Osteosarcoma is the most common primary bone malignancy in children. MAP-based chemotherapy (Methotrexate, Doxorubicin, Cisplatin) increases 5-year overall survival from 10% to 70%. However, the prognosis of metastatic, relapsed or refractory disease is much poorer, with a survival of less than 20%. This poor survival is primarily due to intrinsic or acquired chemotherapy-resistance. Resistance to chemotherapy is well demonstrated to be a key, independent predictor of lower overall survival, and shorter time to relapse or death. However, the mechanisms by which chemotherapy-resistance occurs in osteosarcoma is poorly understood, with no reliable biomarkers to predict resistance, and no effective medical therapies to offer in these cases.
High content pooled CRISPR activation screening is a powerful, efficient and unbiased method to interrogate the whole genome to identify which genes are involved in a specific cellular function. We have utilised this technique to identify the genetic mediators of cisplatin resistance in osteosarcoma in vitro. We determined the sensitivity to cisplatin across 15 osteosarcoma cell lines, and also visualised the differences in DNA damage response using gammaH2AX immunofluorescence. A highly cisplatin-sensitive cell line, OHS, was transduced with dCas9-VP64 and validated with qPCR and flow cytometry. OHS dCas9-VP64 cells were transduced with the Calabrese P65-HSF human CRISPR activation pooled library (56762 gRNAs targeting 18,8885 genes, 3 gRNAs/gene) at an MOI of 0.3. Following puromycin selection of transduced cells for 7 days, cells were treated with 0.15ug/ml cisplatin or vehicle control, in duplicate, at a minimum representation of 1000X, until cisplatin-resistant cells had clearly emerged. Surviving cells underwent next generation sequencing to determine gRNA abundance, indicating the role of these genes in cisplatin resistance. Future work will seek to validate our findings in a larger panel of cisplatin-resistant cell lines and animal models, and identify drug targets that enhance tumour response to therapy.