Radiation-induced chronic injuries increase the risk of sarcomagenesis. A, The final injury scores of the control and p53KD mice that received 30 or 40 Gy are plotted. The final injury scores of mice that did not develop a radiation-induced sarcoma (blue) are compared with the scores of mice that did develop a radiation-induced sarcoma (red). P value is from a t test. B, Kaplan–Meier curves show radiation-induced sarcoma-free survival of the control and p53KD mice irradiated with 30 or 40 Gy to the hind limb. Mice with chronic injury scores equal to or less than 3 are compared with mice with chronic injury scores greater than 3. P value is from a cox proportional hazards model.

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.

Abstract Approximately half of patients with cancer receive radiotherapy and, as cancer survivorship increases, the low rate of radiation-associated sarcomas is rising. Pharmacologic inhibition of p53 has been proposed as an approach to ameliorate acute injury of normal tissues from genotoxic therapies, but how this might impact the risk of therapy-induced cancer and normal tissue injuries remains unclear. We utilized mice that express a doxycycline (dox)-inducible p53 short hairpin RNA to reduce Trp53 expression temporarily during irradiation. Mice were placed on a dox diet 10 days prior to receiving 30 or 40 Gy hind limb irradiation in a single fraction and then returned to normal chow. Mice were examined weekly for sarcoma development and scored for radiation-induced normal tissue injuries. Radiation-induced sarcomas were subjected to RNA sequencing. Following single high-dose irradiation, 21% of animals with temporary p53 knockdown during irradiation developed a sarcoma in the radiation field compared with 2% of control animals. Following high-dose irradiation, p53 knockdown preserves muscle stem cells, and increases sarcoma development. Mice with severe acute radiation-induced injuries exhibit an increased risk of developing late persistent wounds, which were associated with sarcomagenesis. RNA sequencing revealed radiation-induced sarcomas upregulate genes related to translation, epithelial–mesenchymal transition (EMT), inflammation, and the cell cycle. Comparison of the transcriptomes of human and mouse sarcomas that arose in irradiated tissues revealed regulation of common gene programs, including elevated EMT pathway gene expression. These results suggest that blocking p53 during radiotherapy could minimize acute toxicity while exacerbating late effects including second cancers. Significance: Strategies to prevent or mitigate acute radiation toxicities include pharmacologic inhibition of p53 and other cell death pathways. Our data show that temporarily reducing p53 during irradiation increases late effects including sarcomagenesis.

Supplementary table 2 is the injury scoring rubric

Supplementary figure S6 shows that radiation-induced chronic injuries are associated with sarcomagenesis

Temporary reduction of p53 during irradiation increases chronic injuries in subsets of mice. A, Kaplan–Meier curves show acute injury-free survival (score 1+) of control and p53KD male and female mice irradiated with 40 Gy to the hind limb. P value is from a log-rank test. B, Kaplan–Meier curves show acute injury-free survival (score 1+) of control and p53KD male and female mice irradiated with 30 Gy to the hind limb. P value is from a log-rank test. Kaplan–Meier curves show chronic injury-free survival from scores 1+ (C), 2+ (D), 3+ (E), or 4 (F) of control and p53KD male and female mice irradiated with 30 Gy to the hind limb. P value is from a log-rank test.

Supplementary figure S9 shows gene expression analysis of mouse and human sarcomas that arose in irradiated tissue compared to sporadic sarcomas

Supplementary Figure S1. An example of measurements on a staining tissue section. Supplementary Figure S2. The Scripts used for blind analysis of the lung colonization study. Supplementary Figure S3. Example of measurements on a whole lung section. Supplementary Figure S4. All the uncropped images. Supplementary Figure S5. RNA sequencing data showing that NEAT1 is significantly upregulated in lung metastases compared to paired KP primary tumors. DESeq2 method was used for statistical analysis. Supplementary Figure S6. Expression of Neat1 in metastatic primary KP tumors and non-metastatic primary KP tumors. Supplementary Figure S7. RNA sequencing data from the Samuel Lunenfeld Research Institute showed that NEAT1 is upregulated in lung metastases compared to paired human UPS/MFS. Supplementary Figure S8. NEAT1 RNA in situ hybridization showed that the expression of NEAT1 is not significantly different in lung metastases compared to unpaired human primary UPSs. Supplementary Figure S9. Deletion of Neat1_1 region has no effect on cell proliferation in both KP and KI cell lines.

Radiation-induced sarcomas exhibit an increase in proliferative gene programs and a decrease in myogenic differentiation programs. A, Heat map of the top 75 differentially expressed genes in radiation-induced sarcomas (n = 16) versus normal muscle (n = 7). Genes (rows) are colored by scaled, normalized expression values. Both rows and columns are clustered. B, Volcano plot of log2 fold change for all genes with base mean greater than 50 in tumor (n = 16) versus normal (n = 7). Labeled genes have a nominal −log10P value greater than or equal to 30. C, Normalized enrichment scores for all FDR significant pathways for GSEA of Hallmark pathways. Points are colored by −log10 (nominal P value). D, Scatter plot comparing the differentially expressed pathways between tumors that arose in irradiated tissues (Rad) versus sporadic (Spor) tumors in mouse (y-axis) and human (x-axis) datasets. The EMT pathway is highlighted with a green dot. E, Boxplot showing gene expression of EMT pathway targets in mouse radiation-induced UPS compared with p53/RB tumors (top) and in human radiation-associated undifferentiated sarcomas compared with sporadic undifferentiated sarcomas.

Supplementary figure S5 shows mice without dox treatment sustained radiation-induced injuries in the hind limb

Comparison of CNV and expression of specific oncogenes. Boxplot showing gene expression of Yap1 (A), Met (B), and Cdk4 (C) in radiation-induced sarcomas where the gene amplification status is known to be amplified (pink), not amplified (orange), or unknown (gray) compared with normal muscle (blue).

Supplementary table S1 is the experimental mice table

Supplementary figure S1 shows irradiated hind limbs of control and p53KD mice

Supplementary figure S4 shows high dose irradiation induces chronic injuries in mouse hind limbs

Supplementary figure S7 shows gene expression analysis of radiation-induced sarcomas compared to normal muscles

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.

Supplementary figure S2 shows that temporary reduction in p53 expression during irradiation increases sarcomagenesis