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Y. Shi et al.
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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 con- structs targeting selected pre-T-cell receptor (pTCR)/TCR signaling complex components (PTCRA, CD3E, FYN, ZAP70, LCK, LAT), positive (PTEN, RPS29, RPL9), nega- tive 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 con- structs 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.
good viability (≥75%) and proliferation potential (Td = 2.8 days) in co-culture with human mesenchymal stem cells (hMSC), hence subjected to shRNA screening. ShRNA sequencing 30 days after transduction confirmed all con- structs targeting LCK were significantly depleted (Figure 1A, Online Supplementary Table S3 and Online Supplementary Figure S1F).
Knockdown of lymphocyte cell-specific kinase in T-cell acute lymphoblastic leukemia cell lines confirms an essential role for lymphocyte cell-specific kinase
in vitro propagation
To confirm the role of LCK and other components of the pTCR/TCR signaling complex in cell proliferation, com- petitive outgrowth assays were performed. SUPT1, MOLT4 and CUTLL1 cells were transduced with lentiviral shRNAs targeting LCK, ZAP70, FYN, PTCRA or non-tar- geting control shRNAs. Successfully transduced cells expressing green fluorescence protein (GFP) were seeded in a 1:1 ratio with parental cells.
Three shRNAs were used to silence LCK, of which shLCK#3 achieved the greatest degree of knockdown. Lentiviral knockdown with shLCK#3 led to significant reduction in mRNA in SUPT1 (75%KD), MOLT4 (55% KD), and CUTLL1 (45% KD) cells (Figure 2A). In general, greater knockdown was associated with more pro- nounced impairment of in vitro proliferation (Online Supplementary Figure S2A). LCK expression was confirmed
at protein level, demonstrating ubiquitous expression of LCK in cell lines (Online Supplementary Figure S1G). In line with mRNA downregulation, knockdown of LCK led to a decrease in total LCK protein expression (Figure 2A). Non- transduced cells consistently outcompeted LCK knock- down cells resulting in a pronounced loss of over 70% transduced GFP+ cells in all three cell lines, underlining the critical and universal role of LCK in T-ALL cell line main- tenance (Figure 2B and C, and Online Supplementary Figure S2A and C).
A similar but less significant observation was made for ZAP70 knockdown in SUPT1, MOLT4 and CUTLL1 cells. Efficient ZAP70 knockdown correlated with a pronounced proliferation defect (Online Supplementary Figure S2A and C). Knockdown of PTCRA affected proliferation in pTCRα+ MOLT4 and SUPT1, but not in pTCRα- CUTLL1 (Online Supplementary Figure S2A and C). Moreover, FYN knockdown did not affect proliferation in any of the cell lines despite efficient knockdown (Online Supplementary Figure S2B).
Knockdown of lymphocyte cell-specific kinase in T-cell acute lymphoblastic leukemia cell lines and patient-derived xenograft samples impairs leukemia propagation in vivo
To confirm a functional role for LCK in vivo, PDX L963 cells were transduced with our shRNA library and trans- planted into six NSG mice (Figure 3A and Online
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