Extended Data Fig. 4: LCK signaling is essential for dasatinib sensitivity in T-ALL.
a, Phospho-flow of LCK, ZAP70 and CD247 in dasatinib-sensitive vs –resistant T-ALL cell lines. Cells were treated with increasing concentrations of dasatinib for 1 h and then subjected to intracellular staining for phosphorylated LCK, ZAP70, and CD247, as described in Method. Phospho-protein was quantified by flow cytometry and normalized with samples not exposed to dasatinib as 100%. HSB-2 and KOPT-K1 (sensitive to dasatinib) is plotted in darker colors while CEM (resistant) is plotted in lighter colors. P-values were derived by ANOVA. b-d, LCK T316M mutation confers dasatinib resistance in KOPT-K1 cells. LCK T316M mutation was ectopically expressed in dasatinib-sensitive T-ALL KOPT-K1 cells. KOPT-K1 cells with wildtype LCK overexpression or empty vector control remained sensitive to dasatinib and ponatinib while overexpression of T316M LCK resulted in resistance to dasatinib (b) and ponatinib (c). Meanwhile, all three lines were resistant to ABL-specific inhibitor imatinib (d). For each leukemia sample at each drug concentration, cells were tested in duplicates. e. In KOPT-K1 cells with expressing wildtype LCK or empty vector control, LCK phosphorylation was inhibited by dasatinib in a dose-dependent manner, whereas LCK phosphorylation was unablated by dasatinib in cells expressing the T136M mutant LCK. Standard deviation is derived from biologically independent samples (N = 3) and is plotted as error bar. P value was estimated using Wilcoxon test. f,g, Genome-wide CRISPR screen identifies preTCR pathway genes as dependencies in T-ALL. F. LCK, ZAP70 and CD247 dependency score (x-axis) versus gene dependency probability (y-axis) demonstrates that a subset of T-ALL lines (blue, N = 3) show dependency on these preTCR pathway genes compared to all other cell lines screened (hematologic cancer cell lines in black, N = 73 and other cancer cell lines in gray, N = 613). Gene dependency of greater than 0.5 indicates a high probability that a cell line is dependent and corresponds to an approximate dependency score of −0.5. More negative gene dependency scores indicate greater effect on cell line survival. g, Gene dependency score (x-axis) versus gene dependency probability (y-axis) demonstrates that none of the T-ALL lines (blue, N = 3) show dependency on SRC kinase family genes (other than LCK shown in Fig. 2J) compared to all other cell lines screened (hematologic cancer cell lines in black, N = 73, and other cancer cell lines in gray, N = 613). Gene dependency of greater than 0.5 indicates a high probability that a cell line is dependent and corresponds to an approximate dependency score of -0.5. More negative gene dependency scores indicate greater effect on cell line survival.
