Figure 1.

A targeted shRNA screen reveals lymphocyte cell-specific kinase (LCK) is essential for in vitro proliferative potential. (A) T-cell acute lymphoblastic leukemia (T-ALL) cell lines (SUPT1, MOLT4, HBP-ALL, CUTLL1) and PDX LK203 were subjected to a functional screen using a pLKO5-shRNA library containing 36 constructs targeting selected pre-T-cell receptor (pTCR)/TCR signaling complex components (PTCRA, CD3E, FYN, ZAP70, LCK, LAT), positive (PTEN, RPS29, RPL9), negative controls (KLHL7, CD19, DDB2, ERGIC3, FLG, RUNX1-ETO, SESN2, TRPM7) and a non-targeting control (NTC). Genomic DNA was sampled and barcoded. Enriched and depleted shRNAs were identified by next generation sequencing (NGS). The heatmap depicts statistically significant gains (red) or losses (green) of shRNA constructs after in vitro culture of four T-ALL cell lines (40 days) and PDX LK203 (30 days). (B) Relative gene expression of LCK in seven cell lines and 12 PDX samples. LCK expression was determined in four T-ALL cell lines, 697 and REH (B-lineage ALL cell lines [B-ALL]), TK6 (lymphoblastoid cell line), and 12 PDX samples by real time quantitative-polymerase chain reaction. GAPDH served as reference gene for normalization.