Kinase requirements in human cells: I. Comparing kinase requirements across various cell types

Abstract

shRNA loss-of-function screens were used to identify kinases that were rate-limiting for promoting cell proliferation and survival. Here, we study the differences in kinase requirements among various human cells, including freshly prepared primary cells, isogenic cells, immortalized cells, and cancer cell lines. Closely related patterns of kinase requirements among the various cell types were observed in three cases: (i) in repeat experiments using the same cells, (ii) with multiple populations of freshly prepared primary epithelial cells isolated from the same tissue source, and (iii) between nearly isogenic cells that differ from each other by the expression of a single gene. Other commonly used cancer cell lines were distinct from one another, even when they were isolated from similar tumor types. Even primary cells of different lineages isolated from the same tissue source showed many differences. The differences in kinase requirements among cell lines observed in this study suggest that the control of proliferation and survival may be significantly different between cell lines and that simple comparisons from any one cell to another may be misleading. Although the regulation of cell proliferation and survival are heavily studied areas, we did not see a bias in these screens toward the identification of previously known and well studied kinases, suggesting that our knowledge of molecular events in these areas is still meager.

Fig. 1.

Proliferation and survival screen. Relative cell proliferation and survival was measured by alamarBlue assay after transfection of ≈2,000 shRNAs targeting 430 kinases into HeLa cells. Fluorescence readings from two independent HeLa experiments are compared on the x- and y axis with a correlation coefficient of 0.89 demonstrating reproducibility between replicate screens. See Table S1 for a full list of kinases assayed in the primary screen.

Fig. 2.

Differential kinase requirements comparing cell lines derived from different tissue origins and the same tissue origin. (A and B) Relative cell proliferation and survival was measured by alamarBlue assay 5–6 days after transfection of HeLa (cervical) and 293T (renal) cell lines, and after transduction of A549 (lung), H358 (lung), H1975 (lung), and H23 (lung) cell lines with ≈2,000 shRNAs targeting 430 kinases. Kinase requirements were defined as a 50% or more growth inhibition relative to the mean value of examined shRNAs. Total number of essential kinases that scored per cell line is shown in parentheses. Hatch bars show similarities in kinase requirements between (A) two cell lines for HeLa and 293T (B) and four pairs of lung cell lines. (C) Of 278 essential kinases that scored among four lung lines, 109 kinases scored in any two cell lines, 44 kinases scored in any three cell lines, and 14 in all four cell lines.

Fig. 3.

Rank order of HeLa-293T hits across a large panel of cell lines. Relative cell proliferation and survival was measured by alamarBlue assay 6 days after transduction of lentiviruses expressing 89 shRNAs targeting 80 kinases and controls into 19 different cell lines. The RKO-pc and RKO-E7 cells were run in replicate, and separate experiments were listed as RKO-pc-1 and RKO-pc-2 and RKO-E7–1 and RKO-E7–2, respectively. Reduction in viability induced by each shRNA was calculated relative to a lentiviral vector expressing GFP and assembled by rank order. Color scales represent the greatest decrease in viability by an shRNA (red) to the least (green). Columns display 21 different screens (19 cell lines tested + two duplicates). Rows display shRNAs. Data were analyzed by hierarchical clustering with Euclidean distance to link shRNAs and cell lines with related growth inhibition properties.

Fig. 4.

Viral transduction of five NSCLC cell lines using additional shRNAs targeting the same kinase. Presented here are five kinases whose two shRNAs show the most similar patterns across the five NSCLC lines. The plots for all 75 pairs of shRNAs are in Fig. S7.