Osteosarcoma (OS) is the most common primary bone tumour affecting teenagers and young adults. Incorporation of pre-surgical chemotherapy has increased 5-year survival rates from 10% to ~70% for patients with localised disease. However, ~20% of patients present with metastasis at diagnosis and a further 25%-50% will develop metastatic disease during their treatment. Furthermore, the development of metastatic or recurrent disease presents with a 5-year survival rate of 20% often related to the generation of treatment resistance to agents such as Cisplatin (key backbone treatment of paediatric OS).
We have determined the response of 14 human osteosarcoma cell lines to cisplatin in vitro and in vivo to identify treatment-sensitive or resistant models. To determine mechanisms of cisplatin resistance we have performed whole genome CRISPR cut pooled screens in 3 treatment-resistant OS models in the presence and absence of cisplatin. Cas12a-expressing cisplatin-resistant OS cell lines were transduced with the Humagne Set C and Humagne Set D (20,355 constructs per library, each containing 2 gRNAs targeting 19,755 genes) and the Draetta (17,032 constructs, each containing 3-4 gRNAs, targeting 16,977 genes) pooled human genome-wide knockout libraries at an MOI of 0.3 and a minimum representation of 1000x. Comparison of gRNA abundance between cisplatin treatment and vehicle control revealed genes that sensitise to (decreased representation) or enhance resistance to (increased representation) cisplatin.
The results of this screen revealed that loss of genes associated with the Fanconi anaemia pathway and anti-oxidant defence, enhanced sensitivity whereas inactivation of genes associated with cell cycle regulation, division and ribosomal biogenesis enhanced resistance to cisplatin. Ongoing harmonisation of functional data with whole genome transcriptomic data from cisplatin-sensitive and resistant OS models will allow for the prioritization of functional gene dependencies and targetable pathways to be further validated using arrayed CRISPR screens and in vitro and in vivo drug studies. This data will provide insights into cisplatin resistance mechanisms in OS, reveal biomarkers of predictive cisplatin response, and identify new therapeutic strategies to improve tumour response to cisplatin.