HTS384 NCI60: The Next Phase of the NCI60 Screen

Abstract

The NCI60 human tumor cell line screen has been in operation as a service to the cancer research community for more than 30 years. The screen operated with 96-well plates, a 2-day exposure period to test agents, and following cell fixation, a visible absorbance endpoint by the protein-staining dye sulforhodamine B. In this study, we describe the next phase of this important cancer research tool, the HTS384 NCI60 screen. Although the cell lines remain the same, the updated screen is performed with 384-well plates, a 3-day exposure period to test agents, and a luminescent endpoint to measure cell viability based upon cellular ATP content. In this study, a library of 1,003 FDA-approved and investigational small-molecule anticancer agents was screened by the two NCI60 assays. The datasets were compared with a focus on targeted agents with at least six representatives in the library. For many agents, including inhibitors of EGFR, BRAF, MEK, ERK, and PI3K, the patterns of GI50 values were very similar between the screens with strong correlations between those patterns within the dataset from each screen. However, for some groups of targeted agents, including mTOR, BET bromodomain, and NAMPRTase inhibitors, there were limited or no correlations between the two datasets, although the patterns of GI50 values and correlations between those patterns within each dataset were apparent. Beginning in January 2024, the HTS384 NCI60 screen became the free screening service of the NCI to facilitate drug discovery by the cancer research community. Significance: The new NCI60 cell line screen HTS384 shows robust patterns of response to oncology agents and substantial overlap with the classic screen, providing an updated tool for studying therapeutic agents. See related commentary by Colombo and Corsello, p. 2397.

Figure 1.

A COMPARE correlation map, which comingles 1,003 FDA-approved and investigational oncology agents, run in the classic NCI60 screen and the HTS384 NCI60 screen (2003 GI₅₀ patterns). Nodes represent GI₅₀ determinations across the cell lines of the classic NCI60 screen (small symbols) and the NCI60 HTS384 screen (large symbols). The line length between nodes indicates the magnitude of the Pearson correlation between the GI₅₀ patterns, with lines only rendered for correlations >0.75. Compact clusters are apparent from compounds sharing a molecular target (some are labeled).

Figure 2.

Box and whisker plot distributions (see Supplementary Fig. S2C) of positive correlations from pairwise combinations of mean GI₅₀ values for compounds grouped by target for targets with at least six assigned agents. Blue, correlations within the classic NCI60 GI₅₀ dataset; orange, correlations within the HTS384 NCI60 GI₅₀ dataset; gray, correlations for the comingled mean GI₅₀ values from the classic and HTS384 NCI60 datasets.

Figure 3.

A, Scatter plot of the mean GI₅₀ values from the HTS384 NCI60 screen plotted vs. the mean GI₅₀ values from the classic NCI60 screen for individual compounds run in both assays (r = 0.93). B, A scatter plot of the Pearson correlations between the HTS384 NCI60 dataset and classic NCI60 dataset for GI₅₀ data for individual compounds run in both assays plotted against the difference in the mean GI₅₀ values. A total of 228 compounds correlated at 0.75 or greater and 470 compounds correlated at 0.6 or better. C, Mean GI₅₀ for 1003 compounds by cell line across the compounds.

Figure 4.

A, Mean GI₅₀ values for compounds targeting the EGFR. Dark blue, data from the classic NCI60 screen; orange, data from the HTS384 NCI60 screen. Error bars indicate SDs serving as surrogates for the range of individual cell line GI₅₀ values relative to the mean of responses across the 60 cell lines (see Supplementary Fig. S1C). B, Box and whisker representation (see Supplementary Fig. S2C) of all possible pairwise combinations of EGFR-targeted agents. The box encompasses the second and third quartiles. Associated correlation maps for correlations >0.75 are shown in Supplementary Fig. S4A and S4B. C, Mean GI₅₀ values for compounds targeting BRAF, MEK, and ERK. Dark blue, data from the classic NCI60 screen; orange, data from the HTS384 NCI60 screen. Error bars indicate SDs serving as surrogates for the range of individual cell line GI₅₀ values relative to the mean of responses across the 60 cell lines (see Supplementary Fig. S1C). D, Box and whisker representation (see Supplementary Fig. S2C) of all possible pairwise combinations of the agents that target BRAF, MEK, and ERK. The box encompasses the second and third quartiles. Associated correlation maps for correlations >0.75 are shown in Supplementary Fig. S5A and S5B.

Figure 5.

A, Mean GI₅₀ values for the PI3K-targeted agents. Dark blue, mean GI₅₀ values from the classic NCI60 screen; orange, those from the HTS384 NCI60 screen. Error bars indicate SDs serving as surrogates for the range of individual cell line GI₅₀ values relative to the mean of responses across the 60 cell lines (see Supplementary Fig. S1C). B, Box and whisker representation (see Supplementary Fig. S2C) of all possible pairwise combinations of PI3K-targeted agents. The box encompasses the second and third quartiles. Associated correlation maps for correlations >0.75 are shown in Supplementary Fig. S6A and S6B. C, Mean GI₅₀ values for the DNA interacting group of drugs and compounds. Dark blue, mean GI₅₀ values from the classic NCI60 screen; orange, mean GI₅₀ values from the HTS384 NCI60 screen. Error bars indicate SDs serving as surrogates for the range of individual cell line GI₅₀ values relative to the mean of responses across the 60 cell lines (see Supplementary Fig. S1C). D, Box and whisker representation (see Supplementary Fig. S2C) of all possible pairwise combinations of PI3K-targeted agents. The box encompasses the second and third quartiles.

Figure 6.

A, Mean GI₅₀ values for mTOR-targeted agents. Dark blue, mean GI₅₀ values from the classic NCI60 screen ; orange, those from the HTS384 NCI60 screen. Error bars indicate SDs serving as surrogates for the range of individual cell line GI₅₀ values relative to the mean of responses across the 60 cell lines (see Supplementary Fig. S1C). B, Box and whisker representation (see Supplementary Fig. S2C) of all possible pairwise combinations of mTOR-targeted agents. The box encompasses the second and third quartiles. Associated correlation maps for correlations >0.75 are shown in Supplementary Fig. S7A and S7B. C, Mean GI₅₀ values for agents targeting the BET bromodomain. Dark blue, data from the classic NCI60 screen; orange, data from the HTS384 NCI60 screen. Error bars are SDs serving as surrogates for the range of individual cell line GI₅₀ values relative to the mean of responses across the 60 cell lines (see Supplementary Fig. S1C). D, Box and whisker representation (see Supplementary Fig. S2C) of all possible pairwise combinations of agents that target the BET bromodomain. The box encompasses the second and third quartiles. Associated correlation maps for correlations >0.75 are shown in Supplementary Fig. S8A and S8B.

Figure 7.

A, Mean GI₅₀ values for the NAMPRTase-targeted agents. Dark blue, data from the classic NCI60 screen; orange, data from the HTS384 NCI60 screen. Error bars are SDs serving as surrogates for the range of individual cell line GI₅₀ values relative to the mean of responses across the 60 cell lines (see Supplementary Fig. S1C). B, Box and whisker representation (see Supplementary Fig. S2C) of all possible pairwise combinations of NAMPRTase-targeted agents. The box encompasses the second and third quartiles. C, At a Pearson correlation threshold of >0.75, NAMPRTase-targeted agents form two independent clusters even when mean GI₅₀ values from the classic and HTS384 NCI60 datasets are allowed to comingle.