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The goal of targeted cancer therapies is to specifically block oncogenic signalling, thus maximising efficacy, while reducing side-effects to patients. The gamma-secretase (GS) complex is an attractive therapeutic target in haematological malignancies and solid tumours with major pharmaceutical activity to identify optimal inhibitors. Within GS, nicastrin (NCSTN) offers an opportunity for therapeutic intervention using blocking monoclonal antibodies (mAbs). Here we explore the role of anti-nicastrin monoclonal antibodies, which we have developed as specific, multi-faceted inhibitors of proliferation and invasive traits of triple-negative breast cancer cells. We use 3D in vitro proliferation and invasion assays as well as an orthotopic and tail vail injection triple-negative breast cancer in vivo xenograft model systems. RNAScope assessed nicastrin in patient samples. Anti-NCSTN mAb clone-2H6 demonstrated a superior anti-tumour efficacy than clone-10C11 and the RO4929097 small molecule GS inhibitor, acting by inhibiting GS enzymatic activity and Notch signalling in vitro and in vivo. Confirming clinical relevance of nicastrin as a target, we report evidence of increased NCSTN mRNA levels by RNA in situ hybridization (RNAScope) in a large cohort of oestrogen receptor negative breast cancers, conferring independent prognostic significance for disease-free survival, in multivariate analysis. We demonstrate here that targeting NCSTN using specific mAbs may represent a novel mode of treatment for invasive triple-negative breast cancer, for which there are few targeted therapeutic options. Furthermore, we propose that measuring NCSTN in patient samples using RNAScope technology may serve as companion diagnostic for anti-NCSTN therapy in the clinic.

High grade serous ovarian cancer (HGSOC) is the leading cause of death among all gynaecological malignancies. Despite recent advances in maximal effort surgery and systematic treatment, the 5-year survival remains as low as 35-40%. The response to treatment is heterogeneous even among the HGSOC population, although most of the patients are treated without stratification. It is therefore crucial to identify novel predictive and prognostic biomarkers to guide appropriate treatment, and discover novel therapeutic options for this lethal disease. Medical images, including computed tomography (CT) scans, are often used as a diagnostic tool during HGSOC treatment. In the first part of this project, we hypothesised that prognosis- relevant information could be hidden within CT scans and may be used as novel biomarker in HGSOC. We thus extracted 657 image features from each CT scan using a customised software; we then successfully generated and validated a prognostic model, based on a weighted four-feature signature. This CT-based prognostic model can accurately predict at least 5% of patients with median overall survival of less than 2 years, as well as 67% of patients with median overall survival over 5 years. Using genetic, transcriptomic, proteomic and histological analyses, we have uncovered stroma-activating pathways as well as proliferation and DNA damage response as the underlying biology of the CT-based prognostic signature. We therefore proposed treatment options accordingly to the CT-stratified patient subgroups. These results suggest a great potential for CT-images as a non-invasive, cost-effective and real-time biomarker platform in HGSOC. OPCML is a tumour suppressor gene that belongs to the IgLON protein family and is frequently silenced through promoter hyper-methylation in HGSOC. OPCML has been demonstrated to negatively regulate a panel of receptor tyrosine kinases in HGSOC and it is currently under development as a therapeutic agent. It has been suggested that other IgLON family members could be transcriptionally induced by TGFb1 and play essential functions in cancer, however, the role of OPCML in the TGFb pathway is not clear. Therefore, in the second part of this project, we investigated the transcriptional regulation and functions of OPCML in the TGFb pathway in HGSOC. We demonstrated that OPCML could be transcriptionally induced by TGFb1 in non-malignant cells via the canonical Smad pathway, while this induction is completely lost in ovarian cancer cells. Through transcriptomic and proteomic profiling, we discovered that OPCML could contribute to DNA double strand break repair, possibly via the upregulation of ATM. In summary, we have discovered novel functions of OPCML as a feedback gene in the TGFb pathway and suggested new functions for OPCML during tumorigenesis of HGSOC. With this study, we have contributed to improve the knowledge of the biology of HGSOC, and identified possible new stratification biomarkers to achieve personalised medicine in the near future. Open Access

Epithelial Ovarian Cancer (EOC) is a complex and lethal gynecological malignancy as demonstrated by the heterogeneity in gene expression molecular subtypes (GEMS). To address the GEMS-specific lethality in ovarian cancer, we highlight the relevance of how a receptor tyrosine kinase (RTK) differs in the signaling pattern, functional consequences, and therapeutic implications in the context of GEMS. An enrichment analysis of the human kinome among the ovarian cancer GEMS shows that AXL is the top-ranking RTK for the EMT-driven, poor prognosis Mes subtype. Interestingly, AXL is also expressed, at a lower rank, in the epithelial, better prognosis Epi-A subtype. Upon ligand stimulation with Gas6, specific to Mes, there is recurrent temporal activation of the extracellular regulated kinase (ERK) signaling downstream of AXL. Reverse Phase Protein Analysis (RPPA) and proximity ligation assays further show that Gas6/AXL signaling transactivates other RTKs such as cMET, EGFR, and HER2 in Mes, exclusively. This RTK crosstalk in concert with the sustained activation of FRA1 downstream to ERK, coincides with the induction of the EMT driver Slug in Mes but not Epi-A. This suggests that the intrinsic property of GEMS influences the complexity and duration of signal propagation. This signal amplification downstream to the Gas6/AXL axis alludes to pathway addiction in Mes. Functionally, the recurrent ERK activation results in a motile and invasive phenotype synonymous to Mes and EMT. This further sensitizes Mes to a selective AXL inhibitor, R428, by attenuating the RTK-ERK activation, reducing the in vitro motility and invasion, and inhibiting the in ovo tumor growth in the chick chorion-allantoic membrane (CAM) assay at a lower GI50 dose compared to Epi-A. Furthermore, silencing AXL in Mes reverses the mesenchymal phenotype and abolishes tumor formation in SCID mice. Our results imply that the mesenchymal state is more sensitive to AXL inhibition, and the RTK crosstalk along the EMT gradient rewires the system, thereby drastically sensitizing it to RTK node inhibition. The epithelial state might retain a linear signaling axis and shows less therapeutic advantage to targeting AXL. Stratifying ovarian cancer patients based on the GEMS and EMT states followed by targeting AXL is promising in a prospective clinical setting. The second part of this project is modulation of AXL by the GPI-anchored tumor suppressor OPCML (Opioid-binding Protein/Cell-adhesion Molecule Like). OPCML is abrogated in 83% of sporadic EOCs and 44% of all human cancer by somatic methylation. OPCML is a potent tumor suppressor that severely attenuates tumorigenesis by down modulating a spectrum of RTKs, including, but not limited to FGFR1, FGFR3, HER2, HER4 and EPHA2. Here we show that OPCML interacts with and represses AXL. OPCML preferentially binds to the activated form of AXL and alters its membrane distribution, thereby preventing downstream oncogenic signaling and Gas6/AXL-mediated increase in migration and invasion. OPCML potentiated the effect of AXL inhibitor R428, increasing signaling inhibition and cell death. This completely novel mechanism of regulation of RTKs may be a general mechanism for coordinated regulation of signaling, and may contribute to pro-oncogenic cancer signaling through epigenetic inactivation of OPCML. Open Access