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Inactivation of the tumor suppressor genes tumor protein p53 ( TP53 ) and cyclin-dependent kinase inhibitor 2A ( CDKN2A ) occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, because of a paucity of GEJ-specific disease models, cancer-promoting consequences of TP53 and CDKN2A inactivation at the GEJ have not been characterized. Here, we report the development of a wild-type primary human GEJ organoid model and a CRISPR-edited transformed GEJ organoid model. CRISPR-Cas9–mediated TP53 and CDKN2A knockout ( TP53/CDKN2A KO ) in GEJ organoids induced morphologic dysplasia and proneoplastic features in vitro and tumor formation in vivo. Lipidomic profiling identified several platelet-activating factors (PTAFs) among the most up-regulated lipids in CRISPR-edited organoids. PTAF/PTAF receptor (PTAFR) abrogation by siRNA knockdown or a pharmacologic inhibitor (WEB2086) reduced proliferation and other proneoplastic features of TP53/CDKN2A KO GEJ organoids in vitro and tumor formation in vivo. In addition, murine xenografts of Eso26, an established human esophageal adenocarcinoma cell line, were suppressed by WEB2086. Mechanistically, TP53/CDKN2A dual inactivation disrupted both the transcriptome and the DNA methylome, likely mediated by key transcription factors, particularly forkhead box M1 (FOXM1). FOXM1 activated PTAFR transcription by binding to the PTAFR promoter, further amplifying the PTAF-PTAFR pathway. Together, these studies established a robust model system for investigating early GEJ neoplastic events, identified crucial metabolic and epigenomic changes occurring during GEJ model tumorigenesis, and revealed a potential cancer therapeutic strategy. This work provides insights into proneoplastic mechanisms associated with TP53/CDKN2A inactivation in early GEJ neoplasia, which may facilitate early diagnosis and prevention of GEJ neoplasms.

Inactivation of the tumor suppressor genes TP53 and CDKN2A occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, due to a paucity of GEJ-specific disease models, cancer-promoting consequences of TP53 and CDKN2A inactivation at the GEJ have been incompletely characterized. Here we report the development of the first wild-type primary human GEJ organoid model, as well as a CRISPR-edited transformed GEJ organoid model. CRISPR/Cas9 engineering to inactivate TP53 and CDKN2A (TP53/CDKN2AKO) in GEJ organoids induced morphologic dysplasia as well as pro-neoplastic features in vitro and tumor formation in vivo. Notably, lipidomic profiling identified several Platelet-Activating Factors (PTAFs) among the most upregulated lipids in CRISPR-edited organoids; and importantly, PTAF/PTAFR abrogation by siRNA knockdown or a pharmacologic inhibitor (WEB2086) significantly blocked proliferation and other pro-neoplastic features of TP53/CDKN2AKO GEJ organoids in vitro and tumor formation in vivo. In addition, murine xenografts derived from Eso26, an established esophageal adenocarcinoma (EAC) cell line, were suppressed by WEB2086. Mechanistically, TP53/CDKN2A dual inactivation disrupted both the transcriptome and the DNA methylome, likely mediated by key transcription factors, particularly Forkhead Box M1 (FOXM1). Importantly, FOXM1 activated PTAFR transcription by binding to the PTAFR promoter, further amplifying the PTAF-PTAFR pathway. In summary, we established a robust model system for investigating early GEJ neoplastic events, identified crucial metabolic and epigenomic changes occurring during GEJ model tumorigenesis, and discovered a potential cancer-therapeutic strategy, while providing insights into pro-neoplastic mechanisms associated with TP53/CDKN2A inactivation in early GEJ neoplasia.One Sentence SummaryNovel tumorigenic FOXM1-PTAFR-PTAF axis revealed by multi-omic profiling in TP53/CDKN2A-double knockout human gastroesophageal junction organoid model.Graphic Abstract

Mutation-associated neoantigens (MANAs) are exquisitely cancer-specific therapeutic targets. However, MANAs are present at ultra-low densities on the cancer cell surface (as few as 1-2 copies per cell), leading to the challenge of eliciting a sufficiently robust therapeutic effect. We combined components of both T cell receptors (TCRs) and chimeric antigen receptors (CARs) to create a new receptor with improved potency against an ultra-low-density MANA. From CARs, we utilized the antibody-based antigen recognition domain (i.e. the single chain variable fragment, scFv) and the integrated co-stimulation that amplifies T cell activation. From TCRs, we utilized the multi-chain signaling platform that facilitates high antigen sensitivity. This new receptor, termed a TCR Embedded ScFv for Long-term Activation (TESLA), showed promising characteristics when tested with the H2-scFv which targets the p53 R175H mutation presented on HLA-A*02:01 (R175H/A2). Using CRISPR-based homology directed repair in primary human T cells, we tested 15 configurations of appending the H2-scFv to subunits of the TCR complex to identify a design that maximized T cell cytotoxicity and interferon gamma release in co-cultures with cancer cells expressing endogenous levels of the R175H/A2 antigen. In this system, we showed that the optimal TCR-embedded configuration of the H2-scFv produced similar levels of cytotoxicity and interferon gamma secretion as patient-derived TCRs targeting the same R175H/A2 MANA, while conventional H2-CARs were unable to produce any T-cell activation. We then used a multiple stimulation co-culture system to identify a co-stimulation domain combination (MyD88 and CD40) that improved serial cytotoxicity and proliferation of H2-TESLAs when incorporated on the intracellular side of the TCRbeta chain. Finally, we compared the H2-TESLA receptor to patient-derived TCRs modified with the same MyD88 and CD40 co-stimulation domains. In vivo, H2-TESLAs cured all mice in a tumor model, while co-stimulation-modified TCRs produced only temporary tumor control. Moreover, in vivo, H2-TESLAs elicited 100-fold greater T cell expansion than co-stimulation-modified TCRs. In conclusion, we demonstrated that by combining aspects of both CARs and TCRs, the TESLA receptor improved T cell reactivity against an ultra-low-density neoantigen compared to conventional CARs and patient-derived TCRs. Citation Format: Brian J. Mog, Sarah R. DiNapoli, Michael S. Hwang, Tushar D. Nichakawade, Jacqueline Douglass, Emily Han-Chung Hsiue, Katharine M. Wright, Alexander H. Pearlman, Maximilian F. Konig, Suman Paul, Nicolas Wyhs, Nikita Marcou, Stephanie Glavaris, Jiaxin Ge, Michelle S. Miller, P. Aitana Azurmendi, Evangeline Watson, Drew M. Pardoll, Sandra B. Gabelli, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. Hybrid TCR-CAR design surpasses conventional CARs and patient-derived TCRs in targeting an ultra-low-density neoantigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB095.

Background Resistance to androgen deprivation therapies is a major driver of mortality in advanced prostate cancer. Therefore, there is a need to develop new preclinical models that allow the investigation of resistance mechanisms and the assessment of drugs for the treatment of castration-resistant prostate cancer. Methods We generated two novel cell line models (LAPC4-CR and VCaP-CR) which were derived by passaging LAPC4 and VCaP cells in vivo and in vitro under castrate conditions. We performed detailed transcriptomic (RNA-seq) and proteomic analyses (SWATH-MS) to delineate expression differences between castration-sensitive and castration-resistant cell lines. Furthermore, we characterized the in vivo and in vitro growth characteristics of these novel cell line models. Results The two cell line derivatives LAPC4-CR and VCaP-CR showed castration-resistant growth in vitro and in vivo which was only minimally inhibited by AR antagonists, enzalutamide, and bicalutamide. High-dose androgen treatment resulted in significant growth arrest of VCaP-CR but not in LAPC4-CR cells. Both cell lines maintained AR expression, but exhibited distinct expression changes on the mRNA and protein level. Integrated analyses including data from LNCaP and the previously described castration-resistant LNCaP-abl cells revealed an expression signature of castration resistance. Conclusions The two novel cell line models LAPC4-CR and VCaP-CR and their comprehensive characterization on the RNA and protein level represent important resources to study the molecular mechanisms of castration resistance. ISSN:0270-4137 ISSN:1097-0045

We describe the creation of an isogenic cell line panel representing common cancer pathways, with features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cell lines were generated using CRISPR technologies. Surprisingly, most of these lines did not result in complete gene inactivation despite integration of sgRNA at the desired genomic site. A subset of the lines harbored biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterized lines covering 19 frequently lost cancer pathways. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of

SummaryWe describe the creation and characterization of an isogenic cell line panel representing common cancer pathways, with multiple features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cells were generated using CRISPR-technologies. Surprisingly, we discovered most of these lines did not result in complete gene inactivation, despite integration of sgRNA at the desired genomic site. However, a subset of the lines harbored true, biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterize lines covering 19 pathways frequently subject to loss of function in cancers. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a multiplexed high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation ofTP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

BACKGROUNDResistance to androgen deprivation therapies is a major driver of mortality in advanced prostate cancer. Therefore, there is a need to develop new pre-clinical models that allow the investigation of resistance mechanisms and the assessment of drugs for the treatment of castration resistant prostate cancer.METHODSWe generated two novel cell line models (LAPC4-CR and VCaP-CR) which were derived by passaging LAPC4 and VCaP cells in vivo and in vitro under castrate conditions. We performed detailed transcriptomic (RNA-seq) and proteomic analyses (SWATH-MS) to delineate expression differences between castration-sensitive and castration-resistant cell lines. Furthermore, we characterized the in vivo and in vitro growth characteristics of the novel cell line models.RESULTSThe two cell line derivatives LAPC4-CR and VCaP-CR showed castration resistant growth in vitro and in vivo which was only minimally inhibited by AR antagonists, enzalutamide and bicalutamide. High-dose androgen treatment resulted in significant growth arrest of VCaP-CR but not in LAPC4-CR cells. Both cell lines maintained AR expression, but exhibited distinct expression changes on the mRNA and protein level. Integrated analyses including data from LNCaP and the previously described castration resistant LNCaP-abl cells revealed an expression signature of castration resistance.CONCLUSIONSThe two novel cell line models LAPC4-CR and VCaP-CR and their comprehensive characterization on the RNA and protein level represent important resources to study the molecular mechanisms of castration resistance.

DNA methylation can mediate epigenetic silencing of tumor suppressor and cancer protective genes. The protein ubiquitin-like containing PHD and ring finger domains 1 (UHRF1) is an essential component in cells for DNA methylation maintenance. The SET- and RING-associated (SRA) domain of UHRF1 can bind hemimethylated DNA, and mediate recruitment of DNA methyltransferases to copy the methylation pattern to the newly synthesized daughter strand. Loss of UHRF1 function can lead to demethylation and re-expression of epigenetically silenced tumor suppressor genes and can reduce cancer cell growth and survival. We created a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to screen for inhibitors capable of disrupting the interaction between the UHRF1-SRA domain and hemimethylated DNA. Using this assay (Z' factor of 0.74 in 384-well format) we screened the Library of Pharmacologically Active Compounds (LOPAC) for UHRF1-SRA inhibitors, and validated 7 hit compounds. These compounds included the anthracycline derivatives idarubicin and mitoxantrone, which are commonly used chemotherapeutic drugs known to mediate cytotoxicity by acting as class II topoisomerase (TOP2) poisons. In a panel of additional known topoisomerase poisons, only the anthracycline derivatives showed dose responsive inhibition of UHRF1-SRA. Additionally, mitoxantrone and doxorubicin showed dose-responsive global DNA demethylation and demonstrated a synergistic growth inhibition of multiple cancer cell lines when combined with the DNA methyltransferase (DNMT) inhibitor decitabine. These data validate a novel TR-FRET assay for identification of UHRF1 inhibitors, and revealed unexpected epigenetic properties of commonly used chemotherapeutic drugs that showed synergistic cytotoxicity of cancer cells when combined with a demethylating agent.

Adapting the clinical residency training model can help postdoctoral research fellows navigate and personalize their training experience for their unique career goals.

Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z' factor = 0.58) emerged as a superior screening strategy compared with FP (Z' factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET-based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions.

BACKGROUND The goal of the Prostate Cancer Biorepository Network (PCBN) is to develop a biorepository with high-quality, well-annotated specimens obtained in a systematic, reproducible fashion using optimized and standardized protocols, and an infrastructure to facilitate the growth of the resource and its wide usage by the prostate cancer research community. An emerging area of concern in the field of prostate cancer biobanking is an apparent shift in the proportion of surgical procedures performed for prostate cancer treatment from radical retropubic prostatectomy (RRP) to robot-assisted laparoscopic prostatectomy (RALP). Our study aimed to determine the potential impact of the RALP procedure on the detection of known prostate cancer biomarkers, and the subsequent suitability of RALP-derived specimens for prostate cancer biomarker studies. METHODS DNA and RNA were extracted from RRP and RALP specimens. Quality assessment was conducted using spectrophotometric analysis and RNA was analyzed for RNA integrity number (RIN) and by real-time reverse-transcription PCR (qRT-PCR) for racemase, hepsin, ERG, TMPRSS2-ERG gene fusions, and the microRNAs miR-26a, miR-26b, miR-141, and miR-221. RESULTS We demonstrate that extraction of derivatives from frozen tissues from RRP and RALP specimens yields samples of equally high quality as assessed by spectrophotometric and RIN analysis. Likewise, expression levels of genes analyzed by qRT-PCR did not differ between RRP and RALP-derived tissues. CONCLUSIONS Our studies indicate that samples obtained from RALP specimens may be suitable for prostate cancer biomarker studies—an important finding given the current shift in surgical procedures for prostate cancer treatment. Prostate 74:61–69, 2014. © 2013 Wiley Periodicals, Inc.

DNA hypermethylation can trigger silencing of tumor suppressor genes during cancer development and progression, partly through binding by methylated-DNA binding proteins (MBD), such as MBD2, that function as “epigenetic readers” and recruit co-repressor complexes to promote gene repression. Inhibiting MBD2-mediated repression represents an attractive cancer therapeutic strategy. Here, we used a cell-based screen to identify small molecules capable of reactivating hypermethylated promoter sequences. We used biochemical, molecular biologic, and pharmacologic approaches to characterize mechanism of action of identified MBD2 inhibitors. A subset of these compounds represent a new class of inhibitors capable of selectively antagonizing interactions between MBD2 and methylated DNA, leading to reactivation of the hypermethylated gene GSTP1 and the epigenetically silenced retinoic acid signaling pathway. Combinations of one of the newly identified MBD2 inhibitors, KCC-08, with the retinoic acid receptor agonist, isotretinoin, significantly reduced cancer cell growth/survival in vitro and in vivo. These novel MBD2 inhibitors are thus positioned for further pharmacologic lead development for use as probes to interrogate epigenetic gene silencing mechanisms and as cancer therapeutics. Citation Format: Hugh Giovinazzo, Zachary R. Reichert, Andries Bergman, Xiaohui Lin, Nicolas Wyhs, David Esopi, Ajay Vaghasia, Jianyong Liu, Yash Jain, Akshay Bhamidipati, Ruchama Steinberg, Traci Speed, Matthew Vaughn, Yonggang Zhang, Nate Brennen, Theodore Deweese, Srinivasan Yegnasubramanian, William G. Nelson. Novel inhibitors of the epigenetic reader protein MBD2 [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 5881.

A defining hallmark of primary and metastatic cancers is the migration and invasion of malignant cells. These invasive properties involve altered dynamics of the cytoskeleton and one of its major structural components β-actin. Here we identify AIM1 (absent in melanoma 1) as an actin-binding protein that suppresses pro-invasive properties in benign prostate epithelium. Depletion of AIM1 in prostate epithelial cells increases cytoskeletal remodeling, intracellular traction forces, cell migration and invasion, and anchorage-independent growth. In addition, decreased AIM1 expression results in increased metastatic dissemination in vivo. AIM1 strongly associates with the actin cytoskeleton in prostate epithelial cells in normal tissues, but not in prostate cancers. In addition to a mislocalization of AIM1 from the actin cytoskeleton in invasive cancers, advanced prostate cancers often harbor AIM1 deletion and reduced expression. These findings implicate AIM1 as a key suppressor of invasive phenotypes that becomes dysregulated in primary and metastatic prostate cancer., Invasion of malignant cells involves changes in cytoskeleton dynamics. Here the authors identify absent in melanoma 1 as an actin binding protein and show that it regulates cytoskeletal remodeling and cell migration in prostate epithelial cells, acting as a metastatic suppressor in cancer cells.

DNA 5’ cytosine methylation at CpG dinucleotides, particularly at gene promoters and regulatory elements, can mediate epigenetic silencing of key tumor suppressor and caretaker genes in human cancers. This is largely mediated through the activity of methyl binding domain (MBD) “reader” proteins that bind methylated DNA and recruit chromatin repression complexes. Of the MBD family of proteins, MBD2 is a particularly attractive therapeutic target since targeted genetic disruption of this protein is well tolerated and can greatly attenuate tumor formation in cancer prone mice and growth of cancerous cells lines. Therefore development of pharmacological approaches for targeting MBD2 may have utility in cancer therapeutics. In this work we describe a time resolved fluorescence energy transfer (TR-FRET) based assay to find small molecule inhibitors of MBD2. First we optimized conditions to establish an assay that was amenable for high throughput screening (HTS) in 384 well plates, obtaining a Z’ factor of 0.58 (HTS target >0.5). To validate our assay we applied it to the 1280 compound Sigma® LOPAC library. Among 10 primary hits, four (Mitoxantrone, idarubicin, aurintricarboxylic acid, and NF449) showed dose responsive inhibition of MBD2 binding to methylated DNA. However, a counter screen showed these 4 compounds also inhibited interaction of the transcription factor SP-1 to DNA, suggesting their mode of action was not specific to MBD2. This pilot screen provided proof principle that our assay format could identify MBD2 inhibitors and that a counter screen strategy could evaluate the specificity of identified hit compounds. In collaboration with the Scripps Institute in Florida, we further miniaturized, refined, and deployed the MBD2 TR-FRET assay to screen the NCI small molecule library of 370,276 compounds. The primary screening effort identified 1,149 active compounds (able to disrupt MBD2-DNA interaction) at 4.4µM. In the secondary screen each of these were run in triplicate at 4.4µM, with 271 (26.2%) of them validating as active hits. Next, a TR-FRET assay to evaluate inhibition of binding of the ubiquitin-like with PHD and ring finger domains 1 (UHRF1) protein to methylated DNA was utilized to evaluate specificity of the 271 active compounds that passed secondary screening. This yielded 48 small molecules that appeared to specifically inhibit the MBD2-DNA binding interaction. These 48 were subjected to the TR-FRET assay in a dose response from ∼10nM to 50µM on both MBD2 and UHRF1, yielding 18 active compounds which specifically inhibit MBD2-DNA interactions in a dose responsive manner with IC50's between 1µM and 17µM. Work is currently underway to test these compounds for biological activity. Acknowledgments/Funding- The authors would like to thank Peter Hodder, Virneliz Fernandez-Vega and Tom Bannister at the Scripps Institute-Florida for their efforts with this project. Funding provided by Department of Defense award W81XWH-11-1-0618 (to NW), NIH/NCI grants CA70196 and CA58236 (to WGN and SY), and the Prostate Cancer Foundation. Citation Format: Nicolas A. Wyhs, Hugh Giovinazzo, David Walker, Srinivasan Yegnasubramanian, William G. Nelson. Small molecular library screening to find methyl binding domain 2 protein inhibitors. [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 LB-129. doi:10.1158/1538-7445.AM2014-LB-129

Alterations in DNA methylation are a hallmark of prostate carcinogenesis. UHRF1 is an essential component in the machinery required for the maintenance of DNA methylation in actively dividing cells. In S-phase, UHRF1 binds nascent hemimethylated DNA and recruits DNA methyltransferases to copy the methylation pattern to the daughter DNA strand in a manner analogous to semi-conservative replication. Additionally, it has been shown that UHRF1 interacts with methylated histones and histone methyltransferases, making it an important epigenetic regulator in proliferating cells. Because of its ties to epigenetic gene silencing and cell proliferation, we hypothesized that UHRF1 could serve as a target for prostate cancer treatment. In this study, we have taken steps to credential UHRF1 as a therapeutic target in prostate cancer and have developed a high-throughput UHRF1-DNA binding assay to screen for UHRF1 inhibitors. We show that UHRF1 is overexpressed in a panel of prostate cancer cell lines and it appears to track with proliferation in prostate cancer tissue. Through loss-of-function studies in prostate cancer cell lines PC3 and DU145, we show that long-term UHRF1 depletion leads to a decrease in clonogenic survival and an overall reduction in global levels of DNA methylation. Further, we find that UHRF1 is required to maintain DNA methylation and epigenetic silencing of hypermethylated genes in prostate cancer cells. Additionally, ChIP experiments revealed that UHRF1 can be bound at the promoter CpG islands of epigenetically silenced genes in prostate cancer cells. With the goal of ultimately developing UHRF1 inhibitors, we have developed a time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay, compatible with high-throughput screening, that can detect the binding of the SRA domain of UHRF1 to hemimethylated DNA. In summary, we have shown that UHRF1 plays a critical role in maintaining the DNA methylation of epigenetically silenced genes in prostate cancer and have developed an assay for high-throughput screening of UHRF1 inhibitors. 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 992. doi:1538-7445.AM2012-992