Fas expression by gastrointestinal stromal tumours may serve as a therapeutic target for MegaFasL

Although the tyrosine kinase inhibitor imatinib has been shown to be an active agent in patients with gastrointestinal stromal tumours (GIST), complete remissions are almost never seen and most patients finally experience disease progression during their course of treatment. An alternative therapeutic option is to target death receptors such as Fas. We showed that a panel of imatinib-sensitive (GIST882) and imatinib-resistant (GIST48, GIST430 and GIST430K-) cell lines expressed Fas. MegaFasL, a recently developed hexameric form of soluble Fas ligand (FasL), appeared to be an active apoptosis-inducing agent in these cell lines. Moreover, MegaFasL potentiated the apoptotic effects of imatinib. Immunohistochemical evaluations, in 45 primary GISTs, underscored the relevance of the Fas pathway: Fas was expressed in all GISTs and was expressed strongly in 93%, whereas FasL was expressed at moderate and strong levels in 35 and 53% of GISTs, respectively. Fas and FasL expression were positively correlated in these primary GISTs, but there was no association between Fas or FasL expression and primary site, histological subtype, tumour size, mitotic index, risk classification, and KIT mutation status. The abundant immunohistochemical Fas and FasL expression were corroborated by western blot analysis. In conclusion, our data implicate Fas as a potential therapeutic target in GIST.

ARID1A mutation sensitizes most ovarian clear cell carcinomas to BET inhibitors

Current treatment for advanced stage ovarian clear cell cancer is severely hampered by a lack of effective systemic therapy options, leading to a poor outlook for these patients. Sequencing studies revealed that ARID1A is mutated in over 50% of ovarian clear cell carcinomas. To search for a rational approach to target ovarian clear cell cancers with ARID1A mutations, we performed kinome-centered lethality screens in a large panel of ovarian clear cell carcinoma cell lines. Using the largest OCCC cell line panel established to date, we show here that BRD2 inhibition is predominantly lethal in ARID1A mutated ovarian clear cell cancer cells. Importantly, small molecule inhibitors of the BET (bromodomain and extra terminal domain) family of proteins, to which BRD2 belongs, specifically inhibit proliferation of ARID1A mutated cell lines, both in vitro and in ovarian clear cell cancer xenografts and patient-derived xenograft models. BET inhibitors cause a reduction in the expression of multiple SWI/SNF members including ARID1B, providing a potential explanation for the observed lethal interaction with ARID1A loss. Our data indicate that BET inhibition may represent a novel treatment strategy for a subset of ARID1A mutated ovarian clear cell carcinomas.

Integrative kinome profiling identifies mTORC1/2 inhibition as treatment strategy in ovarian clear cell carcinoma

Purpose: Advanced-stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA. In this study, we aimed to identify currently unknown mutated kinases in patients with OCCC and test druggability of downstream affected pathways in OCCC models. Experimental Design: In a large set of patients with OCCC (n = 124), the human kinome (518 kinases) and additional cancer-related genes were sequenced, and copy-number alterations were determined. Genetically characterized OCCC cell lines (n = 17) and OCCC patient–derived xenografts (n = 3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib, and the mTORC1/2 inhibitor AZD8055. Results: We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy-number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway, or the ERBB family of receptor tyrosine kinases, and 82% in the DNA repair pathway. Strong p-S6 staining in patients with OCCC suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not toward drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient–derived xenograft models. Conclusions: These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Clin Cancer Res; 24(16); 3928–40. ©2018 AACR.

Abstract A37: Dual wavelength near-infrared fluorescence imaging of VEGF-A and IGF-1R in ovarian cancer patient-derived xenografts

Background: Platinum-based chemotherapy in combination with surgery is the cornerstone of treatment for advanced high-grade serous ovarian cancer. Recurrence is common and additional treatments have been developed such as angiogenesis inhibition. Small subgroups of ovarian cancer patients may benefit from upfront selection based on tumor target expression. Here, we focus on near-infrared fluorescence (NIRF) dual imaging of the tumor secreted human vascular endothelial growth factor (VEGF-A) and the membrane-bound insulin-like growth factor 1 receptor (IGF-1R), simultaneously. The aim of our study is to determine tracer uptake and kinetics in relation to target expression in ovarian cancer patient-derived xenograft (PDX) models and the effect of cisplatin treatment on these parameters. Methods: Monoclonal antibody bevacizumab (anti-human VEGF-A) and MAB391 (anti-human IGF-1R) were coupled to NIRF dyes IRDye-800CW and IRDye-680RD, respectively. Specific tumor uptake was determined in a panel of subcutaneous ovarian cancer PDX models (established from 10 patients) in NOD SCID gamma mice during 1 week after intravenous co-injection of these tracers. In addition, two PDX models were treated weekly with cisplatin (4 mg/kg dose intraperitoneal administration) followed by NIRF dual imaging. Imaging results were compared with ELISA and immunostaining of VEGF-A and IGF1R. Results: We found large differences in average radiance for bevacizumab-800CW (range 2.53 - 23.3 x 106) and for MAB391-680RD (range 1.5 - 15.5 x 107) between PDX models at 24 hrs. In vivo kinetics of bevacizumab-800CW displayed a rapid tracer decline in PDX tumors 24 hrs after co-injection. MAB391-680RD, however, resided for over 6 days in PDX tumors depending on the IGF-1R positivity of tumors, indicating receptor-mediated tracer trapping. Moreover, IgG labeled with IRDye-800CW or IRDye-680RD showed similar kinetics as the bevacizumab-800CW. Elevated levels of both tracers were found in cisplatin-treated PDX tumors 24 hrs after co-injection. The higher levels of bevacizumab-800CW corresponded with higher intratumoral human VEGF-A levels in PDX tumors after cisplatin treatment. However, tracer decline kinetics in tumors did not change. Enhanced MAB391-680RD accumulation was related to higher IGF-1R tumor levels in the less responsive PDX model. A rapid decline of MAB391-680RD, similar to bevacizumab-800CW, was observed in the cisplatin-sensitive PDX model, which was related to reduced tumor proliferation and viability. Conclusions: These results indicate that levels and kinetics of targeted NIRF tracers can be used to dissect growth factor receptor-positive from -negative tumors. This encourages further exploration of multi-wavelength NIRF imaging to select targets for personalized medicine in small subgroups of ovarian cancer patients using PDX models. This work was supported by the Dutch Cancer Society (KWF) grants RUG 2010-4833 and RUG 2011-5231. Citation Format: Tushar Tomar, Nicolette G. Alkema, Jolanda A.L. Visser, Anton G. Terwisscha van Scheltinga, Gert Jan Meersma, Harry G. Klip, Evelien W. Duiker, Elisabeth G.E. De Vries, Ate G.J. Van der Zee, Steven De Jong. Dual wavelength near-infrared fluorescence imaging of VEGF-A and IGF-1R in ovarian cancer patient-derived xenografts. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A37.

Abstract B19: Genome-wide integrated epigenomics identifies FZD-X as novel modulator for platinum sensitivity in high-grade serous ovarian cancer

The major hindrance for successful treatment of advanced stage ovarian cancer patients stems from platinum-based chemotherapy resistance. In cancer, the epigenetic alterations like DNA methylation, play a crucial role in regulation of genes related to response towards chemotherapies. Identifying these epigenetically regulated key genes, which modulate platinum response, may improve patient selection, response to therapy, and find novel targeted strategies to overcome platinum resistance. Here we applied integrated epigenomics approach to identify novel genes associated with high grade serous ovarian cancer platinum response. We performed next generation sequencing on methylation-enriched genomic DNA of ovarian cancer patient material from responders (≥18 months progression free survival, n=8) and non-responders (≤6 months progression free survival, n=10) to platinum chemoresponse. Expression data of same patients was also integrated to identify differentially methylated and expressed genes. These genes were validated further on external patients cohort using bisulfite-pyrosequencing and other publically available datasets. Furthermore, candidate genes were functionally tested on ovarian cancer cell lines panel using several in vitro assays. Based on integrated epigenomics analysis, we identified Frizzled receptor (FzdX) as an epigenetically regulated gene, significantly unmethylated (p Conclusively, our integrative epigenomics analysis revealed FzdX as novel epigenetic modulator for platinum-sensitivity in ovarian cancer patients, which might be exploited for predictive and therapeutic approaches. This study has been granted by the Dutch Cancer Society, KWF (RUG 2010-4833). Citation Format: Tushar Tomar, Nicolette G. Alkema, Gert Jan Meersma, Tim De Meyer, Wim van Criekinge, Harry G. Klip, Ate GJ van der Zee, Steven de Jong, G. Bea A. Wisman. Genome-wide integrated epigenomics identifies FZD-X as novel modulator for platinum sensitivity in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B19.

Abstract 646: Identification of novel epigenetic biomarkers for platinum-based chemotherapy resistance in high grade serous ovarian cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Platinum-based chemotherapy has been standard of care for advanced stage ovarian cancer (OC) patients since the last 3 decades. However, majority of patients relapse within 6 months with drug-resistant disease, which entitle OC among the most lethal gynecological malignancy. Besides ‘’classic’’ clinicopathological features of the primary tumor, no biological parameters have shown clinically predictive and prognostic value for chemoresponse in OC. In the present study, we are focusing on the identification of novel DNA methylation markers in response to platinum-based chemotherapy in high grade serous OC. To accomplish this aim, we performed next generation sequencing on methylation-enriched genomic DNA, isolated from frozen OC patient material from responders (more than 18 months progression free survival) and non-responders (less than 6 months progression free survival) to platinum chemoresponse (n=10 in each group). Subsequent robust biostatistics and comparative expression data analysis revealed a list of significantly differentially methylated genes of which a number were checked by methylation specific PCR (MSP) on a large platinum sensitive and resistance OC cell line panel. Gene ontology analysis showed that these genes are frequently associated with cell-fate determination, lineage commitment, transcriptional factor binding and ions-transporter system. Selected candidate methylation markers are currently being validated by pyrosequencing. Moreover, these selected markers are in-silico validated with publically available methylation and expression databases. Next, selected candidate methylation markers will be validated by quantitative-MSP on an independent OC patient cohort. Ultimately, this study will provide a DNA methylation profile for identifying those patients that may benefit from platinum-based chemotherapy in combination with epigenetic treatment modalities. Citation Format: Tushar Tomar, Nicolette G. Alkema, Tim De Meyer, Wim van Criekinge, Harry G. Klip, Gert Jan Meersma, Ate GJ van der Zee, Steven de Jong, G. Bea A. Wisman. Identification of novel epigenetic biomarkers for platinum-based chemotherapy resistance in high grade serous ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2013-646

Abstract 161: Discovery of novel methylation-based biomarkers for epithelial ovarian cancer using oligonucleotide microarrays

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Objective: To improve current screening modalities, additional molecular markers that allow detection of ovarian cancer at an early stage are needed. The aim of the current study was to identify novel methylation-based biomarkers for ovarian cancer. Methods: Genes frequently expressed at low levels were identified by comparing global expression levels from 232 primary ovarian cancers to Universal Reference RNA. Ten genes possessing a CpG island in the promoter region that showed frequent low expression in serous cancers and at least one other histological subtype were selected. The methylation status of candidate genes was verified in 50 sporadic ovarian cancers, 11 hereditary cancers, 13 borderline cancers and 12 cystadenomas using methylation specific PCR (MSP). Results: Three candidate genes (IGFBP1, LIN28 and ZNF582) showed frequent methylation in cancers and were unmethylated in normal leukocyte DNA and human ovarian surface epithelial cells. Promoter methylation of any of the three candidate genes was observed in 88% of stage III/IV cancers and 72% of stage I/II cancers. In contrast, only 9% of hereditary cancers showed evidence of methylation for any of the three genes (p<0.001). IGFBP1 was mainly methylated in stage III/IV cancers (88% vs. 12%, p<0.001), while methylation of LIN28 and ZNF582 was more frequent in stage I/II cancers (8% vs. 44% and 8% vs. 48%, respectively; p<0.05). Conclusions: Using oligonucleotide microarray data, three novel methylation-based biomarkers for sporadic epithelial ovarian cancer were discovered. Further studies should elucidate the methylation status of these genes in larger cohorts of ovarian cancers and investigate their methylation status in serum. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 161.