Legends for Supplementary Figures S1-S10

Correlation plots BET-targeting agents.

Representative conc/resp mTor-targeting agents.

Abstract G-quadruplexes (G4s) are four-stranded nucleic acid structures that help regulate key cellular processes and are attractive therapeutic targets in oncology. To assess the therapeutic potential of three G4 ligands—pidnarulex, APTO-253, and BRACO-19—we performed a high-throughput drug-combination screen across thirty-one multicellular tumor spheroids derived from patient tumors and established cancer cell lines. These 3D spheroids model salient features of the tumor microenvironment, incorporating malignant, endothelial, and mesenchymal components. Combination partners were selected for mechanistic relevance to G4 biology, including inhibitors of DNA damage response (DDR), replication stress, and chromatin regulation, based on the proposed roles of G4s in replication and genome stability. Responses were quantified using viability assays complemented by longitudinal brightfield imaging to track spheroid morphology and growth. Drug interactions were evaluated by Bliss independence and volume under the viability surface, providing complementary metrics of synergy and global response. Among the ligands, pidnarulex showed the broadest single-agent activity, whereas APTO-253 and BRACO-19 produced more limited effects. Combination screening with PARP inhibitors, DDR-kinase inhibitors (ATM, ATR, DNA-PK), and cell-cycle regulators (WEE1, PIM1) revealed model-specific synergy. Notably, pidnarulex exhibited consistent synergy in one of eight pancreatic adenocarcinoma models (966289-007-R4-J1) across multiple DDR-targeted combinations. Additional interactions were observed with HDAC inhibitors in a subset of models. Brightfield imaging corroborated enhanced suppression of spheroid growth in synergistic combinations. These data highlight the context-dependent activity of G4 ligands and support integrated functional plus imaging-based approaches to define therapeutic combinations in physiologically relevant 3D cancer models. This project was funded with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024. Citation Format: Thomas S. Dexheimer, Nathan P. Coussens, Thomas Silvers, Poorva Juneja, Eric Jones, Steven D. Gore, Mark W. Kunkel, James H. Doroshow, Beverly A. Teicher. Combinatorial profiling of pidnarulex and other G-quadruplex ligands in 3D multicellular tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6504.

Table listing NCI60 cell lines, disease of origin and key genetic alterations

Diagram outlining COMPARE calculations and data presentation formats.

Diagram outlining processing of assay data

Aggregate correlations of compound pairs grouped by target for 91 targets with more than a single representative compound

Correlation plots agents targeting BRAF, MEK or ERK.

G-quadruplexes (G4s) are four-stranded nucleic acid structures that regulate key cellular processes and represent promising therapeutic targets in oncology. To investigate the therapeutic potential of three G4 ligands—pidnarulex, APTO-253, and BRACO-19—a high-throughput drug combination screen was conducted in thirty-one multi-cell type tumor spheroids derived from patient tumors and established cancer cell lines. These 3D spheroids mimic key features of the tumor microenvironment, comprising malignant, endothelial, and mesenchymal cell populations. Compounds selected for combination screening included agents with mechanistic relevance to G4 biology, such as inhibitors of DNA damage response (DDR), replication stress, and chromatin regulation, based on the proposed roles of G4s in replication and genome stability. Combination responses were assessed using cell viability assays and supported by longitudinal brightfield imaging to monitor spheroid morphology and growth dynamics. Drug interactions were quantified using Bliss independence scores and the volume under the viability surface, providing complementary metrics of synergy and overall response. Among the G4 ligands, pidnarulex demonstrated the broadest single-agent activity, while APTO-253 and BRACO-19 showed limited effects. Model-specific synergy was observed from combinations with inhibitors of PARP, DDR kinases (ATM, ATR, DNA-PK), and cell cycle regulators (WEE1, PIM1). Interestingly, pidnarulex exhibited consistent synergy in one of eight pancreatic adenocarcinoma models (966289-007-R4-J1) across multiple DDR-targeted combinations. Combination interactions were also observed with HDAC inhibitors in a subset of models. Brightfield imaging corroborated enhanced spheroid growth suppression from synergistic combinations. These findings underscore the context-dependent activity of G4 ligands and support the use of integrated functional and imaging-based approaches to characterize potential therapeutic combinations in physiologically relevant 3D cancer models.

Abstract The NCI60 screen was established in the 1980s and introduced in the 1990s as a screening service for the worldwide cancer research community. The screen features 60 human tumor cell lines that represent 9 cancer types including non-small cell lung, colon, central nervous system, ovarian, renal, prostate and breast, as well as leukemia and melanoma. On a weekly basis, synthetic molecules, natural products, and biologics are screened against the 60-cell line panel to evaluate their anticancer potential. On average, more than 13,000 compounds are screened annually for investigators throughout the world. Cell growth and cytotoxicity were assessed by the sulforhodamine B (SRB) assay, performed in a 96-well format, that measures cellular protein content with an absorbance readout and enabled unprecedented throughput in the 1980s. Although the SRB assay supported a robust screening platform for three decades, its sensitivity and throughput are limited. Recently the NCI’s DCTD introduced the HTS384 NCI60 as a modernized screening platform. While the same 60 cell lines are evaluated, the HTS384 NCI60 is fully automated and performed in a 384-well format to enable a higher throughput. Additionally, the 48-h test agent exposure period of the SRB assay was increased to 72 h, which may result in lower 50% growth inhibition values (GI50) for some agents. A highly sensitive CellTiter-Glo luminescence readout for cell viability, based on cellular ATP content, was selected to enable comparisons of the NCI60 monolayer data to 3D cell culture models where the same assay chemistry can be applied. The HTS384 NCI60 was evaluated by screening a library of 1,003 U.S. Food and Drug Administration-approved and investigational oncology agents at five concentrations. Assay performance of the 60 cell lines was assessed from the controls of 30 - 60 microplates per cell line. The average doubling time of the 60 cell lines during the 72-h exposure period ranged between 19 h for NCI-H460 (17 - 23 h, n = 57) to 117 h for MDA-MB-468 (43 - 272 h, n = 60). The average signal-to-background ratio of all 60 cell lines was 89 (3,413 microplates) and ranged from 6 for OVCAR-5 (3 - 13, n = 54) to 229 for SF-539 (141 - 566, n = 60). The average coefficient of variation for microplate vehicle controls (n = 14) of all 60 cell lines (n = 3,413) was 5.2% and ranged from 3.2% for SK-MEL-28 (1.4 - 7.7%, n = 60) to 11.1% for HL-60(TB) (5.2 - 20.2%, n = 59). The average Z’-factor value of all 60 cell lines (n = 3,413) was 0.82 and ranged between 0.66 for HL-60(TB) (0.39 - 0.84, n = 59) to 0.89 for MALME-3M (0.79 - 0.95, n = 59), SF-295 (0.49 - 0.97, n = 59), SK-MEL-28 (0.74 - 0.95, n = 60), and UO-31 (0.8 - 0.94, n = 54). The development and performance of the HTS384 NCI60 screen will be described. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Nathan P. Coussens, Thomas S. Dexheimer, Eric M. Jones, Thomas Silvers, John R. Britt, Ronald C. Taylor, Mark W. Kunkel, James H. Doroshow, Beverly A. Teicher. Development and performance of the National Cancer Institute’s HTS384 NCI60 screen: A modernized platform to support drug discovery and development by the worldwide cancer research community [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3090.

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

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

The NCI60 human tumor cell line panel is a useful tool in the discovery and development of new anticancer agents. The publicly available cell-line characterization and compound screening data from the NCI60 screen have contributed to identifying cellular mechanisms of potential new anticancer agents. Mean-graph sensitivity/resistance patterns in the NCI60 screen serve as a fingerprint for molecular target identification and mechanism of action (MOA). A new NCI60 resource was developed based on screening of 175 FDA-approved oncology drugs (AOD) plus >825 investigational oncology agents (IOA), representing >250 therapeutic targets and MOAs. Compounds targeting different components in a biochemical pathway tend to show high correlations in their GI50 patterns through COMPARE analysis forming clusters with similar NCI60 mean-graph patterns. COMPARE evaluation of compounds that target components in the PI3K/AKT/mTOR pathway form correlation clusters linked by both target and pathway. Only 3 of 15 AKT inhibitors in the IOA set are not found in the connected cluster with a COMPARE correlation of 0.7 including the multi-kinase inhibitors perifosine and AT-13148. The AKT cluster includes both allosteric (MK-2206) and competitive inhibitors highlighting the NCI60 is a functional cell assay. More than 75% of PI3K inhibitors (32/42 agents), including the selective PI3K alpha and PI3K beta inhibitors, form a highly connected cluster at a 0.7 COMPARE correlation. The heat map view showed that >50% of the non-connected PI3K outliers were inactive in the NCI60 assay. The mTOR inhibitors (12/20) form a connected cluster at 0.7 COMPARE correlation, with half of the non-connected mTOR singletons representative of the rapamycin class of inhibitors. The BRAF/MEK/ERK pathway compounds (37) form a highly connected correlation cluster (13/15 BRAF, 14/15 MEK, 8/9 ERK inhibitors) at a 0.75 COMPARE correlation. The NCI60 heat maps for BRAF inhibitors show a consistent pattern of the melanoma cell lines and 2 of the colon cancer lines (COLO 205, HT29). The ERK inhibitors display sensitivity patterns similar to the BRAF agents with additional activity observed against a leukemia (HL-60), ovarian (OVCAR-5) and renal (A498) cell line and 3 other colon cell lines (HCC-298, HCT-116, SW-620). This data analysis resource will be available to the public (https://ioa.cancer.gov). NCI60 compound suppliers can incorporate their test compound(s) data to use the interactive visualization tools with AOD and IOA agents. This project was funded in part with federal funds from the NCI, NIH, under Contract # 75N91019D00024/75N91020F00003. Citation Format: Joel Morris, Mark W. Kunkel, Stephen L. White, Donn G. Wishka, Omar D. Lopez, Lori Bowles, Penny Sellers, Patricia Ramsey, Julie Grams, Tiffany Rohrer, Karen Martin, Thomas Dexheimer, Dmitriy Sonkin, John D. Williams, Jerry M. Collins, James H. Doroshow, Beverly A. Teicher. Targeted investigational oncology agents (IOA) in the NCI60: a phenotypic systems-based resource. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4884.

The NCI60 human tumor cell line screen, which has operated as a free service to the cancer research community for over 20 years, has proven to be an important drug discovery tool. The screen has operated using cutting-edge technology of the 1990s including a 96-well microplate format, a 2-day drug exposure period and following cell fixation, and a visible absorbance endpoint by the protein-staining dye sulforhodamine B (SRB). The SRB endpoint determines cell density based on protein content with a lower resolution limit of 1,000 – 2,000 cells. This report describes the next phase of this important cancer research tool, the NCI60 HTS384 screen format. While the cell lines remain the same, the screen is performed using 384-well microplates with a 3-day compound exposure period and a CellTiter-Glo luminescence endpoint. The CellTiter-Glo endpoint determines cell number based upon ATP content with a lower resolution limit of <10 cells. To develop a baseline comparison, data were generated from a library of 1,000 FDA-approved and investigational small molecule anticancer agents by the two assay formats. Although the mean-graph patterns and COMPARE analyses are very similar for many compounds, there is a divergence between the two screens for some molecular targets. Within subsets of approved and investigational agents directed toward the same target (e.g., EGFR, BRAF/MEK/ERK, and PI3K inhibitors), the clustering of compound data from the NCI60 HTS384 screen is qualitatively similar to the clustering of compound data from the classic NCI60 SRB screen and there is a strong COMPARE correlation between the two screens. However, for some drug targets there is very little or no clustering between data from the two screens, although the clustering within each screen is moderate-to-good. Examples include mTOR rapalogs, BET bromodomain and NAMPRTase inhibitors. Differences in data between the two screens might be partially attributed to increases in the assay endpoint sensitivity and compound exposure time of the NCI60 HTS384 assay. Compounds submitted for NCI60 screening after September 30, 2023, will be tested in the modernized NCI60 HTS384 format. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I and by the Intramural Research Program of the NIH, National Cancer Institute. Citation Format: Beverly A Teicher, Mark W Kunkel, Joel Morris, Ronald C Taylor, Thomas S Dexheimer, Nathan P Coussens, James H Doroshow. NCI60 HTS384: The next phase of the NCI60 screen [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A21.

Sarcoma cell lines that were in the screen

Abstract The NCI-60 human tumor cell line panel has proved to be a useful tool for the global cancer research community in the search for novel chemotherapeutics. The publicly available cell line characterization and compound screening data from the NCI-60 assay have significantly contributed to the understanding of cellular mechanisms targeted by new oncology agents. Signature sensitivity/resistance patterns generated for a given chemotherapeutic agent against the NCI-60 panel have long served as fingerprint presentations that encompass target information and the mechanism of action associated with the tested agent. We report the establishment of a new public NCI-60 resource based on the cell line screening of a large and growing set of 175 FDA-approved oncology drugs (AOD) plus >825 clinical and investigational oncology agents (IOA), representing a diverse set (>250) of therapeutic targets and mechanisms. This data resource is available to the public (https://ioa.cancer.gov) and includes the raw data from the screening of the IOA and AOD collection along with an extensive set of visualization and analysis tools to allow for comparative study of individual test compounds and multiple compound sets.