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Evaluation of pharmacodynamic (PD) markers in vivo.

Supplemental Table S1. IC50 values for Nultin-3a, Carboplatin, and 1:1 Combination; Supplemental Material and Methods; Supplemental Figure Legends

The Human Cancer Models Initiative (HCMI) is an international consortium founded by National Cancer Institute (NCI), Cancer Research UK, Wellcome Sanger Institute, and the foundation Hubrecht Organoid Technology. The initiative has generated patient derived Next-generation Cancer Models (NGCMs) from diverse tumor types and subtypes including rare adult and pediatric cancers as a community resource. HCMI addresses deficiencies in traditional cell lines models by collecting patients’ clinical data, as well as the genomes and transcriptomes of the parent tumor, case-matched normal tissue, and the derived next-generation cancer model. NCI’s Center for Cancer Genomics (CCG) sponsors four Cancer Model Development Centers (CMDCs) who are managed by Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. CCG also supports the downstream model development pipeline. The CMDCs are tasked with generating HCMI NGCMs. The model-associated clinical data are submitted to the Clinical Data Center. The models, their associated tumor, and normal samples are processed at the Biospecimen Processing Center (BPC). The nucleic acids isolated at BPC are sent to the Genomic Characterization Centers for molecular characterization. All biospecimen, clinical, and molecular characterization data are quality controlled and submitted to NCI’s Genomic Data Commons (GDC) for the research community. The HCMI models and culture protocols are made available to the research community through a single third-party distributor. The HCMI Searchable Catalog (https://hcmi-searchable-catalog.nci.nih.gov/) is an online resource that allows users to query and identify available models using various data elements including clinical and molecular characterization data, including WGS, WXS, RNA-seq, and methylation array. To date, over 250 HCMI models are available to query on the Searchable Catalog and are available to the research community through the NCI designated model distributor, ATCC. These models have been derived from several cancer types including glioblastoma, colorectal, pediatric, gastroesophageal, pancreatic, and more. Biospecimen, clinical, and molecular characterization data are available for over 100 models at NCI’s GDC, with additional cases released as the data completes the HCMI pipeline. Data, tools, and resources generated by CCG initiatives are made publicly available via the CCG website and GDC. The CCG website also provides available data types, data usage policies and guides to access data (https://www.cancer.gov/about-nci/organization/ccg). Citation Format: Eva Tonsing-Carter, Rachana Agarwal, Cindy W. Kyi, Julyann Perez-Mayoral, Conrado T. Soria, Jean Claude Zenklusen. Human Cancer Models Initiative (HCMI): A community resource of next-generation cancer models and associated data. [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 4681.

Background Non-ER nuclear receptor activity can alter estrogen receptor (ER) chromatin association and resultant ER-mediated transcription. Consistent with GR modulation of ER activity, high tumor glucocorticoid receptor (GR) expression correlates with improved relapse-free survival in ER+ breast cancer (BC) patients. Methods In vitro cell proliferation assays were used to assess ER-mediated BC cell proliferation following GR modulation. ER chromatin association following ER/GR co-liganding was measured using global ChIP sequencing and directed ChIP analysis of proliferative gene enhancers. Results We found that GR liganding with either a pure agonist or a selective GR modulator (SGRM) slowed estradiol (E2)-mediated proliferation in ER+ BC models. SGRMs that antagonized transcription of GR-unique genes both promoted GR chromatin association and inhibited ER chromatin localization at common DNA enhancer sites. Gene expression analysis revealed that ER and GR co-activation decreased proliferative gene activation (compared to ER activation alone), specifically reducing CCND1, CDK2, and CDK6 gene expression. We also found that ligand-dependent GR occupancy of common ER-bound enhancer regions suppressed both wild-type and mutant ER chromatin association and decreased corresponding gene expression. In vivo, treatment with structurally diverse SGRMs also reduced MCF-7 Y537S ER-expressing BC xenograft growth. Conclusion These studies demonstrate that liganded GR can suppress ER chromatin occupancy at shared ER-regulated enhancers, including CCND1 (Cyclin D1), regardless of whether the ligand is a classic GR agonist or antagonist. Resulting GR-mediated suppression of ER+ BC proliferative gene expression and cell division suggests that SGRMs could decrease ER-driven gene expression. Electronic supplementary material The online version of this article (10.1186/s13058-019-1164-6) contains supplementary material, which is available to authorized users.

The mission of the Office of Cancer Genomics (OCG) at the National Cancer Institute (NCI) is to advance the understanding of cancers to improve clinical outcomes. To accomplish this goal, OCG supports innovative and complementary initiatives that characterize and generate comprehensive genomic datasets from rare and pediatric cancers, develop bioinformatics tools to analyze large-scale genomic datasets and identify the underlying etiology of cancer, generate biologically relevant human tumor-derived next-generation cancer models (NGCMs), and advance technologies to better expand and utilize these NGCMs. The Cancer Genome Characterization Initiative (CGCI) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) programs aim to identify therapeutic targets and biomarkers in understudied, rare and high-risk adult and pediatric cancers. Clinically annotated tumor and normal matched tissues are characterized by whole-genome, whole-exome and transcriptome sequencing. The research community can access these datasets via the CGCI Data Matrix and TARGET Data Matrix, as well as through NCI's Genomic Data Commons (GDC). The Cancer Target Discovery and Development (CTD2) Network and Human Cancer Models Initiative (HCMI) are functional genomics programs. The CTD2 Network aims to understand tumor development, heterogeneity, drug resistance and metastasis to develop optimal combinations of treatments. The Network develops analytical tools, generates diverse raw/primary datasets that can be accessed through the CTD2 Data Portal and further validates subsets of these data which are shared on the CTD2 Dashboard. The Human Cancer Models Initiative (HCMI) is an international consortium generating patient-derived NGCMs from diverse tumor subtypes including rare adult and pediatric cancers as a community resource. The biospecimen, clinical and molecular characterization data from the derived model, originating tumor and normal tissue can be accessed through NCI's GDC. The HCMI Searchable Catalog allows users to query and identify available models using various data elements including clinical data and masked somatic MAF variants. The Next-Gen Technologies for Next-Gen Cancer Models (NGTfNGCM) program supports the development of technologies and tools that will enhance the use of HCMI and other models in research. The new tools and broader use of NCGMs will contribute to identification of mechanisms of resistance, novel therapeutic targets and development of diagnostic and/or predictive biomarkers for clinical applications. Data, tools, protocols and resources generated by the OCG initiatives are made publicly available via the OCG website (https://ocg.cancer.gov/). The OCG website also provides data usage policies, data access guides, experimental methods, standard operating procedures and helpful links. Citation Format: Cindy W. Kyi, Pamela C. Birriel, Tanja M. Davidsen, Martin L. Ferguson, Patee Gesuwan, Nicholas B. Griner, Yiwen He, Julyann Perez-Mayoral, Eva Tonsing-Carter, Daniela S. Gerhard. NCI Office of Cancer Genomics: Multidisciplinary genomic research initiatives drive discoveries towards the clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2224.

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein–protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein–protein interactions.

In estrogen receptor (ER)–negative breast cancer, high tumor glucocorticoid receptor (GR) expression has been associated with a relatively poor outcome. In contrast, using a meta-analysis of several genomic datasets, here we find that tumor GR mRNA expression is associated with improved ER+ relapse-free survival (RFS; independently of progesterone receptor expression). To understand the mechanism by which GR expression is associated with a better ER+ breast cancer outcome, the global effect of GR-mediated transcriptional activation in ER+ breast cancer cells was studied. Analysis of GR chromatin immunoprecipitation followed by high-throughput sequencing in ER+/GR+ MCF-7 cells revealed that upon coactivation of GR and ER, GR chromatin association became enriched at proximal promoter regions. Furthermore, following ER activation, increased GR chromatin association was observed at ER, FOXO, and AP1 response elements. In addition, ER associated with GR response elements, suggesting that ER and GR interact in a complex. Coactivation of GR and ER resulted in increased expression (relative to ER activation alone) of transcripts that encode proteins promoting cellular differentiation (e.g., KDM4B, VDR) and inhibiting the Wnt signaling pathway (IGFBP4). Finally, expression of these individual prodifferentiation genes was associated with significantly improved RFS in ER+ breast cancer patients. Together, these data suggest that the coexpression and subsequent activity of tumor cell GR and ER contribute to the less aggressive natural history of early-stage breast cancer by coordinating the altered expression of genes favoring differentiation. Implications: The interaction between ER and GR activity highlights the importance of context-dependent nuclear receptor function in cancer. Mol Cancer Res; 14(8); 707–19. ©2016 AACR.

Early-stage ER+ breast cancer with high tumor glucocorticoid receptor (GR) expression is associated with improved long term relapse-free survival compared to tumors with low GR expression. In addition, activation of GR inhibits estradiol-mediated ER+ BC cell proliferation. This finding led us to hypothesize that GR and ER engage in nuclear receptor cross-talk to affect pro-proliferative gene expression, thus contributing to a better outcome in ER+/GR+ BC patients. To better understand the mechanisms by which ER/GR co-activation contributes to this more indolent phenotype, we performed ChIP-sequencing and gene expression analyses in ER+/GR+ BC cell lines (MCF-7 and T47D). We found that co-activation of ER and GR led to decreased ER+ BC cell proliferation in vitro. Furthermore, following co-activation of ER/GR, there was decreased gene expression of key cell cycle genes (e.g. CDK6, CDK2 and CCNE1) compared to ER-activation alone. Studies are underway to determine if this decrease in gene expression is associated with less CDK activity as well as a slowing of cell cycle progression. We also wanted to know if GR activation with a pure agonist versus a mixed GR modulator was required for decreasing ER-mediated cell proliferation. Based on our previous work (DC West et al. MCR 2016), we hypothesized that ligand-mediated GR activation could be working by preventing access of ER to regulatory regions of pro-proliferative genes rather than causing direct GR agonist-mediated gene expression. To examine this question, we used a GR modulator, mifepristone (expected to alter ER-mediated gene expression indirectly). Indeed we found that a GR modulator also reduced ER-mediated cell proliferation. We are currently testing the mechanisms by which GR modulators affect estradiol-mediated cell cycle gene expression. Taken together, these studies suggest that modulation of GR activity could be an effective approach to decrease ER+ tumor cell proliferation. Xenograft studies are underway to examine whether the addition of GR modulation to tamoxifen will improve tumor shrinkage compared to tamoxifen alone. Citation Format: Tonsing-Carter E, Bowie KR, West DC, Harkless RV, Hernandez KM, Conzen SD. Glucocorticoid receptor modulation affects ER+ breast cancer cell proliferation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-09.

The National Cancer Institute's (NCI) Office of Cancer Genomics' (OCG) mission is to advance the molecular understanding of cancers to improve clinical outcomes. To accomplish this goal, OCG develops and collaboratively manages molecular characterization and translational genomics research initiatives. OCG currently supports four innovative and complementary initiatives. These programs generate comprehensive genomic datasets, develop bioinformatics tools to analyze these datasets, create biologically-relevant human tumor-derived Next-Generation Cancer Models (NGCMs) and valuable experimental reagents and resources. The Cancer Genome Characterization Initiative (CGCI) (https://ocg.cancer.gov/programs/cgci) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) (https://ocg.cancer.gov/programs/target) are the molecular characterization programs. These programs aim to identify therapeutic targets and biomarkers in understudied, rare, and high-risk adult and pediatric cancers. Clinically annotated tumor and normal matched tissue samples are collected and analyzed by whole-genome, whole-exome, and transcriptome sequencing. The datasets are available to the research community through program-specific Data Matrices: CGCI Data Matrix and TARGET Data Matrix. CGCI and TARGET data are also available through NCI's Genomic Data Commons (GDC)(https://portal.gdc.cancer.gov/). The Pediatric Genomic Data Inventory (PGDI) is a global inventory of pediatric molecular and clinical datasets. The CTD2 Network (https://ocg.cancer.gov/programs/ctd2) aims to understand cancer metastasis, tumor heterogeneity and drug resistance, and develop optimal combinations of pharmacologic and immunological agents. The CTD2 Network develops bioinformatics tools, generates diverse raw/primary datasets that can be accessed through the CTD2 Data Portal and further validates subsets of these data listed at the CTD2 Dashboard. The Human Cancer Models Initiative (HCMI) (https://ocg.cancer.gov/programs/HCMI) is an international consortium that is generating human tumor-derived NGCMs from diverse tumor subtypes including rare and pediatric cancers. The models, together with related clinical and genomic data, are available as a community resource. The HCMI Searchable Catalog allows users to search the list of available models and associated clinical and molecular data. The clinical and molecular data are stored at the NCI's GDC. Data, analytical tools, and resources generated by OCG initiatives are made publicly available to further the collaborative goal of enabling precision oncology to improve patient care. The OCG website (https://ocg.cancer.gov/) also provides data usage policies, guides to access data, experimental methods, standard operating procedures, and educational and helpful links for the community. Citation Format: Cindy W. Kyi, Pamela C. Birriel, Tanja M. Davidsen, Martin L. Ferguson, Patee Gesuwan, Nicholas B. Griner, Yiwen He, Lauren M. Hurd, Subhashini Jagu, Eva Tonsing-Carter, Daniela S. Gerhard. NCI Office of Cancer Genomics supports multidisciplinary genomics research initiatives to advance precision oncology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5862.

Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein–protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC. Mol Cancer Ther; 14(12); 2850–63. ©2015 AACR.

Purpose: Although high glucocorticoid receptor (GR) expression in early-stage estrogen receptor (ER)-negative breast cancer is associated with shortened relapse-free survival (RFS), how associated GR transcriptional activity contributes to aggressive breast cancer behavior is not well understood. Using potent GR antagonists and primary tumor gene expression data, we sought to identify a tumor-relevant gene signature based on GR activity that would be more predictive than GR expression alone. Experimental Design: Global gene expression and GR ChIP-sequencing were performed to identify GR-regulated genes inhibited by two chemically distinct GR antagonists, mifepristone and CORT108297. Differentially expressed genes from MDA-MB-231 cells were cross-evaluated with significantly expressed genes in GR-high versus GR-low ER-negative primary breast cancers. The resulting subset of GR-targeted genes was analyzed in two independent ER-negative breast cancer cohorts to derive and then validate the GR activity signature (GRsig). Results: Gene expression pathway analysis of glucocorticoid-regulated genes (inhibited by GR antagonism) revealed cell survival and invasion functions. GR ChIP-seq analysis demonstrated that GR antagonists decreased GR chromatin association for a subset of genes. A GRsig that comprised n = 74 GR activation-associated genes (also reversed by GR antagonists) was derived from an adjuvant chemotherapy-treated Discovery cohort and found to predict probability of relapse in a separate Validation cohort (HR = 1.9; P = 0.012). Conclusions: The GRsig discovered herein identifies high-risk ER-negative/GR-positive breast cancers most likely to relapse despite administration of adjuvant chemotherapy. Because GR antagonism can reverse expression of these genes, we propose that addition of a GR antagonist to chemotherapy may improve outcome for these high-risk patients. Clin Cancer Res; 24(14); 3433–46. ©2018 AACR.

Increased glucocorticoid receptor (GR) expression and activity following androgen blockade can contribute to castration-resistant prostate cancer (CRPC) progression. Therefore, we hypothesized that GR antagonism will have therapeutic benefit in CRPC. However, the FDA-approved nonselective, steroidal GR antagonist, mifepristone, lacks GR specificity, reducing its therapeutic potential. Here, we report that two novel nonsteroidal and highly selective GR modulators (SGRM), CORT118335 and CORT108297, have the ability to block GR activity in prostate cancer and slow CRPC progression. In contrast to mifepristone, these novel SGRMs did not affect androgen receptor (AR) signaling, but potently inhibited GR transcriptional activity. Importantly, SGRMs decreased GR-mediated tumor cell viability following AR blockade. In vivo, SGRMs significantly inhibited CRPC progression in high GR–expressing, but not in low GR–expressing xenograft models. Transcriptome analysis following AR blockade and GR activation revealed that these SGRMs block GR-mediated proliferative gene expression pathways. Furthermore, GR-regulated proliferation-associated genes AKAP12, FKBP5, SGK1, CEBPD, and ZBTB16 are inhibited by CORT108297 treatment in vivo. Together, these data suggest that GR-selective nonsteroidal SGRMs potently inhibit GR activity and prostate cancer growth despite AR pathway inhibition, demonstrating the therapeutic potential of SGRMs in GR-expressing CRPC. Mol Cancer Ther; 16(8); 1680–92. ©2017 AACR.

The mission of the National Cancer Institute’s (NCI) Office of Cancer Genomics (OCG) is to advance the molecular understanding of cancers in order to improve clinical outcomes through precision medicine. Although vast amounts of genomic data are available for many types of cancers, identifying genetic alterations in rare and pediatric cancers is still a challenge. Efficient bioinformatics tools to analyze, manage, store, and access data are also necessary for the research community. To develop effective and targeted treatments, clinically accurate genotypic and phenotypic research models are much needed. OCG’s programs focus on addressing these challenges through multidisciplinary, collaborative research efforts. The four initiatives of OCG support research on structural, functional, and translational genomics, as well as development of next-generation cancer models. The Cancer Genome Characterization Initiative (CGCI) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) programs use transcriptomic, genomic, and epigenomic approaches to examine genetic alterations between various tumors and matched normal tissues. CGCI projects focus on HIV-associated and rare cancers such as Burkitt lymphoma, while TARGET focuses on high-risk childhood cancers. The goals of these programs are to attain insights into key mutations that drive tumors and genetic abnormalities specific to cancer subtypes and to develop effective and less toxic therapies for patients. CGCI and TARGET data are available to the research community through data matrices on the OCG website. OCG also recently launched the Pediatric Genomic Data Inventory (PGDI) as a new resource for investigators to access molecular characterization data.The Cancer Target Discovery and Development (CTD2) Network advances cancer research by bridging the knowledge gap between cancer genomics and precision oncology. The Network aims to understand the cancer metastasis, tumor heterogeneity, and drug resistance to develop optimal combinations of small-molecules or immunotherapy with small molecules. As a community resource program, the CTD2 Network develops and provides access to data, tools, methods, and reagents through the Data Portal and the Dashboard. The Human Cancer Models Initiative (HCMI) is an international consortium that is generating novel human tumor-derived culture models from a wide variety of cancer types including rare and understudied cancers. The models, together with related clinical and genomic data, will be available as a resource to the world-wide research community. OCG’s policies on data usage, as well as guides to accessing data, are explained on the OCG website (https://ocg.cancer.gov/). Researchers, potential collaborators, and interested members of the public are encouraged to visit the OCG webpages or contact OCG at ocg@mail.nih.gov. Citation Format: Cindy W. Kyi, Pamela C. Birriel, Tanja M. Davidsen, Martin L. Ferguson, Patee Gesuwan, Nicholas B. Griner, Yiwen He, Subhashini Jagu, Eva Tonsing-Carter, Daniela S. Gerhard. NCI Office of Cancer Genomics: Promoting multidisciplinary research to translate findings into the clinic and advance precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4341.

Background: Despite the recent discovery of several potent androgen receptor (AR)-targeted therapies, progressive castration-resistant prostate cancer (CRPC) remains the second leading cause of male cancer deaths in the United States. While androgen-deprivation therapy (ADT) and AR antagonism continue to be the standard of care for progressive metastatic CRPC, identifying alternative molecular mechanisms is crucial. Our lab and others have shown that increased glucocorticoid receptor (GR) expression and activity can contribute to CRPC progression; this discovery has led to the development of selective GR modulators (SGRMs) as potential therapies for CRPC. Although GR clearly contributes to cell growth in AR-blocked PC cells, it is unclear whether GR serves solely to recapitulate AR-driven cancer-related mechanisms or if GR also regulates critical gene expression pathways unique to GR (and different from AR) allowing a novel growth advantage for tumor cells. Methods: Primary PC and metastatic CRPC samples were normalized and utilized for gene expression analysis. LAPC4 and CWR-22Rv1 PC cell lines were treated with androgen +/- enzalutamide +/- dexamethasone and +/- SGRMs in order to activate or inhibit GR function in both AR active and AR-blocked conditions. For transcriptome analysis we performed next generation sequencing (NGS) of RNA followed by gene expression pathway analysis. To determine GR and AR chromatin binding sites, we performed GR and AR ChIP followed by DNA NGS. Results: Transcriptome analysis of GR-mediated gene expression in LAPC4 PC cells following AR antagonism revealed that the majority of GR-regulated genes were unique to GR activity rather than overlapping with AR target genes.. Furthermore, GR and AR ChIP-sequencing indicated that while important overlap existed between GR and AR ChIP-seq, the majority of GR binding sites were distinct from AR binding sites. Analysis of the GR-unique transcriptomes of both enzalutamide-treated LAPC4 and CWR-22Rv1 cells predicted that cAMP-mediated signaling was activated by GR. Antagonism of GR with SGRMs blocked GR-unique cAMP pathway gene expression. Gene expression analysis of patient samples indicated that GR signaling is also activated in mCRPC and correlated with increased cAMP pathway gene expression. Conclusions: These data suggest that GR activity in progressive CRPC results in novel GR-specific mechanisms of CRPC progression involving signaling pathways distinct from those driven by AR activation. Citation Format: Tiha M. Long, Eva Y. Tonsing-Carter, Wen-Ching Chan, Donald Vander Griend, Suzanne D. Conzen, Russell Z. Szmulewitz. Glucocorticoid receptor (GR)-mediated activation of cyclic-adenosine monophosphate (cAMP) pathway gene expression following androgen receptor (AR) antagonism of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 944.

Background: A recent effort to distinguish early-stage TNBC based on outcome and to expose driver pathways using gene profiling/cluster analysis divided TNBC into four subtypes- basal-like, mesenchymal, immunomodulatory, and luminal androgen receptor (LAR). The clinical AR+ TNBC subtype (>10% AR by IHC) comprises up to 35% of metastatic disease. The discovery of this TNBC subtype and the concomitant development of potent new anti-androgens for prostate cancer have led to several clinical trials evaluating AR antagonists in TNBC. In parallel, glucocorticoid receptor (GR) expression and activation have been shown to mediate anti-apoptotic gene expression in TNBC. However, to our knowledge, no one has examined the coordinate expression of GR and AR activity and the potential crosstalk downstream of dual GR and AR activation. We hypothesized that AR/GR cooperative gene expression pathways may induce TNBC therapy resistance and tumor progression. Methods and Results: We analyzed GR+ TNBC primary tumor gene expression from TCGA samples and found a wide range of AR expression. To examine whether GR-associated gene expression differed in high versus low AR+ TNBC, we divided TCGA TNBC primary tumors (N=123) at the AR median into “high” versus “low” AR expression. We then identified relative GR target gene expression levels in those genes identified from a previous TNBC meta-analysis. Invasion, motility and proliferation pathways were found to be most significantly expressed among GR-associated genes in AR high TNBC (top 5 significant pathways). Similarly, we found that GR and AR co-activation in AR+ MDA-MB-453 TNBC cells significantly increased cell migration compared to GR or AR activation alone. In addition, when MDA-MB-453 cells were exposed to the AR antagonist enzalutamide (enza) for more than 30 days (long-term, LT), the magnitude of increase in cell migration following GR activation increased, suggesting that increased GR activity may compensate for long-term AR blockade. Furthermore, long-term enza exposure (LTenza) in AR+ MFM-223 and MDA-MB-453 cells resulted in increased GR mRNA and protein levels. The magnitude of GR-mediated target gene expression was also significantly increased in LTEnza versus control-treated cells. In vivo, LTenza-treated MDA-MB-453 xenografts were relatively insensitive to paclitaxel chemotherapy treatment, consistent with increased GR activity compared to control cells. Conclusions: We conclude that coordinate GR and AR expression in TNBC contributes to a particularly poor outcome in TNBC. Downstream AR/GR gene expression favoring cell survival and invasive phenotypes may contribute to this outcome. Consideration of dual AR/GR inhibition in TNBC may increase the effectiveness of anti-androgen therapy. Citation Format: Deniz N. Dolcen, Eva Tonsing-Carter, Ryan Harkless, Caroline Kim, Kathleen Bowie, Gini Fleming, Suzanne Conzen. Combined androgen and glucocorticoid receptor (AR/GR) activity drives TNBC progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2541.

An elusive goal in the treatment of patients with glioblastoma multiforme (GBM) is the development of therapeutic strategies that prevent emergence of therapy-resistant tumor cells. Our laboratory has identified a novel combination therapy that targets the Akt-Mdm2 signaling network in the context of genotoxic temozolomide (TMZ) therapy. Inhibitors were specifically selected based on structural properties related to blood-brain barrier permeability, and pharmacokinetic profiles of plasma, normal brain, flank/ectopic brain tumors, and intracranial tumors. Isobolograms and combination indices indicated that Akt (GDC0068) and Mdm2 (RG7112) inhibitors either together or in combination with TMZ, resulted in synergistic inhibition of cell growth in both mtp53 and wt53 GBM primary patient cell lines (GBM10, GBM43, and MHBT32). When RG7112 and GDC0068 (at a ratio of 0.5:1) were combined with TMZ, strong synergism was evident at multiple dose-ratios. As downstream measures of blocking Akt and Mdm2, activation of the Forkhead box O-class 1/3a (FoxO) transcription factors and increased p53/p73 proteins were evident following TMZ + RG7112 + GDC0068. For in vivo studies, an intermittent dosing regimen of TMZ + RG7112 + GDC0068 was developed to avoid normal tissue toxicity. Mice with TMZ-resistant GBM43 ectopic tumors were treated 3 × weekly for 3 weeks. Combination of TMZ + RG7112 + GDC0068 significantly delayed tumor progression compared to other treatments (n = 8-9 per group, p < 0.05). TMZ + GDC0068 and TMZ + RG7112 + GDC0068 significantly reduced tumor volume relative to TMZ, and on the last day with surviving TMZ + GDC0068-treated mice (2500 mm3 endpoint), tumor volumes were significantly reduced in TMZ + RG7112 + GDC0068 compared to TMZ + GDC0068 (p < 0.05). In mice with GBM43 intracranial tumors, survival was significantly increased by GDC0068 + RG7112 (13 days), and further increased by TMZ + RG7112 + GDC0068 (24 days) compared to TMZ alone. These data suggest that blocking the Mdm2-Akt signaling network in the context of TMZ-mediated DNA damage holds therapeutic promise. Studies to identify mechanism and appropriate biomarkers of response are in progress.

Hematopoietic stem cells (HSCs) are a rare population of pluripotent cells that predominantly reside in the bone marrow. Under the appropriate microenvironmental cues, HSCs can undergo self-renewal, expansion, and differentiation into all types of progenitor and terminally differentiated blood cells required for survival of the host (Figure 1). Due to the importance of this cell population for survival, protection of its genome from endogenous and exogenous genotoxic insults is a necessity. However, the intracellular molecular signaling network in hematopoietic cells that control surveillance of the genome as well as maintain genome stability is still largely unexplored. As more is learned regarding how these cells detect a genotoxic event and seek to repair the damaged nucleotides (i.e. DNA adducts), it will become even more feasible to design strategies to protect these life-sustaining cells when the host is exposed to a genotoxic event. Maintenance of genome stability in both the hematopoietic stem and progenitor cell (HSPC) populations is essential for the sustainment of normal hematopoiesis. For example, transient depletion of bone-marrow derived HSC induced by irradiation or chemotherapy can induce these primitive cells to expand so that the bone marrow can be fully reconstituted; blood-cell development can then continue with minimal disruption. However, once therapy-mediated DNA damage is too high, a DNA-damage threshold is reached resulting in subsequent cell death, myelosuppression, and if not treated, life-threatening bone-marrow failure (Figure 1). With the basal level of DNA repair relatively low in these cells, this does present a challenge to maintain normal hematopoiesis in individuals exposed to prolonged or high levels of genotoxic stress. The reduced ability to repair DNA damage in HSPCs that give rise to multiple mature blood-cell lineages can cause detrimental and long-lasting effects to the host resulting in abnormal cell function, cell death, cellular transformation, and eventually leukemogenesis (Figure 1). Numerous studies have shown that HSPCs are intrinsically more sensitive than other cell types and tissues mostly due to intrinsic limitations in DNA-repair capacity. Buschfort-Papewalis et al previously demonstrated that when human HSPCs (phenotypically defined as CD34+ cells) or differentiated cells (phenotypically defined as CD34cells) from the same donor were exposed to alkylating agents, an overall decrease in repair capacity of the more primitive CD34+ cells compared the more differentiated cells CD34cells was observed. When human CD34+ cells were exposed to a variety of chemotherapeutic drugs, single-strand DNA breaks as well as DNA adducts were found at higher levels and persisted for longer time periods than in CD34cells (Buschfort-Papewalis et al., 2002), providing evidence that the kinetics of DNA repair are slower overall in the

A multi-targeted approach will be necessary to eradicate glioblastoma multiforme (GBM) cells due to the immense genetic heterogeneity associated with GBM. Mouse double minute-2 (MDM2) regulates multiple signaling pathways and is a promising therapeutic target in GBM. In wild type (wt) p53 cells, MDM2 binds to wtp53, ubiquitinates it, and negatively regulates p53-mediated downstream events. In wtp53 and mutant (mt) p53 cells, MDM2 binds to and sequesters p73α thereby blocking p73α-mediated signaling. Our objective in the present studies was to determine if the p73α-MDM2 axis could be exploited to increase death of mtp53 GBM cells. We utilized MDM2 antagonists nutlin3a or RG7112 to block protein-protein interactions between MDM2-p53 and MDM2-p73α. In a panel of GBM cell lines, TMZ resistance was reduced in both wt53 and mt53 cells in the presence of MDM2 antagonists. In mtp53 cells, siRNA knockdown of p73α indicated that sensitivity to treatment was dependent on p73α levels. Isobologram analysis indicated that while dose-ratios of TMZ to MDM2 antagonists were additive to synergistic in inhibiting growth of wtp53 GBM cells, this was not the case in mtp53 GBM cells (SF118, GBM43, gain-of-function-mtp53 R273H U373 and MHBT32). Analysis of intracellular targets in mtp53 GBM cells exposed to TMZ and MDM2 antagonists indicated that p73α and MDM2 expression increased by 24 hours post-treatment. In addition, AKT activity was increased or sustained in mtp53 GBM cells following treatment with TMZ in the absence or presence of MDM2 antagonists. Since increased AKT activity may render cells resistant to therapy, the AKT inhibitor GDC0068 was evaluated in combination with TMZ and RG7112. As a measure of AKT-downstream target modulation, phosphorylation status of the Forkhead box O-class (FoxO) transcription factors (TFs) was determined. In the non-phosphorylated state, FoxO TFs upregulate expression of proteins involved in cell-death pathways. While phospho-FoxO1/FoxO3a TFs were increased in TMZ/RG7112-treated mtp53 GBM cells compared to controls, it was decreased in GDC0068-, TMZ/GDC0068- and TMZ/RG7112/GDC0068-treated mtp53 GBM cells which is consistent with inactivation of AKT and activation of FoxO TFs. Isobologram analysis of mtp53 GBM cell growth indicated that combination RG7112 and GDC0068 inhibited growth in a synergistic manner even in the absence of TMZ. For in vivo studies, an intermittent dosing regimen of TMZ/RG7112/GDC0068 was developed to avoid normal tissue toxicity. GBM43 flank tumor growth was significantly inhibited in mice with tumors treated with RG7112/GDC0068 and inhibited to a larger extent by the triple combination TMZ/RG7112/GDC0068 compared to vehicle and single-agent exposure (n=9-10 mice per group; single agent vs GDC0068/RG7112 or TMZ/RG7112/GDC0068, p Citation Format: Mohammad Reza Saadatzadeh, Haiyan Wang, Jixin Ding, Barbara J. Bailey, Eva Tonsing-Carter, Shanbao Cai, Nimita Dave, Harlan E. Shannon, Aaron Cohen- Gadol, Karen E. Pollok. Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A26.

Breast cancer will affect 1 in 8 women in their lifetime. Approximately 20-30% of all breast cancer cases become metastatic. This study was designed to evaluate the effect of tumor met knockdown with individual and combination drug therapy. In this mouse model, with 3 per group, we used the following chemotherapeutic agents: a vehicle control, carboplatin (20mg/kg i.p.), Nutlin-3a (200mg/kg p.o.) and a combination group with the same doses. Nutlin-3 is a small-molecule inhibitor that binds to the p53-binding pocket of MDM2, and activates the p53 pathway. This has been shown to be effective in mouse models of cancer that have a wild-type p53, including prostate, leukemia and multiple myeloma. The lungs were removed and examined for metastatic lung lesions, fixed in 10% neutral buffered formalin (NBF), processed into a paraffin block, microtomed, and slides were stained with H&E. Ki67 immunostain was performed to clearly define the met lesions and the whole slide was imaged with an Aperio Digital Imaging System. The Image Scope positive pixel algorithm was used to quantitate the met lesions in the lungs. The data revealed upon drug treatment the number of mets in the lungs were unchanged in the carboplatin and nutlin-3a individual treatment groups compared to the vehicle group. The lungs in the combination group had the lowest number of tumors with both visual and quantitative image analysis compared to the vehicle control group. The Aperio automated positive pixel count for Ki67 immunostain was consistent with the H&E pathology read. The values decreased with combination treatment (control vs. combination 20mg/kg carboplatin, 200mg/kg nutlin 3a) and were 6% and 1.7% respectively. It was concluded that mice treated with carboplatin and Nutlin-3a combination therapy had fewer metastatic breast cancer mets in the lungs. The individual drug doses were low compared to previous studies and did not have an effect as was expected. This finding suggests that the combination of a traditional chemotherapy with a small protein inhibitor could inhibit the spread of metastatic breast cancer. Citation Format: Margaret Strack, Eva Tonsing-Carter, Tony Sinn, Tiaishia Spragin, Kacie Peterson, Barbara Bailey, Karen Pollok, George Sandusky. Histologic and whole slide quantitative image analysis of lung metastases in a mouse breast cancer model using carboplatin and Nutlin 3A alone and in combination. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5196. doi:10.1158/1538-7445.AM2015-5196

Metastatic breast cancer is highly refractive to current treatment strategies, and new multi-targeted treatments need to be elucidated. In metastatic disease, inhibiting key protein-protein interactions with the murine double minute 2 (MDM2) could be beneficial for developing new treatment modalities since this signaling pathway is a critical regulatory point in cancer progression. Inhibition of protein binding to the hydrophobic pocket of MDM2 by Nutlin-3a can activate pro-apoptotic proteins such p73 and E2F1 as well as decrease pro-angiogenic Hif-1α. Since the DNA damaging agent carboplatin is currently being studied in clinical trials of triple-negative breast cancers (TNBCs), our objective was to evaluate the effects of carboplatin and Nutlin-3a in combination in TNBC in a mutant p53 background. Using a TNBC cell line TMD231 derived from the MDA-MB-231 human breast cancer cell line, we performed combination studies using different ratios of carboplatin to Nutlin-3a in vitro to evaluate the range of carboplatin-mediated DNA damage required to obtain synergism with inhibition of MDM2 function. A fixed ratio of 1:1 carboplatin:Nutlin-3a was strongly synergistic with a combination index of Citation Format: Eva Tonsing-Carter, Harlan E. Shannon, Barbara J. Bailey, Anthony L. Sinn, Kacie M. Peterman, Lindsey D. Mayo, Karen E. Pollok. Modulation of MDM2 in context of DNA damage enhances cell death in a metastatic breast-to-lung xenograft model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1680. doi:10.1158/1538-7445.AM2014-1680

Digital whole slide imaging is the technique of digitizing a microscope slide at the highest resolution to produce a “digital virtual microscope slide”. This digital image can be viewed in three or four fields, from low to high power fields, which can be commonly used to evaluate the tissue. Many of these systems have a whole slide software image analysis system. The goal of this study was to determine if the Aperio positive pixel algorithm (image analysis) could effectively quantitate metastatic mouse lung tumors in a lung section using a H&E stain. Lung sections from a mouse lung metastasis model with 8 mice in each group were evaluated: control, 50mg/kg, and 75mg/kg carboplatin. H&E and Ki67 immunostain slides were scanned using the Aperio whole slide scanning system (Scanscope CS). A single field of view from each slide representing a whole lung lobe with multiple lung metastases was selected for image analysis. The standard positive pixel algorithm was altered to read the H&E slides. Various histology slides were used to validate the altered algorithm. The immunostain (Ki67) was generated using the standard positive pixel algorithm analysis. The Aperio automated positive pixel count for a Ki67 immunostain was consistent with the H&E image analysis. The values went down with a dose dependent treatment (control vs. 50mg/kg, and 75mg/kg Carboplatin) and were (H&E) 37%, 28%, 22%, and (Ki67) 9%, 5%, 3%, respectively. The analysis had decreasing values for both the H&E and Ki67 analysis on a dose dependent drug treatment. The metastases decreased in both treatment groups compared to controls with both the H&E and Ki67 analyses. The Aperio Image Analysis positive pixel algorithm allows large areas of the lung tissue section to be examined and NOT just a single 25x or 40x field like many common image analyses. Citation Format: George E. Sandusky, Ronne Surface, Eva Tonsing-Carter, jayne Silver, Tony Sinn, Karen Pollok. Whole slide image analysis quantification in a mouse lung metastasis model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4309. doi:10.1158/1538-7445.AM2014-4309

Metastatic breast cancer is highly refractive to current treatment strategies and new multi-targeted treatments need to be elucidated. In metastatic disease, blocking key protein-protein interactions with the murine double minute 2 (MDM2) could be beneficial for developing new treatment modalities since this signaling pathway is a critical regulatory point in cancer progression. Inhibition of protein binding to the hydrophobic pocket of MDM2 by Nutlin-3a can result in increases in pro-apoptotic proteins such as p53, p73, and E2F1. Since the DNA damaging agent carboplatin is being studied in clinical trials of triple-negative breast cancers (TNBCs), our objective was to evaluate the effects of carboplatin and Nutlin-3a in combination in TNBC cancer in a mutant p53 background. We utilized TMD231 breast cancer cells derived from the MDA-MB-231 human TNBC cancer line. TMD231 cells are highly metastatic in vivo and readily metastasize from the primary tumor to the lung. Combination studies were performed using different ratios of carboplatin to Nutlin-3a to evaluate the range of carboplatin-mediated DNA damage required to obtain synergism with inhibition of MDM2 function. A fixed ratio of 1:1 carboplatin:Nutlin-3a was strongly synergistic with a combination index of < 0.5. In cell proliferation assays there was increased sensitivity to the drugs when given together and a significant reduction in number of colonies in colony formation assays. In order to understand how DNA damage and inhibition of MDM2 signaling leads to enhanced cell death, dose-response and time-course studies were performed. Western analyses demonstrated increases in E2F1 when treated with carboplatin and Nutlin-3a alone, and a time-dependent increase in E2F1 following combination treatment at a fixed 1:1 ratio. Following DNA damage, E2F1 has been shown to promote the transcription of pro-apoptotic genes and repression of survival genes once E2F1 is released from interaction with Rb. Here, E2F1 levels correlated with Rb phosphorylation at serine 780, a phosphorylation site which inhibits binding of E2F1 to Rb. The E3 ubiquitin ligase Itch, which ubiquitinates p73, increased following treatment with carboplatin alone or in combination with Nutlin-3a in a dose- and time-dependent manner but appeared not to regulate p73 since p73 levels remained unchanged following combination treatment. Studies are ongoing to further understand how modulation of DNA damage by blockade of MDM2 results in a robust synergistic response and how upregulation of E2F1 affects downstream targets. The impact of the combination of DNA damaging carboplatin and MDM2 blockade by Nutlin-3a on DNA repair kinetics is currently being evaluated in an orthotopic TMD231 model. These studies will lead to a better understanding of how to potentiate DNA damage and may lead to new clinical therapies in the future. Citation Format: Eva Tonsing-Carter, Harlan E. Shannon, Barbara J. Bailey, Lindsey D. Mayo, Karen E. Pollok. Blockade of MDM2-mediated signaling in context of DNA damage increases E2F1 expression and enhances cell death in triple-negative breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4639. doi:10.1158/1538-7445.AM2013-4639

Orthotopic mouse models of human disease are absolutely essential for validating therapeutic targets and assessing efficacy of emerging therapies. Early detection of in vivo tumor growth is a critical benchmark in humanized mouse models, for it reveals tumor take at a time of reasonable tumor burden. To develop an optimal mouse model of human metastatic breast cancer, we utilized TMD231 breast cancer cells (derived from MDA-MB-231) that have a high propensity to metastasize to the lung. TMD231 tumor take and improved kinetics of tumor growth and metastatic foci formation appeared more consistently and at a faster rate in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) versus NOD/SCID mice. For in vivo detection of small tumor burden at study initiation and metastatic lesions over time, TMD231 breast cancer cells were transduced with lentiviral vectors that express either mCherry or E2-Crimson fluorescent proteins (FPs). Flow cytometric analysis indicated transduction efficiencies for the lentiviral vectors expressing mCherry and E2-Crimson in the TMD231 cells was >99% and >80%, respectively. TMD231 cells expressing either the mCherry or E2-Crimson FPs were implanted into NSG mice and were imaged weekly using the Optix MX3 (ART Technologies, Montreal, Canada) to assess to what degree fluorescently tagged cells could be longitudinally and non-invasively visualized at primary and secondary sites within the NSG mice. While photon emission from TMD231 tumors expressing mCherry could be detected, levels were low and did not correlate with tumor growth overtime. In contrast, high levels of photon emission were detected using TMD231-E2-Crimson cells. Non-palpable tumors expressing the E2-Crimson FP could be detected in NSG mice as early as 7 days post-implant into the mammary fat pad. Additionally, metastatic foci in the lung were detected via optical imaging as early as 2-3 weeks post-implant. Following imaging analysis, TMD231-E2-Crimson tumor size correlated to increased fluorescent intensity, tumor depth, and fluorescent concentration longitudinally. In vitro imaging analysis of TMD231-E2-Crimson cells confirmed the in vivo imaging results showing a linear relationship between cell number and fluorescent intensity. The discrepancy in the in vivo detection of mCherry versus E2-Crimson proteins is most likely due to the greater brightness of E2-Crimson, as well as the closer alignment of the excitation/emission spectra of E2-Crimson with the laser/filter set of the Optix MX3 imaging system. To confirm optical imaging results, H&E staining indicated the presence of numerous foci in the lungs post-mortem. The optimized orthotopic model described here is being used to investigate mechanisms of metastasis as well as novel therapeutic strategies in metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1409. doi:1538-7445.AM2012-1409