Whole genome siRNA screen reveals novel proliferation inhibitors in melanoma

Introduction: Metastatic melanoma, arising from pigment producing melanocytes, is the most lethal form of skin cancer. Great advances in the treatment of metastatic melanoma have been achieved in recent years, with immunotherapy being the most promising strategy. Nevertheless, many obstacles still exist such as the inability to predict response and lack of appropriate biomarkers, toxicity (including life-threatening side effects) and development of resistance. Targeted therapy has also emerged for the treatment of metastatic melanoma, mainly inhibition of the BRAF/MAPK pathway. However, even in responding patients, anti-tumorigenic effect and clinical benefit are only temporary, and the majority of tumors often relapse within a year. Thus, there is still need for novel improved and safe therapies for advanced melanoma.

Materials and methods: High-throughput genome-wide primary siRNA screen, consisting of ~8,300 genes, was run on two patient-derived melanoma cell systems. Effect on proliferation was assessed 96 hours post siRNA transfection using LDH-based proliferation assay. Successful knockdown of positive control was assessed by flow cytometry. The confirmatory screen, consisting of 46 genes, was validated on 8 melanoma cell systems (including the two cell systems screened in the primary siRNA screen) using the same design as the primary screen. Positive hits were defined based on their Z-score and percent of proliferation inhibition.

Results and discussion: The genome-wide primary screen resulted in 523 hits with a Z-score ≤-1.5 and ≥ 20% reduction in proliferation compared to control. 46 hits were further tested in the confirmatory screen. Combining hits from the primary screen and confirmatory screen revealed 9 hits with a Z-score ≤-1.5 and ≥ 30% reduction in proliferation in at least four different melanoma cell systems, out of eight. Among these 9 hits - BRAF, PLK1 and LDHA - have established roles in cancer cells proliferation including melanoma, attesting for the credibility of the screen. Remarkably, four additional hits have known roles in proliferation of non-melanoma cancerous cells while two hits are novel proliferation inhibitor proteins.

Conclusion: Our results revealed novel potential targets, paving the way to the development of new therapeutics for metastatic melanoma.