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Although immune-checkpoint inhibitors (ICIs) have been a remarkable advancement in bladder cancer treatment, the response rate to single-agent ICIs remains suboptimal. There has been substantial interest in the use of epigenetic agents to enhance ICI efficacy, although precisely how these agents potentiate ICI response has not been fully elucidated. We identified entinostat, a selective HDAC1/3 inhibitor, as a potent antitumor agent in our immune-competent bladder cancer mouse models (BBN963 and BBN966). We demonstrate that entinostat selectively promoted immune editing of tumor neoantigens, effectively remodeling the tumor immune microenvironment, resulting in a robust antitumor response that was cell autonomous, dependent upon antigen presentation, and associated with increased numbers of neoantigen-specific T cells. Finally, combination treatment with anti-PD-1 and entinostat led to complete responses and conferred long-term immunologic memory. Our work defines a tumor cell-autonomous mechanism of action for entinostat and a strong preclinical rationale for the combined use of entinostat and PD-1 blockade in bladder cancer.

BACKGROUND Historically, African American (AA) patients with renal cell carcinoma (RCC) have had inferior survival compared with Caucasian patients. Recent studies suggest that the survival disparity between races may be worsening since the advent of targeted therapies for RCC. In this study, survival rates among AA and Caucasian patients with advanced RCC are examined over time to determine whether a disparity in survival persists in the targeted therapy era. METHODS The authors identified patients with stage IV RCC in the National Cancer Data Base and compared survival between AA and Caucasian patients during the periods before (1998-2004) and after (2006-2011) the advent of targeted therapy. RESULTS In total, 48,846 patients were identified, and 10% were AA. Three-year survival among both AA and Caucasian patients improved between the 2 periods (P < .01 for both), with no interaction observed between race and improved survival over time (P = .15). The adjusted hazard ratio (HR) for death among AAs compared with Caucasians was 1.13 (95% confidence interval, 1.08-1.19) in the post-targeted therapy era, which was unchanged from the pretargeted therapy era (adjusted HR, 1.10; 95% confidence interval, 1.04-1.15). The adjusted HR was similar when the analysis was restricted to those who received systemic therapy. CONCLUSIONS Both AA and Caucasian patients with advanced RCC have had a significant improvement in survival since the advent of targeted therapy. However, AA patients maintain a survival disadvantage compared with Caucasians independent of treatment received, potentially related to unmeasured comorbidities, disease burden, or tumor biology. Cancer 2016;122:2988-2995. © 2016 American Cancer Society.

Bladder cancer harbors a high frequency of somatic mutations that occur randomly across the genome. A fraction of these mutations can be expressed and processed into peptides called neoantigens. When presented on the surface of tumor cells by the major histocompatibility complexes (MHC), these neoantigens can invoke an adaptive immune response. In general, a high neoantigen burden is associated with a significant increase in overall survival. However, tumor cells can avoid the detection of immune cells by silencing the expression of the neoantigens. The post-translational acetylation of histone tails is catalyzed by histone acetyltransferases (HATs) and is counterbalanced by histone deacetylases (HDACs), which mediate their removal. Histone acetylation is associated with open chromatin and active transcription. HDAC inhibition would, therefore, promote histone acetylation and restore the expression of suppressed genes. Many of the frequently mutated genes in bladder cancer are involved in chromatin remodeling, such as KMT2D, KDM6A, and KMT2C, suggesting the contribution of epigenetic dysregulation to disease progression. Therefore, therapy that targets epigenetic processes such as HDAC inhibition is a rational treatment approach for bladder cancer. Here, we evaluated the antitumor efficacy and mechanisms of entinostat, a selective HDAC1 and HDAC3 inhibitor, in a preclinical murine model of high-grade muscle-invasive bladder cancer, BBN963. In this study, BBN963 cells were implanted subcutaneously into immunocompetent C57BL/6 (B6) mice or immunodeficient NOD/scid/gamma (NSG) mice. Entinostat exhibited robust in vivo activity in B6 mice implanted with BBN963 cells with a number of complete responses. However, the effect was much weaker in BBN963 cells grown in NSG mice. RNAseq analysis demonstrated increased immune gene signature expression in entinostat treated tumors from B6 (but not NSG) mice. Flow cytometry verified the increased numbers of CD8+ effector memory T cells. Most interestingly, the expressions of predicted neoantigens were almost completely lost in the entinostat-treated tumors in B6 mice but were unchanged in tumors from NSG mice. Additionally, the in vivo antitumor effect of entinostat in the BBN963 cells lacking MHC class 1 was significantly reduced. Finally, the combination of anti-PD1 therapy and entinostat robustly out-performed each agent alone in inhibiting BBN963 tumor growth with 67% incident of complete response. These data support the hypothesis that entinostat mediates antitumor effect through enhanced neoantigen expression and antigen-driven immune response. Citation Format: Andrew S. Truong, Mi Zhou, Takanobu Utsumi, Bhavani Krishnan, Kyle G. Stewart, Ryoichi Saito, Ujjawal Manocha, Sean T. Bailey, William Y. Kim. Entinostat enhances antitumor immune responses and cooperates with PD-1 blockade in bladder cancer mouse models [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A13.

We report the discovery of a claudin-low molecular subtype of high-grade bladder cancer that shares characteristics with the homonymous subtype of breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features including increased rates of RB1, EP300, and NCOR1 mutations; increased frequency of EGFR amplification; decreased rates of FGFR3, ELF3, and KDM6A mutations; and decreased frequency of PPARG amplification. While claudin-low tumors showed the highest expression of immune gene signatures, they also demonstrated gene expression patterns consistent with those observed in active immunosuppression. This did not appear to be due to differences in predicted neoantigen burden, but rather was associated with broad upregulation of cytokine and chemokine levels from low PPARG activity, allowing unopposed NFKB activity. Taken together, these results define a molecular subtype of bladder cancer with distinct molecular features and an immunologic profile that would, in theory, be primed for immunotherapeutic response.

SUMMARY Renal cell carcinoma (RCC) is not a single disease, but several histologically defined cancers with different genetic drivers, clinical courses, and therapeutic responses. The current study evaluated 843 RCC from the three major histologic subtypes, including 488 clear cell RCC, 274 papillary RCC, and 81 chromophobe RCC. Comprehensive genomic and phenotypic analysis of the RCC subtypes reveals distinctive features of each subtype that provide the foundation for the development of subtype-specific therapeutic and management strategies for patients affected with these cancers. Somatic alteration of BAP1, PBRM1, and PTEN and altered metabolic pathways correlated with subtype-specific decreased survival, while CDKN2A alteration, increased DNA hypermethylation, and increases in the immune-related Th2 gene expression signature correlated with decreased survival within all major histologic subtypes. CIMP-RCC demonstrated an increased immune signature, and a uniform and distinct metabolic expression pattern identified a subset of metabolically divergent (MD) ChRCC that associated with extremely poor survival., Graphical abstract In Brief Ricketts et al. find distinctive features of each RCC subtype, providing the foundation for development of subtypespecific therapeutic and management strategies. Somatic alteration of BAP1, PBRM1, and metabolic pathways correlates with subtype-specific decreased survival, while CDKN2A alteration, DNA hypermethylation, and Th2 immune signature correlate with decreased survival within all subtypes.

e20568 Background: Many targeted therapy clinical trials require a somatic gene mutation/alteration for eligibility. We assessed the feasibility of leveraging Real-World Data (RWD) to enrol NSCLC patients into clinical trials. Methods: US insurance claims data were extracted to identify lung cancer patients. These data were matched with EMR data also containing NSCLC patients’ details regarding the occurrence and results of molecular testing for EGFR, ALK, ROS1, JAK2, HER2 and RET somatic alterations, achieving a level of granular detail beyond that available in each individual dataset. A one-year extraction period was applied, with no gender or age restrictions. Results: Results for the matched dataset are summarised in the table below - the overall patient record match was 89.6%. Conclusions: The observed prevalence correlated reasonably well with literature reported prevalence for the molecular biomarkers associated commercially available targeted therapies in NSCLC (EGFR, ALK, ROS1). The sample size for the remaining biomarkers was too small to draw conclusions, though the presence of data correlating to these is of interest, considering that there are no currently approved targeted therapies in NSCLC tailored to these predictive biomarkers. This approach could be expanded upon to recruit patients into targeted therapy clinical trials as the dataset is fully linkable to sites and investigators. With the emergence of broad genomic profiling, the availability of molecular data to support clinical trial enrolment is also expected to grow.[Table: see text]

e19511 Background: The global burden of multiple myeloma (MM) has increased steadily in last 3 decades. The IARC reports there were ~160,000 new cases worldwide in 2018. There has been an increase in the development and approval of more effective targeted therapy options (new immunomodulatory agents, proteasome inhibitors, and monoclonal antibodies); this, coupled with high adoption of these therapies presents a major challenge in enrollment of current ongoing clinical trials. We assessed the impact of near-term regulatory approvals on clinical trial enrollment. Methods: Public domain clinical trial enrollment data from MM studies (Phase I, I/II, II) closed/completed between 2011-2018 were used to determine enrollment trends pre and post 2014. We leveraged real-world medical claims data to project/model adoption of recently approved drugs in the U.S.; additionally, drug sales volume data was used to evaluate ex-US national adoption trends. The utilization rates of 5 recently approved drugs by regimen and line of treatment was determined. Results: In the U.S., there is a 13% increase in median enrollment duration with a corresponding 25% decrease in median p/s/m enrollment in MM studies, irrespective of phase, where enrollment was completed between 2015 -2018 compared to 2011-2014. We hypothesize that one of the factors for this increase in enrollment timeline is the approval and adoption of newly approved therapies post 2014. Two of the five recently approved drugs show a steady increase in the number of patients treated over 2016, 2017 and 2018, while one of the drugs plateaus over the same period. Outside of the U.S., our analysis confirms the existence of gaps in time to approval /adoption of recently approved drugs; for example, we observe a two-year delay in approval/adoption for one of the drugs in France compared to the other countries in Western Europe. Conclusions: There are over 10 investigational MM drugs currently in development which are anticipated to be granted approval between 2019-2021. Factoring the real-world adoption of near-term drug approval into global clinical and operational strategies offers insights into mitigating potential future enrollment challenges.

Importance There are well-documented racial disparities in outcomes for African American patients with clear cell renal cell carcinoma (ccRCC). Despite a dramatic improvement in overall survival in white patients since the advent of targeted therapy, survival for African Americans with advanced ccRCC has not changed. There is little known about potential racial differences in tumor biology of ccRCC. Objective To determine if there are racial differences in the somatic mutation rate and gene expression of ccRCC tumors from white and African American patients. Design, Setting, and Participants Overall, 438 patients with ccRCC were identified through The Cancer Genome Atlas (TCGA) clear cell kidney (KIRC) dataset (419 white and 19 African American patients). The GSE25540 dataset containing 135 patients (125 white and 10 African American patients) was used for validation. Tumor samples were collected from numerous cancer centers and were examined for racial differences in somatic mutation rates and RNA expression. Racial differences in somatic mutation rates and RNA expression were examined. Main Outcomes and Measures The comparison of somatic mutation rates and differences in RNA expression in white and African American patients with ccRCC. Results Overall, 419 ccRCC tumor data sets from non-Hispanic white patients and 19 from non-Hispanic African American patients were identified through the publically available TCGA KIRC data set, and a validation set of 125 white and 10 African American ccRCC patient tumors was identified from the publicly available GSE25540 data set. African American patients were significantly less likely than white patients to have VHL mutations (2 of 12 [17%] vs 175 of 351 [50%], respectively; P = .04) and were enriched in the ccB molecular subtype (79% in African American vs 45% in white patients ; P = .005), a molecular subtype that carries a worse prognosis. It was found that RNA expression analysis revealed relative down-regulation of hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF)-associated pathways in African American patients compared with white patients. Conclusions and Relevance African American patients have less frequent VHL inactivation, are enriched in the ccB molecular subtype, and have decreased up-regulation of HIF-associated gene signatures than white patients. These genomic differences would predict decreased responsiveness to VEGF-targeted therapy and are a biologically plausible contributing factor to the worse survival of African American patients with ccRCC, even in the targeted therapy era.

BACKGROUND Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, heptapeptide hormone with anti-proliferative and anti-angiogenic properties. The primary objective of this study was to determine whether Ang-(1-7) effectively reduces prostate cancer metastasis in mice. METHODS Human PC3 prostate cancer cells were injected into the aortic arch via the carotid artery of SCID mice pre-treated with Ang-(1-7) or injected into the tibia of athymic mice, administered Ang-(1-7) for 5 weeks beginning 2 weeks post-injection. Tumor growth and volume were determined by bioluminescent and magnetic resonance imaging. The presence of tumors was confirmed by hematoxylin and eosin staining; TRAP histochemistry was used to identify osteolytic lesions. The effect of Ang-(1-7) on osteoclastogenesis was assessed in differentiated bone marrow cells. RESULTS Pre-treatment with Ang-(1-7) prevented metastatic tumor formation following intra-aortic injection of PC3 cells, while 83% of untreated mice developed tumors in metastatic sites. Circulating VEGF was significantly higher in control mice compared to mice administered Ang-(1-7). A 5-week regimen of the heptapeptide hormone attenuated intra-tibial tumor growth; Ang-(1-7) was significantly higher in the tibia of treated mice than in control animals. Osteoclastogenesis was reduced by 50% in bone marrow cells differentiated in the presence of Ang-(1-7), suggesting that the heptapeptide hormone prevents the formation of osteolytic lesions to reduce tumor survival in the bone microenvironment. CONCLUSIONS These findings suggest that Ang-(1-7) may serve as an anti-angiogenic and anti-metastatic agent for advanced prostate cancer. By extension, the heptapeptide hormone may provide effective therapy for bone metastasis produced from primary tumors of the lung and breast. Prostate 73: 71–82, 2013. © 2012 Wiley Periodicals, Inc.

This paper explores the feasibility of and challenges in developing methods for black-box monitoring of the power usage of a virtual machine (VM) at run-time, on shared virtualized compute platforms, including those with complex memory hierarchies. We demonstrate that VM-level power utilization can be accurately estimated, or estimated with accuracy with bound error margins. The use of bounds permits more lightweight online monitoring of fewer events, while relaxing the fidelity of the estimates in a controlled manner. Our methodology is evaluated on the Intel Core i7 and Core2 x86-64 platforms, running synthetic and SPEC benchmarks.

Aberrant chromatin remodeling by epigenetic modifier proteins such as histone deacetylases (HDACs) is common within many types of cancer, including muscle-invasive bladder cancer (MIBC). The removal of acetyl groups from the lysine residues of histones leads to transcriptional silencing that promotes tumor growth and potentially enhances cancer cells' ability to evade the host immune response. Therefore, HDACs present themselves as attractive targets for cancer therapy. Histone deacetylase inhibitors (HDACi) demonstrate broad anti-cancer activity. Many proposed mechanisms exist to explain their effects, including those that involve the immune system. Here, we evaluated the efficacy and transcriptional effects of entinostat, a selective class 1 HDAC inhibitor (HDAC1 and HDAC3), in a novel preclinical murine model of high-grade MIBC. BBN963 cells were derived from a primary bladder cancer GEM, cultured, and implanted subcutaneously into immunocompetent C57BL/6 mice and immunodeficient NOD scid gamma (NSG) mice. Animals were randomized to control or entinostat treatment. Tumor volume was recorded weekly and fresh tissue collected for RNAseq and Ingenuity Pathway Analysis (IPA). Entinostat exhibited in vivo activity in the BBN963 model in both C57BL/6 and NSG mice, however the anti-tumor response in B6 mice was significantly greater than the observed response in NSG mice. RNAseq analysis on tumor tissue collected from NSG mice indicated that entinostat treated tumors had distinct gene expression changes. Comparison of control to entinostat treated tumors revealed 4987 genes were significantly upregulated and 4112 genes were significantly downregulated (p-value < 0.05). IPA contextualized these changes as an enhanced inflammatory response, as indicated by increased expression of upstream regulators TGFβ, LPS, and IFNγ. Additionally, hierarchical clustering using established immune gene signatures stratified entinostat treated and untreated tumors into two distinct groups. Our results suggest that the anti-cancer properties of entinostat are in part immunologically mediated. BBN963 tumors in B6 mice displayed a more robust therapeutic response compared to their NSG counterparts. Transcriptional analysis of the tumor tissue indicated significant differences in immune gene signature levels as well as inflammatory pathway activation. Therefore, an intact immune system appears critical to achieve a maximal therapeutic response observed with HDAC inhibition. Together these results lay the foundation for further elucidating entinostat's mechanism of action in the context of high-grade MIBC. Citation Format: Kyle G. Stewart, Andrew S. Truong, Bhavani Krishnan, Mi Zhou, Ryoichi Saito, Jordan Kardos, Ujjawal Manocha, Sean T. Bailey, William Y. Kim. Antitumor activity of HDAC inhibition in bladder cancer mouse models correlates with enhanced immune response [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 2748.

In patients with bladder cancer, programmed death-ligand-1 (PD-L1) and programmed death-1 (PD-1) inhibitors have been shown to be effective in around twenty percent of patients, and there is evidence indicating that the level of immune infiltration and immune suppression within the tumor microenvironment correlates with response to these treatments. We have previously shown that there are intrinsic subtypes of bladder cancer, with the basal subtype characterized by high levels of immune infiltration, and the luminal subtype characterized by immune exclusion. Here we show that treatment with current standard of care chemotherapeutic agents has a subtype specific effect on the tumor microenvironment. Cisplatin-based chemotherapeutic treatment of luminal tumors induces a mesenchymal phenotype, immune infiltration, and a transition to a more basal-like tumor, while not altering these characteristics of the tumor microenvironment in basal tumors. Two of the most widely used standard of care chemotherapeutic regiments are Cisplatin-Gemcitabine (GemCis) and Methotrexate-Vinblastine-Doxorubicin-Cisplatin (MVAC), and we show that MVAC treatment induces significant immune infiltration within the luminal subtype while GemCis treatment does not, indicating there are treatment specific effects on the immune microenvironment. Furthermore, using mouse models of bladder cancer previously developed by our lab that accurately reflect the basal and luminal subtypes of bladder cancer, treatment with a representative chemotherapeutic regimen induced immune infiltration in the luminal mouse model while not affecting the tumor immune microenvironment in the basal mouse model. These results indicate chemotherapeutic regimens have subtype specific effects on the tumor microenvironment which could potentially be used to increase the efficacy of immune checkpoint inhibitors. Citation Format: Jordan Kardos, Lisa Bixby, Andrew Truong, Bhavani Krishnan, Kyle Stewart, Benjamin Vincent, William Kim. Effects of subtype specific chemotherapeutic immunomodulation in bladder 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 2749.

e14517Background: Many basket trials in immunotherapy and targeted therapy utilize Simon two-stage design, independently in each basket. Each basket represents a different indication with its own f...

Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist.We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis.Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH).Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).

Introduction: High-grade, muscle-invasive bladder cancer has recently been shown to harbor intrinsic molecular subtypes with distinct biologic features. Current murine models of bladder cancer, including the prominent carcinogen induced model MB49, do not account for subtype specific characteristics, leaving a gap in available tools for understanding subtype specific differences in bladder cancer. We have developed and validated immunocompetent, subtype specific models of bladder cancer, and we have used these models to assess differential responses to immune checkpoint inhibition. Methods: Two distinct models of murine bladder cancer were developed in a C57BL/6 background. The UPPL models were generated through Pten/Trp53 conditional knockout in Uroplakin3a expressing cells. BBN models were generated through exposure of wild-type C57BL/6 mice to the carcinogen N-Butyl-N-(4-hydmoxybutyl)nitrosamine and subsequent generation of cell lines from spontaneous tumors. RNAseq was performed on several BBN and UPPL tumors and cell lines, with findings validated with flow cytometry and T/B cell receptor (TCR/BCR) amplicon sequencing of tumor infiltrating lymphocytes (TILs). Results: BBN and UPPL models reflected characteristics of human basal and luminal bladder cancers, respectively. BBN (basal) models demonstrated higher immune gene signature expression, with concordantly higher numbers of TILs compared to the UPPL (luminal) model (p < 0.0001). Two BBN and two UPPL models were assessed for response to anti-PD-1 therapy in vivo as syngeneic tumors grown in wild type C57BL/6 mice. One of the BBN lines (BBN963) demonstrated robust control of tumor growth in some animals, including multiple complete responses (p = 0.0003), but also tumors that progressed, leading us to characterize BBN963 as a mixed response model. The marked response to PD-1 blockade in BBN963 was associated with significantly higher sharing of TCR CDR3 sequences among TILs compared to sequences of the other tumors (p = 0.003). In addition, analysis of BBN963 tumors by flow cytometry demonstrated naïve and memory T cell phenotypes correlated with increased and decreased tumor sizes, respectively. Closer examination of individual BBN963 tumor responses to PD-1 blockade revealed distinct responder and non-responder infiltrating immune cell phenotypes. Responders demonstrated a less diverse B cell repertoire (p = 0.0043) with increased BCR CDR3 sequence sharing (p < 0.0001). Discussion: We have developed two unique classes of murine bladder cancer lines, UPPL and BBN, with gene expression and TIL profiles that closely correlate with human luminal and basal bladder cancers, respectively. The BBN and UPPL subtype specific models can serve as a tool for elucidating bladder cancer responses to immunotherapy. The mixed response of BBN963 tumors to PD-1 blockade should be an asset for assessing pathways mediating response to checkpoint blockade as well as the value of combination therapy. [C.S., R.S, B.V, W.K contributed equally to this work] Citation Format: Christof C. Smith, Ryoichi Saito, Lisa M. Bixby, Takanobu Utsumi, Jordan Kardos, Shengjie Chai, Sara E. Wobker, Bhavani Krishnan, Jeffrey S. Damrauer, Jonathan S. Serody, David Darr, Benjamin G. Vincent, William Y. Kim. Development of subtype specific mouse models of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1654. doi:10.1158/1538-7445.AM2017-1654

Rapamycin derivatives allosterically targeting mTOR are currently FDA approved to treat advanced renal cell carcinoma (RCC), and catalytic inhibitors of mTOR/PI3K are now in clinical trials for treating various solid tumors. We sought to investigate the relative efficacy of allosteric versus catalytic mTOR inhibition, evaluate the crosstalk between the mTOR and MEK/ERK pathways, as well as the therapeutic potential of dual mTOR and MEK inhibition in RCC. Pharmacologic (rapamycin and BEZ235) and genetic manipulation of the mTOR pathway were evaluated by in vitro assays as monotherapy as well as in combination with MEK inhibition (GSK1120212). Catalytic mTOR inhibition with BEZ235 decreased proliferation and increased apoptosis better than allosteric mTOR inhibition with rapamycin. While mTOR inhibition upregulated MEK/ERK signaling, concurrent inhibition of both pathways had enhanced therapeutic efficacy. Finally, primary RCC tumors could be classified into subgroups [(I) MEK activated, (II) Dual MEK and mTOR activated, (III) Not activated, and (IV) mTOR activated] based on their relative activation of the PI3K/mTOR and MEK pathways. Patients with mTOR only activated tumors had the worst prognosis. In summary, dual targeting of the mTOR and MEK pathways in RCC can enhance therapeutic efficacy and primary RCC can be subclassified based on their relative levels of mTOR and MEK activation with potential therapeutic implications.

Malignant melanoma is characterized by a propensity for early lymphatic and hematogenous spread. The hypoxia-inducible factor (HIF) family of transcription factors is upregulated in melanoma by key oncogenic drivers. HIFs promote the activation of genes involved in cancer initiation, progression, and metastases. Hypoxia has been shown to enhance the invasiveness and metastatic potential of tumor cells by regulating the genes involved in the breakdown of the ECM as well as genes that control motility and adhesion of tumor cells. Using a Pten -deficient, Braf -mutant genetically engineered mouse model of melanoma, we demonstrated that inactivation of HIF1α or HIF2α abrogates metastasis without affecting primary tumor formation. HIF1α and HIF2α drive melanoma invasion and invadopodia formation through PDGFRα and focal adhesion kinase–mediated (FAK-mediated) activation of SRC and by coordinating ECM degradation via MT1-MMP and MMP2 expression. These results establish the importance of HIFs in melanoma progression and demonstrate that HIF1α and HIF2α activate independent transcriptional programs that promote metastasis by coordinately regulating cell invasion and ECM remodeling.

504 Background: African American (AA) patients with RCC have historically had inferior survival relative to Caucasians. There is little data on racial differences in tumor biology between AA and Caucasians to explain this disparity. In addition, it is not known if racial differences may result in differential response to targeted therapy and if the disparity has changed over time. Our study aims to define racial differences in tumor biology between AA and Caucasians with clear cell RCC (ccRCC) and to evaluate if observed differences in tumor biology are reflected in survival rates in the era of targeted therapy. Methods: We identified AA and Caucasian patients with stage IV clear cell RCC (ccRCC) in the National Cancer Data Base (NCDB) and analyzed survival of AA and Caucasians in the pre- and post-targeted therapy eras (1998-2004 and 2006-2011). We then analyzed The Cancer Genome Atlas (TCGA): Renal Clear Cell Carcinoma dataset to determine differences in somatic mutation rate, molecular subtype, and RNA expression between AA and Caucasians. Results: Overall survival (OS) is significantly longer in Caucasians than AA (9.2 vs 6.5 months, p < 0.01). The adjusted HR for death for AA compared with Caucasian patients was 1.08 (95% CI 1.01-1.14) in the post-targeted therapy era, which was unchanged from the pre-targeted therapy era (HR 1.07, 95% CI 1.01-1.14). AA patients were less likely than Caucasians to have VHL mutations (17% vs 50%, p = 0.04) and were enriched in the ccB molecular subtype (79% in AA vs 45% in Caucasians, p < 0.01). RNA expression revealed upregulation of several HIF-associated pathways in Caucasian compared with AA patients. Conclusions: ccRCC in AA have less frequent VHL mutations, are enriched in the ccB molecular subtype, and have less upregulation of HIF-associated pathways than Caucasians. These molecular features predict for decreased responsiveness to VEGF-targeted therapy and inferior survival in AA compared to Caucasians. Data from a large national database confirm that AA patients maintain inferior survival compared to Caucasians in the era of targeted therapy, potentially related to underlying tumor biology.

BACKGROUND Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. The purpose of this study was to determine the anti-proliferative and anti-angiogenic efficacy of angiotensin-(1-7) [Ang-(1-7)], an endogenous peptide hormone, in human prostate cancer xenografts. METHODS Human LNCaP prostate cancer cells were injected into the flank of athymic mice and tumors were treated with Ang-(1-7) for 54 days. Tumor growth and angiogenesis were determined by immunohistochemistry and western blot hybridization. RESULTS Ang-(1-7) markedly reduced the volume and wet weight of LNCaP xenograft tumors. Histological analysis of tumor sections from saline-treated mice showed increased Ki67 immunoreactivity and enhanced phosphorylation of the MAP kinases ERK1/2 compared to tumors from Ang-(1-7)-treated mice, suggesting that the heptapeptide reduces cell proliferation. Intratumoral vessel density was decreased in Ang-(1-7)-treated mice with an associated reduction in vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), suggesting that the heptapeptide attenuates vascularization by reducing angiogenic factors. Ang-(1-7) administration markedly increased the soluble fraction of VEGF receptor 1 (sFlt-1), with a concomitant reduction in VEGF receptors 1 and 2. sFlt-1 serves as a decoy receptor that traps VEGF and PlGF, making the ligands unavailable to membrane-bound VEGF receptors and preventing activation of pro-angiogenic signaling. CONCLUSIONS The decrease in PlGF and VEGF coupled with the increase in sFlt-1 suggests that Ang-(1-7) may serve as a novel anti-angiogenic therapy for prostate cancer. Further, the pleiotropic mechanisms of action by Ang-(1-7) may limit angiogenic resistance that occurs with VEGF inhibitors or receptor blockers. Prostate 73: 60–70, 2013. © 2012 Wiley Periodicals, Inc.

The continuing, unsustainable increase in datacenter power consumption is causing researchers in industry and academia to be heavily invested in addressing power management challenges. This chapter presents the basic elements of a measurement-based approach toward managing distributed datacenter and cloud computing systems to meet both application and end-user needs and to obtain improved efficiency and sustainability in their operation. The main components of the approach presented include (1) continuous online monitoring, measurement and assessment of systems and applications behaviors and power consumption, including for online estimation of the power usage of virtual machines running application components in these virtualized systems; (2) the ability to perform these tasks efficiently at scale, so as to deal with the ever-increasing sizes and complexity of modern datacenter infrastructures; and (3) the importance of “coordinated” management methods that operate across multiple levels of abstraction and multiple layers of the management stack in an orchestrated manner.

Concurrency and exchanging design knowledge among thermal and IT management are required to achieve an energy efficient operational data center. In this paper, a design approach is presented to bring adaptability and concurrency for coordinated minimization of cooling and IT power consumption in data centers. The presented approach is centered on a proper orthogonal decomposition based reduced order thermal modeling approach, and power profiling of the IT equipment to identify the optimal parameters of the air cooling systems along with optimal dynamic workload distribution among the servers. The method is applied to a data center cell with different rack and server architectures. The results show that the design approach results in 12–70% saving in the total energy consumption of the data center cell for various scenarios, compared with a baseline design.

Activation of the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway is a mechanism of cytotoxic drug resistance in ovarian cancer, and inhibitors of this pathway can sensitize to cytotoxic drugs. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) depletes some proteins involved in PI3K/AKT signaling, e.g., ERBB2, epidermal growth factor receptor (EGFR), and phosphorylated AKT (p-AKT). 17-AAG and paclitaxel were combined (at a fixed 1:1 ratio of their IC50) in four ovarian cancer cell lines that differ in expression of p-AKT, EGFR, and ERBB2. The EGFR-overexpressing A431 and KB epidermoid cell lines were also included. Combination indices (CI) were calculated using the median-effect equation and interpreted in the context of 17-AAG-mediated inhibition of PI3K signaling. Synergy was observed in IGROV-1- and ERBB2-overexpressing SKOV-3 ovarian cancer cells that express a high level of constitutively activated p-AKT [CI at fraction unaffected (fu)0.5 = 0.50 and 0.53, respectively]. Slight synergy was observed in A431 cells (moderate p-AKT/overexpressed EGFR; CI at fu0.5 = 0.76) and antagonism in CH1 (moderate p-AKT), HX62 cells (low p-AKT), and KB cells (low p-AKT/overexpressed EGFR; CI at fu50 = 3.0, 3.5, and 2.0, respectively). The observed effects correlated with changes in the rate of apoptosis induction. 17-AAG induced a decrease in HSP90 client proteins (e.g., C-RAF, ERBB2, and p-AKT) or in downstream markers of their activity (e.g., phosphorylated extracellular signal-regulated kinase or p-AKT) in SKOV-3, IGROV-1, and CH1 cells at IC50 concentrations. A non–growth-inhibitory concentration (6 nmol/L) reduced the phosphorylation of AKT (but not extracellular signal-regulated kinase) and sensitized SKOV-3 cells to paclitaxel. In conclusion, 17-AAG may sensitize a subset of ovarian cancer to paclitaxel, particularly those tumors in which resistance is driven by ERBB2 and/or p-AKT. [Mol Cancer Ther 2006;5(5):1197–208]

Human adenovirus Ad-36 causes adiposity in animal models and shows association with human obesity. Ad-36 enhances differentiation of 3T3-L1 and human preadipocytes, without cell lysis, a characteristic that may contribute to its adipogenic effect observed in vivo. Ad-2, another human adenovirus is nonadipogenic in animals and in 3T3-L1 cells and shows no correlation with human obesity. The objective of this study was to determine the adipogenic roles of viral mRNA and DNA, which may explain the differential effects of Ad-36 and Ad-2 on preadipocyte differentiation.This study determined the duration of selected Ad-36 gene expression in 3T3-L1 cells, and the effect on preadipocytes differentiation, when Ad-36 gene expression was attenuated by Cidofovir, an antiadenoviral agent.The results showed that Ad-36, but not Ad-2, expresses viral mRNA. Ad-36 gene expression peaked at 2-4 days postinoculation and very low levels persisted after day 7. Despite the viral mRNA expression, Ad-36 infection of 3T3-L1 cells was abortive as indicated by a progressive decrease in viral DNA quantity. Attenuation of Ad-36 mRNA expression by Cidofovir reduced the adipogenic effect of the virus.In conclusion, viral mRNA expression, although transient, is a prerequisite for enhancing differentiation of preadipocytes by Ad-36. Viral DNA replication was not required for the effect. This is the first evidence for the role of gene expression of an adipogenic human virus in enhancing preadipocytes differentiation. This study provides the basis for further understanding novel regulatory modulators of preadipocytes differentiation.

Pediatric sarcomas are rare tumors encompassing a diversity of histology and can arise in almost any part of the body. Despite overall advances in pediatric cancer treatment, outcomes in sarcomas have improved little in the past 2 decades. Based on the rarity of these tumors and the growing body of knowledge on late effects of conventional therapies in pediatric cancer survivors, it is imperative to develop new targeted agents for the treatment of these childhood diseases. In published studies, we showed that angiotensin-(1-7) [Ang-(1-7)], an endogenous seven amino acid peptide hormone, inhibited the proliferation of human lung cancer cells through activation of a unique AT(1-7) receptor and significantly reduced the size of human A549 lung tumor xenografts, in association with a decrease in the density of blood vessels and pro-angiogenic factors. These studies are in agreement with our Phase I clinical trial in which adult patients displaying clinical benefit had reduced circulating levels of the angiogenic factor PlGF; two of the four patients showing a clinical response to Ang-(1-7) had a primary sarcoma. The purpose of this study was to determine whether Ang-(1-7) may serve as a targeted chemotherapeutic agent for the treatment of pediatric sarcoma. Ang-(1-7) significantly inhibited the growth of A673 cells, a pediatric rhabdomyosarcoma cell line which expresses the AT(1-7) receptor mas. More importantly, the heptapeptide significantly reduced the proliferation of human A673 tumor xenograft growth in vivo. A673 rhabdomyosarcoma cells were injected into the flank of athymic male mice; the tumors grew to approximately 100 mm3 followed by infusion of saline or 24 µg/kg/h Ang-(1-7) for 18 days. The average volume of the tumors from mice treated with the heptapeptide was approximately 6-fold less than the size of the tumors from control animals (4770.6 ± 860.4 mm3 versus 664.8 ± 190.9 mm3; n = 6, p < 0.001). In addition, Ang-(1-7) administration markedly reduced tumor weight, from 7.2 ± 1.5 g in the saline-treated mice to 2.2 ± 0.8 g in Ang-(1-7)-treated mice (n = 6, p < 0.02). The decrease in tumor growth from Ang-(1-7)-medicated mice was associated with an approximate 50% reduction in immunoreactive Ki67, suggesting that the heptapeptide reduces tumor cell proliferation. Tumors from mice treated with Ang-(1-7) also showed a marked decrease in vessel density as assessed by CD34 immunoreactivity and morphology (7.6 ± 0.9 to 3.6 ± 0.6 vessels/per field, p < 0.001), demonstrating that the observed reduction in tumor growth was due, in part, to inhibition of angiogenesis. These studies suggest that the heptapeptide has both anti-proliferative and anti-angiogenic properties and that Ang-(1-7) may be a new, first-in-class compound for the treatment of pediatric sarcoma targeting a specific AT(1-7) receptor mas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5349. doi:10.1158/1538-7445.AM2011-5349

Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. We previously showed that angiotensin-(1-7) [Ang-(1-7)], a seven amino acid peptide hormone, significantly inhibited the growth of human lung cancer cells and tumors, with an associated reduction in angiogenesis. Since previous epidemiological studies suggest that administration of anti-hypertensive drugs which increase Ang-(1-7) reduces the risk of sex-specific cancers, we investigated the effects of the heptapeptide on prostate cancer. Ang-(1-7) markedly reduced human LNCaP prostate tumor xenograft size by 72% in association with a decrease in Ki67 and CD34, markers of tumor proliferation and angiogenesis, respectively. Ang-(1-7) significantly decreased both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) with a concomitant 12-fold increase in the soluble fraction of VEGF receptor 1 (sFlt-1); sFlt-1 is a decoy receptor that traps PlGF and VEGF, rendering the ligands unavailable to membrane-associated VEGF receptors. Ang-(1-7) also inhibits metastasis of prostate cancer to bone, which is the primary cause of mortality in prostate cancer patients. Human prostate cancer cells were injected into the circulation of SCID mice pretreated with Ang-(1-7), to determine the effect of the heptapeptide on the migration of cells to the metastatic environment. Six weeks following the injection of stably transfected luciferase tagged PC3 (PC3Luc) cells, 5 of the 6 untreated mice developed metastatic bone tumors, measured by bioluminescence and MRI imaging; in contrast, no detectable tumors were observed in mice administered Ang-(1-7). Circulating VEGF was significantly higher in untreated mice compared to mice treated with the heptapeptide. Ang-(1-7) also significantly reduced metastatic tumor formation in athymic mice injected with PC3Luc cells in the tibia as determined by bioluminescence, MRI imaging and immunohistochemistry. Osteolytic lesions as assessed by tartrate resistant acid phosphatase (TRAP) staining were observed surrounding the tibial tumors in control animals. A 50% reduction in osteoclastogenesis was observed when bone marrow cells were differentiated with RANK ligand and colony-stimulating factor in the presence of Ang-(1-7) [from 78.6 ± 8.0 TRAP+-multinucleated cells/field to 33.6 ± 4], suggesting that Ang-(1-7) hinders tumor survival in the bone microenvironment and prevents the formation of osteolytic lesions. Since VEGF is known to facilitate tumor growth and osteolytic disease by enhancing osteoclast survival, the inhibition of VEGF coupled with the reduction in osteoclastogenesis may mediate the inhibition of metastatic skeletal tumor formation. These results suggest that Ang-(1-7) may serve as an anti-proliferative, anti-angiogenic, and anti-metastatic agent for the treatment of prostate cancer that targets the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 550. doi:10.1158/1538-7445.AM2011-550

Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. We showed that angiotensin-(1-7) [Ang-(1-7)], a seven amino acid peptide hormone of the renin-angiotensin system, significantly inhibited the proliferation of human lung cancer cells and attenuated the growth of non-small cell adenocarcinoma tumors in a xenograft model, with an associated reduction in angiogenesis. Since previous epidemiological studies suggest that treatment with anti-hypertensive drugs which increase Ang-(1-7) reduces the risk of sex-specific cancers, we investigated the effects of the heptapeptide on prostate cancer. Ang-(1-7) significantly reduced the growth of both androgen-responsive LNCaP human prostate cancer cells and PC3 cells derived from a bone metastasis of a patient with prostate cancer. Athymic mice with xenograft LNCaP human prostate tumors were treated with Ang-(1-7) for 54 days. Treatment with the heptapeptide resulted in an 84.5% reduction in tumor volume and a 72% decrease in tumor weight. The reduction in tumor size was associated with a 78% decrease in Ki67 immunoreactivity, suggesting that Ang-(1-7) inhibits tumor cell proliferation. Treatment with the heptapeptide also caused a 50% decrease in the density of tumor vessels, identified by morphology using an antibody to CD34 to label endothelial cells, suggesting that Ang-(1-7) reduces angiogenesis. The reduction in angiogenesis by heptapeptide administration was associated with a decrease in the mRNA and protein of vascular endothelial growth factor (VEGF) (a 48% reduction in protein and an 82% reduction in mRNA) and placental growth factor (PlGF) (a 72% reduction in both protein and mRNA), key factors involved in angiogenesis. Incubation of LnCaP cells with 100 nM Ang-(1-7) for 24 h also caused a significant reduction in the secretion of both VEGF (64%) and PlGF (71%). The decrease in PlGF and VEGF correlated with a 12-fold increase in the soluble fraction of VEGF receptor 1 (sFLT1) [from 0.12 ± 0.06 relative density units in tumors from saline-treated mice compared to 1.41 ± 0.30 relative density units in tumors from Ang-(1-7)-treated mice, n = 5, p < 0.001]. sFLT1 is a decoy receptor that traps PlGF and VEGF, making the ligands unavailable to membrane-associated VEGF receptors and blocking activation of key angiogenic signaling pathway. While anti-angiogenic therapies targeting VEGF revolutionized cancer treatment, tumors develop resistance to these agents and patients have an increased risk of developing metastases, which may result from production of alternate pro-angiogenic factors, such as PlGF. Since treatment with Ang-(1-7) reduces both VEGF and PlGF and concomitantly increases sFLT1 to attenuate angiogenic signaling, the heptapeptide may represent a novel anti-angiogenic therapeutic in the treatment of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1299.