ROLE OF STAT1 AND CXCL10 IN THE TUMOUR IMMUNE MICROENVIRONMENT OF HIGH-GRADE SEROUS OVARIAN CANCER

High-grade serous ovarian cancer (HGSC) is the most prevalent epithelial ovarian cancer characterized by late detection, metastasis and resistance to chemotherapy. Previous studies on the tumour immune microenvironment in HGSC identified STAT1 and CXCL10 as the most differentially expressed genes between treatment naïve chemotherapy resistant and sensitive tumours. Interferon-induced STAT1 is a transcription factor, which induces many genes including tumour suppressor genes and those involved in recruitment of immune cells to the tumour immune microenvironment (TME), including CXCL10. CXCL10 is a chemokine that recruits tumour infiltrating lymphocytes (TILs) and exhibits angiostatic function. The current study was performed to determine the effects of differential STAT1 and CXCL10 expression on HGSC disease progression and TME. STAT1 expression and intratumoural CD8+ T cells were evaluated as prognostic and predictive biomarkers via immunohistochemistry on 734 HGSC tumours accrued from the Terry Fox Research Institute-Canadian Ovarian Experimental Unified Resource. The combined effect of STAT1 expression and CD8+ TIL density was confirmed as prognostic and predictive companion biomarkers in the second independent biomarker validation study. Significant positive correlation between STAT1 expression and intratumoral CD8+ TIL density was observed. The effects of enforced CXCL10 expression on HGSC tumour growth, vasculature and immune tumour microenvironment were studied in the ID8 mouse ovarian cancer cell engraftment in immunocompetent C57BL/6 mice. Significant decrease in tumour progression in mice injected with ID8 CXCL10 overexpressing cells compared to mice injected with ID8 vector control cells was observed. Multiplexed cytokine analysis of ascites showed differential expression of IL-6, VEGF and CXCL9 between the two groups. Endothelial cell marker staining showed differences in tumour vasculature between the two groups. Immune transcriptomic profiling identified distinct expression profiles in genes associated with cytokines, chemokines, interferons, T cell function and apoptosis between the two groups. These findings provide evidence that STAT1 is an independent biomarker and in combination with CD8+ TIL density could be applied as novel immune-based biomarkers in HGSC. These results provide the basis for future studies aimed at understanding mechanisms underlying differential tumour STAT1 and CXCL10 expression and its role in pre-existing tumour immunologic diversity, thus potentially contributing to biomarker guided immune modulatory therapies.

Kinome-wide CRISPR-Cas9 screen to identify kinases involved in Taxol resistance in breast cancer

Breast cancer is the most frequently diagnosed cancer in women, accounting for over 25% of cancer diagnoses and 13% of cancer-related deaths in Canadian women. There are many types of therapies for treatment or management of breast cancer, with chemotherapy being one of the most widely used. Taxol (paclitaxel) is one of the most extensively used chemotherapeutic agents for treating cancers of the breast and numerous other sites. Taxol stabilizes microtubules during mitosis, causing the cell cycle to arrest until eventually the cell undergoes apoptosis. Although Taxol has had significant benefits in many patients, response rates range from only 25-69%, and over half of Taxol-treated patients eventually acquire resistance to the drug. Drug resistance remains one of the greatest barriers to effective cancer treatment, yet little has been discerned regarding resistance to Taxol, despite its widespread clinical use. Kinases are known to be heavily involved in cancer development and progression, and several kinases have been linked to resistance of Taxol and other chemotherapeutic agents. However, a systematic screen for kinases regulating Taxol resistance is lacking. Thus, in this study, a set of kinome-wide screens was conducted to interrogate the involvement of kinases in the Taxol response. Positive-selection and negative-selection CRISPR-Cas9 screens were conducted, whereby a pooled library of 5070 sgRNAs targeted 507 kinase-encoding genes in MCF-7 breast cancer cells that were Taxol-sensitive (WT) or Taxol-resistant (TxR) which were then treated with Taxol. Next generation sequencing (NGS) was performed on cells that survived Taxol treatment, allowing identification and quantitation of sgRNAs. STK38, Blk, FASTK and Nek3 stand out as potentially critical kinases for Taxol-induced apoptosis to occur. Furthermore, kinases CDKL1 and FRK may have a role in Taxol resistance. Further validation of these candidate kinases will provide novel pre-clinical data about potential predictive biomarkers or therapeutic targets for breast cancer patients in the future.