Androgen deprivation therapy (ADT) is an effective treatment for metastatic prostate cancer (mPCa), but patients eventually relapse with ADT resistant disease. Well-characterized mechanisms of ADT resistance include AR amplification, intra-tumoral androgen synthesis, AR splice variants, and growth receptor bypass. All of these mechanisms function to maintain sufficient AR signaling for tumor growth and survival. Improved ADT like abiraterone acetate (AA) and enzalutamide (Enza) were developed to combat such resistance mechanisms associated with alterations in androgen receptor or androgen metabolism. While AA and Enza extend survival, clinical benefits are short-lived. A new form of resistance is increasingly appreciated in patients relapsing from AA or Enza, histologic transformation of prostate adenocarcinoma (PADC) to neuroendocrine prostate cancer (NEPC) variants. NEPC is lethal and the survival time is less than a year as effective targeted therapy is unavailable. NEPC typically exhibits reduced AR expression, increased expression of neuroendocrine markers, and visceral metastasis in the absence of rising PSA. Of note, NEPC possesses the similar genome rearrangements with adjacent PADC cells, indicating they share clonal origin. Thus, NEPC may arise by histologic transformation of PADC. Underlying mechanisms of histologic transformation are not understood and experimental models are limited, hindering development of effective remedies. RB1 loss is common in NEPC, but rare in PADC; genetic profiling shows human NEPC exhibit elevated levels of several epigenetic modifiers. We hypothesize that transdifferentiation from PADC to NEPC in the context of RB1 loss is due to epigenetic alterations and can be reversed or blocked by epigenetic targeted therapies. We established several genetically engineered mouse models (GEMMs) to test the role of Rb1, and we find Rb1 loss causes metastatic progression of PADC initiated by Pten deficiency. This Rb1/Pten deficient (DKO) PADC exhibits expression markers for both PADC and NEPC as seen in human patients. Yet, these tumors are sensitive to ADT but relapse with low AR expression and acquired Trp53 mutations. RNA profiling demonstrates the phenotype of DKO tumors is similar to human NEPC. Both human and mouse NEPC is accompanied by increased expression of epigenetic reprogramming factors like Sox2 and Ezh2. Clinically relevant Ezh2 inhibitors GSK126 and EPZ6438 can restore Enza sensitivity by reversing neuroendocrine transformation. This finding has been genetically validated using short-hairpin RNA(shRNA) in vitro. These results uncover genetic mutations driving prostate cancer lineage plasticity and suggest an epigenetic approach for extending the clinical benefits of ADT. Citation Format: Sheng-Yu Ku, Spencer Rosario, Yanqing Wang, Ping Mu, Mukund Seshadri, Zachary Goodrich, Maxwell Goodrich, David P. Labbé, Eduardo Cortez Gomez, Jianmin Wang, Henry W. Long, Bo Xu, Myles Brown, Massimo Loda, Charles L. Sawyers, Leigh Ellis, David G. Goodrich. Rb1 suppresses prostate cancer metastasis and lineage plasticity underlying castration resistance [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 2170. doi:10.1158/1538-7445.AM2017-2170

Undifferentiated soft tissue sarcomas (UDSTS ) are a group of mesenchymal tumors that remain a diagnostic challenge due to their morphologic heterogeneity and unclear histologic origin (Peters et al. 2015b). In this case report, we present the first multi-omics molecular signature for a BCOR-CCNB3 sarcoma (BCS) that includes mutation analysis, gene expression, DNA methylation, and mi-RNA expression. We identify a paucity of additional mutations in this tumor and detail that there is significant dysregulation of gene expression of epigeneic remodeling agents including key members of the PRC, Sin3A/3b, NuRD, and NcoR/SMRT complexes and the DNA methyltransferases DNMT1, DNMT3a, and DNMT3b. This is accompanied by significant DNA methylation changes and dysregulation of multiple miRNA with known links to tumorigenesis. This study significantly increases our understanding of the BCOR effects on fusion positive undifferentiated sarcomas at both the genomic and epigenomic level and suggests that as better-tailored and more refined treatment algorithms continue to evolve, epigenetic modifying agents should be further evaluated for their efficacy against these tumors.

Background: Interactions between the neonate host and its gut microbiome are central to the development of a healthy immune system. However, the mechanisms by which animals alter early colonization of microbiota for their benefit remain unclear. Host-derived carbohydrates, which can serve as metabolic substrates for the expansion of specific commensal and pathogenic bacteria, are one method by which the host may influence interspecies competition in the microbiome. Here, we investigated the role of early-life expression of the α2,6-sialyltransferase ST6GAL1 in microbiome phylogeny and mucosal immunity. Methods: Intestinal sialylation was characterized by RT-qPCR, immunoblot, microscopy, and sialyltransferase enzyme assays in genetic mouse models at rest or with glucocorticoid receptor modulators. The fecal, upper respiratory, and oral microbiomes of pups expressing or lacking St6gal1 were analyzed by 16S rRNA sequencing. Pooled fecal microbiomes from syngeneic donors were transferred to antibiotic-treated wild-type mice, before analysis of recipient mucosal immune responses by flow cytometry, RT-qPCR, microscopy, and ELISA. Results: ST6GAL1 was highly expressed in duodenal enterocytes between birth and weaning, driving temporary sialylation of the associated glycocalyx and secretion of active ST6GAL1 into the intestinal lumen, with subsequent uptake by colonic goblet cells. Expression was mediated by the P1 promoter of the St6gal1 gene, which was efficiently inhibited by intraperitoneal dexamethasone. At weaning, the fecal microbiome of St6gal1-KO pups was unchanged in diversity but exhibited reductions in Clostridium, Coprobacillus, and Adlercreutzia, along with increased Helicobacter and Bilophila. Transfer of St6gal1-KO microbiome induced an AhR-dependent Th17 response, with expression of T-bet and IL-17, and IL-22-dependent gut lengthening. Conclusions: Intestinal sialylation by the sialyltransferase ST6GAL1 in the neonatal period is a developmentally regulated host mechanism coordinating bacterial colonization in the early gut microbiome. The inability to produce α2,6-sialyl ligands results in microbiome-dependent Th17 inflammation, highlighting a pathway by which intestinal epithelium regulates mucosal immunity. Considering the prevalence of intestinal fucosylation in adult animals, sialic acid may promote an early stage of ecological succession in the developing gut. Trial registration: N/A

Background: Disparities in prostate cancer (PrCa) incidence and mortality between African American (AA) and European American (EA) men are partially mediated by underlying disease biology. The goal of this study was to determine how DNA methylation and transcriptomic changes drive PrCa health disparities in AA men. Methods: Transcriptomic alterations potentially mediated by DNA methylation were identified by Illumina arrays and RNA-sequencing in PrCa from 30 AA and 32 EA men. Androgen receptor (AR) protein expression was measured using tissues microarray of matched PrCa tissues from 95 EA and 92 AA men. In vitro, MDA PCa 2a (2a)/MDA PCa 2b (2b) cells derived from an AA man and LNCaP/LaPC-4 cells derived from EA men were stimulated with ionomycin, a calcium ionophore to measure intracellular calcium using fluorescent-based live imaging. Results: Unsupervised hierarchical clustering revealed DNA methylation clusters (Cluster A and Cluster B) with differential methylation of loci that regulate intracellular calcium levels including RYR2, TRPC6, and TRPA1. Increased methylation of calcium regulatory genes in Cluster A was associated with reduced disease-free time (DFT) (21.65 vs 46.71 months, p<0.05) only in AA men with PrCa. RYR2 (-0.122 vs -0.004, p=0.69), TRPC6 (0.006 vs -0.639, p=0.06) and TRPA1 (-0.070 vs -0.269, p<0.05) transcript levels were lower in Cluster A compared to Cluster B. These data suggest DNA methylation can regulate expression of calcium regulating genes. In vitro, we found AA PrCa derived 2b cells have reduced RNA levels of RYR2, TRPV6 and CALB1 compared to EA PrCa derived LNCaP cells. Reduced transcription can result in lower protein expression and thus activity of calcium regulatory genes. To test this, we stimulated cells with ionomycin and found a rapid increase in intracellular calcium in 2a/2b cells (within 60 seconds) compared to LNCaP/LAPC-4 cells (120-300 seconds). This suggests that decreased transcription correlates to reduced buffering capacity of calcium regulatory genes. Others have shown that increased intracellular calcium reduces AR protein levels. Therefore, we analyzed AR protein expression in a subset of tumors from Cluster A and B. AR levels were lower in adjacent non-tumor and tumor tissue in these overlapping samples. AR low PrCa are associated with basal like features and respond poorly to androgen deprivation therapy. Therefore, we analyzed the PAM50 basal/luminal gene sets and AR target genes and found differentially expression of these genes in AA PrCa derived 2b and EA PrCa derived LNCaP cells. Conclusions: Our study shows that AA men with PrCa have epigenetically dysregulated calcium signaling that is associated with worse DFT. Our ongoing work seeks to identify how these alterations regulate AR expression and thus basal/luminal features. Our long- term goal is to establish novel molecular subtypes using calcium-AR-basal/luminal features that can guide design of rationale therapies in a subset of AA men with PrCa. Citation Format: Swathi Ramakrishnan, Xuan Peng, Eduardo C. Gomez, Kristopher Attwood, Ivan V. Maly, Wilma A. Hoffmann, Wendy Huss, Gissou Azabdaftari, Li Yan, Jianmin Wang, Anna Woloszynska. Intracellular calcium regulates androgen receptor expression and basal-luminal features in prostate cancer from African American men [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-134.

Background: Interactions between the neonate host and its gut microbiome are central to the development of a healthy immune system. However, the mechanisms by which animals alter early colonization of microbiota for their benefit remain unclear. Host-derived carbohydrates, which can serve as metabolic substrates for the expansion of specific commensal and pathogenic bacteria, are one method by which the host may influence interspecies competition in the microbiome. Here, we investigated the role of early-life expression of the α2,6- sialyltransferase ST6GAL1 in microbiome phylogeny and mucosal immunity. Methods: Fecal, upper respiratory, and oral microbiomes of pups expressing or lacking St6gal1 were analyzed by 16S rRNA sequencing. Pooled fecal microbiomes from syngeneic donors were transferred to antibiotic-treated wild-type mice, before analysis of recipient mucosal immune responses by flow cytometry, RT-qPCR, microscopy, and ELISA. Intestinal sialylation was characterized by RT-qPCR, immunoblot, microscopy, and sialyltransferase enzyme assays in genetic mouse models at rest or with glucocorticoid receptor modulators. Results: At weaning, the fecal microbiome of St6gal1 -KO mice exhibited reductions in Clostridiodes, Coprobacillus, and Adlercreutzia, but increased Helicobacter and Bilophila . Transfer of St6gal1- KO microbiome induced a mucosal Th17 response, with expression of T-bet and IL-17, and IL-22-dependent gut lengthening. ST6GAL1 was found to be highly expressed in duodenal enterocytes between birth and weaning, driving temporary sialylation of the associated glycocalyx and secretion of active ST6GAL1 into the intestinal lumen. Expression was mediated by the P1 promoter of the St6gal1 gene, which was efficiently inhibited by dexamethasone. Conclusions: Intestinal sialylation by the sialyltransferase ST6GAL1 in the neonatal period is a developmentally regulated host mechanism coordinating bacterial colonization in the early gut microbiome. The inability to produce α2,6-sialyl ligands results in microbiome-dependent Th17 inflammation, highlighting a pathway by which intestinal epithelium regulates mucosal immunity. Considering the prevalence of intestinal fucosylation in adult animals, sialic acid may promote an early stage of ecological succession in the developing gut. Trial registration: N/A