Entinostat induces antitumor immune responses through immune editing of tumor neoantigens.

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.

The Cancer Genome Atlas Comprehensive Molecular Characterization of Renal Cell Carcinoma.

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.

MYC activation cooperates with Vhl and Ink4a/Arf loss to induce clear cell renal cell carcinoma.

Renal carcinoma is a common and aggressive malignancy whose histopathogenesis is incompletely understood and that is largely resistant to cytotoxic chemotherapy. We present two mouse models of kidney cancer that recapitulate the genomic alterations found in human papillary (pRCC) and clear cell RCC (ccRCC), the most common RCC subtypes. MYC activation results in highly penetrant pRCC tumours (MYC), while MYC activation, when combined with Vhl and Cdkn2a (Ink4a/Arf) deletion (VIM), produce kidney tumours that approximate human ccRCC. RNAseq of the mouse tumours demonstrate that MYC tumours resemble Type 2 pRCC, which are known to harbour MYC activation. Furthermore, VIM tumours more closely simulate human ccRCC. Based on their high penetrance, short latency, and histologic fidelity, these models of papillary and clear cell RCC should be significant contributions to the field of kidney cancer research.

Claudin-low bladder tumors are immune infiltrated and actively immune suppressed.

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.

Racial disparities in survival among patients with advanced renal cell carcinoma in the targeted therapy era.

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.

Intrinsic Genomic Differences Between African American and White Patients With Clear Cell Renal Cell Carcinoma.

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.

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.).

mTOR inhibition induces compensatory, therapeutically targetable MEK activation in renal cell carcinoma.

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.

HIF1α and HIF2α independently activate SRC to promote melanoma metastases.

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.

Angiotensin-(1-7) reduces proliferation and angiogenesis of human prostate cancer xenografts with a decrease in angiogenic factors and an increase in sFlt-1.

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.

Angiotensin-(1-7) attenuates metastatic prostate cancer and reduces osteoclastogenesis.

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.

Potentiation of paclitaxel activity by the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin in human ovarian carcinoma cell lines with high levels of activated AKT.

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 IC(50)) 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 fu(0.5) = 0.76) and antagonism in CH1 (moderate p-AKT), HX62 cells (low p-AKT), and KB cells (low p-AKT/overexpressed EGFR; CI at fu(50) = 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 IC(50) 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.