A genome-scale CRISPR-Cas9 loss-of-function screen in ALCL cells treated with crizotinib
We used a CRISPR-Cas9-based genome-wide guide RNA (sgRNA) library to identify genes responsible for driving drug resistance in ALK+ ALCL cells under crizotinib treatment.We screened 4 diffrerent ALK+ ALCL cell lines, TS, SU-DHL1, COST and KARPAS299. Every cell line was transduced with GeCKO A and GeCKO B sgRNA libraries separately. After puromycin selection, cells were splitted into 3 groups: Day 0  (baseline time point), crizotinib treated group (treated for 14 days) and DMSO treated group (treated for 14 days). TS and SU-DHL1 cells were treated with 40nM criztonib for 14 days and at the end of this period crizotinib concentration was increased to 80nM. COST cells received 50nM crizotinib for 14 days. KARPAS299 cells first received 50nM crizotinib for 14 days and at the end of this period crizotinib concentration was increased to 100nM. Crizotinib concentrations were determined based on their sensitivity for crizotinib for each cell line. The screening was repeated twice for TS and SU-DHL1 ALK+ ALCL cells. DNA isolotation was performed from all groups and each sgRNA sequence served as a barcode for Next Generation Illumina-based DNA sequencing. We compared enriched sgRNA sequences between Day 0 and DMSO conditions. We found that while PTPN2 was a top hit for all 4 ALK+ ALCL cells, PTPN1 was a top hit for TS and SU-DHL1 cells. We subsequently validated roles of PTPN1 and PTPN2 in crizotinib resistance separately. We demonstrated that both PTPN1 and PTPN2 can drive crizotinib resistance in ALK+ ALCL cells. In this study, we found two phosphatases, PTPN1 and PTPN2, involved in drug resitance in ALK+ ALCL using a CRSIPR Cas9-based screening approach. These two phosphatases regulate ALK phospharylation and therefore affect downstream signalling pathways involved in tumor growth and proliferation. Overall design: Identify drug resistance mechanisms in ALK+ ALCL