Holme, Harriet Katie; (2017) Identifying Genetic Dependencies and Potential Novel Therapeutic Targets for Osteosarcoma. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

With little recent improvement in osteosarcoma (OS) outcomes, identification of therapeutic targets is critical. RNA interference (RNAi) and drug screens using OS tumour cell lines (TCL) were used to identify novel genetic dependencies and validate tractable therapeutic targets. Cell viability data from RNAi screens in 18 OS TCL was integrated with whole-exome sequencing and protein expression data. Comparison with non-osteosarcoma TCL, demonstrated OS to be more reliant on skeletal morphogenesis pathways and FGFR1/2, with increased sensitivity to FGFR1 inhibitors. OS TCL positive for FGFR1 amplification and polysomy were significantly more sensitive to FGFR1 inhibitors than unknown or non-amplified OS TCL, providing further evidence for a clinical trial in an enriched population. Correlation of RNAi results with the presence of recurrent driver gene alterations revealed that sensitivity to selective silencing of DYRK1A was associated with deficiency of RB1. This finding was validated using RNAi in the OS TCL, an additional 34 breast TCL, and a DYRK1A kinase inactive model. Harmine, a DYRK1A inhibitor, resulted in greater apoptosis in an RB1 deficient OS TCL than in a RB1 wildtype model. DYRK1A has been identified as a protein interaction partner of RB1 and is pharmacologically tractable. Further work is necessary to mechanistically understand this synthetic lethality. The model system was also used as a tool to validate the potential role of BRCAness in OS, recently identified as a potential target in genomic studies. This determined the majority of OS TCL not to be profoundly sensitive to PARP inhibition. However, LM7 (an OS TCL) created by repeated pulmonary murine passage of SAOS2, demonstrated this acquired phenotype. Absence of RAD51 foci in LM7 in contrast to SAOS2, identifies this as a suitable, mechanistically relevant, tool for studying ‘BRCAness’ in OS. Integrated screens provided a framework for pre-clinical identification and validation of tractable therapeutic targets to facilitate translation into development of clinical trials.

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