Loss of LPAR6 and CAB39L dysregulates the basal-to-luminal urothelial differentiation program, contributing to bladder carcinogenesis.

We describe a strategy that combines histologic and molecular mapping that permits interrogation of the chronology of changes associated with cancer development on a whole-organ scale. Using this approach, we present the sequence of alterations around RB1 in the development of bladder cancer. We show that RB1 is not involved in initial expansion of the preneoplastic clone. Instead, we found a set of contiguous genes that we term "forerunner" genes whose silencing is associated with the development of plaque-like field effects initiating carcinogenesis. Specifically, we identified five candidate forerunner genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11) mapping near RB1. Two of these genes, LPAR6 and CAB39L, are preferentially downregulated in the luminal and basal subtypes of bladder cancer, respectively. Their loss of function dysregulates urothelial differentiation, sensitizing the urothelium to N-butyl-N-(4-hydroxybutyl)nitrosamine-induced cancers, which recapitulate the luminal and basal subtypes of human bladder cancer.

Molecular Analysis of Short- versus Long-Term Survivors of High-Grade Serous Ovarian Carcinoma.

Despite having similar histologic features, patients with high-grade serous ovarian carcinoma (HGSC) often experience highly variable outcomes. The underlying determinants for long-term survival (LTS, ≥10 years) versus short-term survival (STS, <3 years) are largely unknown. The present study sought to identify molecular predictors of LTS for women with HGSC. A cohort of 24 frozen HGSC samples was collected (12 LTS and 12 STS) and analyzed at DNA, RNA, and protein levels. OVCAR5 and OVCAR8 cell lines were used for in vitro validation studies. For in vivo studies, we injected OVCAR8 cells into the peritoneal cavity of female athymic nude mice. From RNAseq analysis, 11 genes were found to be differentially expressed between the STS and LTS groups (fold change > 2; false discovery rate < 0.01). In the subsequent validation cohort, transmembrane protein 62 (TMEM62) was found to be related to LTS. CIBERSORT analysis showed that T cells (follicular helper) were found at higher levels in tumors from LTS than STS groups. In vitro data using OVCAR5 and OVCAR8 cells showed decreased proliferation with TMEM62 overexpression and positive correlation with a longevity-regulating pathway (KEGG HSA04213) at the RNA level. In vivo analysis using the OVCAR8-TMEM62-TetON model showed decreased tumor burden in mice with high- vs. low-expressing TMEM62 tumors. Our results demonstrate that restoring TMEM62 may be a novel approach for treatment of HGSC. These findings may have implications for biomarker and intervention strategies to help improve patient outcomes

Gene Body Methylation of the Lymphocyte-Specific Gene CARD11 Results in Its Overexpression and Regulates Cancer mTOR Signaling.

Investigations into the function of nonpromoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated nonpromoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we examined 32 tumor types and identified 57 tumor suppressors and oncogenes out of 260 genes exhibiting a correlation of > 0.5 between gene body methylation and gene expression in at least one tumor type. The lymphocyte-specific gene CARD11 exhibits robust association between gene body methylation and expression across 19 of 32 tumor types examined. It is significantly overexpressed in kidney renal cell carcinoma (KIRC) and lung adenocarcinoma (LUAD) tumor tissues in comparison with respective control samples; and is significantly associated with lower overall survival in KIRC. Contrary to its canonical function in lymphocyte NFκB activation, CARD11 activates the mTOR pathway in KIRC and LUAD, resulting in suppressed autophagy. Furthermore, demethylation of a CpG island within the gene body of CARD11 decreases gene expression. Collectively, our study highlights how DNA methylation outside the promoter region can impact tumor progression. IMPLICATIONS: Our study describes a novel regulatory role of gene body DNA methylation-dependent CARD11 expression on mTOR signaling and its impact on tumor progression.

Overexpression of CD200 is a Stem Cell-Specific Mechanism of Immune Evasion in AML.

BACKGROUND: Acute myeloid leukemia (AML) stem cells (LSCs) are capable of surviving current standard chemotherapy and are the likely source of deadly, relapsed disease. While stem cell transplant serves as proof-of-principle that AML LSCs can be eliminated by the immune system, the translation of existing immunotherapies to AML has been met with limited success. Consequently, understanding and exploiting the unique immune-evasive mechanisms of AML LSCs is critical. METHODS: Analysis of stem cell datasets and primary patient samples revealed CD200 as a putative stem cell-specific immune checkpoint overexpressed in AML LSCs. Isogenic cell line models of CD200 expression were employed to characterize the interaction of CD200+ AML with various immune cell subsets both in vitro and in peripheral blood mononuclear cell (PBMC)-humanized mouse models. CyTOF and RNA-sequencing were performed on humanized mice to identify novel mechanisms of CD200-mediated immunosuppression. To clinically translate these findings, we developed a fully humanized CD200 antibody (IgG1) that removed the immunosuppressive signal by blocking interaction with the CD200 receptor while also inducing a potent Fc-mediated response. Therapeutic efficacy of the CD200 antibody was evaluated using both humanized mice and patient-derived xenograft models. RESULTS: Our results demonstrate that CD200 is selectively overexpressed in AML LSCs and is broadly immunosuppressive by impairing cytokine secretion in both innate and adaptive immune cell subsets. In a PBMC-humanized mouse model, CD200+ leukemia progressed rapidly, escaping elimination by T cells, compared with CD200- AML. T cells from mice with CD200+ AML were characterized by an abundance of metabolically quiescent CD8+ central and effector memory cells. Mechanistically, CD200 expression on AML cells significantly impaired OXPHOS metabolic activity in T cells from healthy donors. Importantly, CD200 antibody therapy could eliminate disease in the presence of graft-versus-leukemia in immune competent mice and could significantly improve the efficacy of low-intensity azacitidine/venetoclax chemotherapy in immunodeficient hosts. CONCLUSIONS: Overexpression of CD200 is a stem cell-specific marker that contributes to immunosuppression in AML by impairing effector cell metabolism and function. CD200 antibody therapy is capable of simultaneously reducing CD200-mediated suppression while also engaging macrophage activity. This study lays the groundwork for CD200-targeted therapeutic strategies to eliminate LSCs and prevent AML relapse.

Methylation-eQTL analysis in cancer research.

MOTIVATION: DNA methylation is a key epigenetic factor regulating gene expression. While promoter methylation has been well studied, recent publications have revealed that functionally important methylation also occurs in intergenic and distal regions, and varies across genes and tissue types. Given the growing importance of inter-platform integrative genomic analyses, there is an urgent need to develop methods to discover and characterize gene-level relationships between methylation and expression. RESULTS: We introduce a novel sequential penalized regression approach to identify methylation-expression quantitative trait loci (methyl-eQTLs), a term that we have coined to represent, for each gene and tissue type, a sparse set of CpG loci best explaining gene expression and accompanying weights indicating direction and strength of association. Using TCGA and MD Anderson colorectal cohorts to build and validate our models, we demonstrate our strategy better explains expression variability than current commonly used gene-level methylation summaries. The methyl-eQTLs identified by our approach can be used to construct gene-level methylation summaries that are maximally correlated with gene expression for use in integrative models, and produce a tissue-specific summary of which genes appear to be strongly regulated by methylation. Our results introduce an important resource to the biomedical community for integrative genomics analyses involving DNA methylation. AVAILABILITY AND IMPLEMENTATION: We produce an R Shiny app (https://rstudio-prd-c1.pmacs.upenn.edu/methyl-eQTL/) that interactively presents methyl-eQTL results for colorectal, breast and pancreatic cancer. The source R code for this work is provided in the Supplementary Material. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Clinical significance of homologous recombination deficiency score testing in endometrial Cancer.

BACKGROUND: Homologous recombination deficiency (HRD) score is related to chemotherapy response in some cancers, but its role in endometrial cancer in not known. We determined frequency and clinical significance of alterations in the HR pathway in endometrial cancer. METHODS: 253 endometrioid endometrial adenocarcinoma (EEA) samples from two independent cohorts (discovery and replication) were tested for HRD score using the Myriad HRD assay, microsatellite instability (MSI) and tumor mutation burden (TMB) using a next generation sequencing assay. HRD scores were also generated on endometrial cancer cell lines and in vivo response to olaparib was assessed. RESULTS: ROC curves were employed to determine optimal cutoffs of HRD in relation to survival impact in endometrial cancer and a cutoff of HRD ≥ 4 was suggested for DFS using the discovery cohort. Patients from two independent cohorts with HRD score ≥ 4 trended toward worse survival as compared to those with HRD score < 4. Both cohorts were further separated into four groups according to molecular subtypes (TMB positive; MSI positive; HRD positive; all others). When grouped by molecular subtype, there was a significant difference between groups using an HRD ≥4 cutoff in the initial (p = 0.0024) and replication (p = 0.042) cohorts. The Hec1a model (HRD score = 19) was highly sensitive to olaparib in in vitro and in vivo experiments. CONCLUSIONS: High HRD score was associated with worse DFS in our patient cohort. These findings suggest that HRD score may have clinical utility in patients with advanced or recurrent endometrial cancer.

Blockade of the Short Form of Prolactin Receptor Induces FOXO3a/EIF-4EBP1-Mediated Cell Death in Uterine Cancer.

Abnormal activity of human prolactin (PRL) and its membrane-associated receptor (PRLR) contributes to the progression of uterine carcinoma. However, the underlying mechanisms are not well understood, and current means of targeting the PRL/PRLR axis in uterine cancer are limited. Our integrated analyses using The Cancer Genome Atlas and Genotype-Tissue Expression (GTEx) databases demonstrated that a short form of PRLR (PRLR_SF) is the isoform predominantly expressed in human uterine cancers; expression of this PRLR_SF was elevated in uterine cancers in comparison with cancer-free uterine tissues. We hypothesized that the overexpression of PRLR_SF in uterine cancer cells contributes, in part, to the oncogenic activity of the PRL/PRLR axis. Next, we employed G129R, an antagonist of human PRL, to block the PRL/PRLR axis in both PTEN wt and PTEN mut orthotopic mouse models of uterine cancer. In comparison with control groups, treatment with G129R as monotherapy or in combination with paclitaxel resulted in a significant reduction of growth and progression of orthotopic uterine tumors. Results from protein profiling of uterine cancer cells and in vivo tumors revealed a set of new downstream targets for G129R. Our results showed that G129R induced sub-G0 population arrest, decreased nascent protein synthesis, and initiated FOXO3a/EIF-4EBP1-mediated cell death in both PTEN wt and PTEN mut uterine cancer cells. Collectively, our results show a unique pattern of PRLR_SF expression predominantly in uterine cancer. Moreover, FOXO3a and EIF-4EBP1 are important mediators of cell death following G129R treatment in uterine cancer models.

Molecular Analysis of Clinically Defined Subsets of High-Grade Serous Ovarian Cancer.

The diversity and heterogeneity within high-grade serous ovarian cancer (HGSC), which is the most lethal gynecologic malignancy, is not well understood. Here, we perform comprehensive multi-platform omics analyses, including integrated analysis, and immune monitoring on primary and metastatic sites from highly clinically annotated HGSC samples based on a laparoscopic triage algorithm from patients who underwent complete gross resection (R0) or received neoadjuvant chemotherapy (NACT) with excellent or poor response. We identify significant distinct molecular abnormalities and cellular changes and immune cell repertoire alterations between the groups, including a higher rate of NF1 copy number loss, and reduced chromothripsis-like patterns, higher levels of strong-binding neoantigens, and a higher number of infiltrated T cells in the R0 versus the NACT groups.

Oral MEK 1/2 Inhibitor Trametinib in Combination With AKT Inhibitor GSK2141795 in Patients With Acute Myeloid Leukemia With RAS Mutations: A Phase II Study.

BACKGROUND: With proven single-agent activity and favorable toxicity profile of MEK-1/2 inhibition in advanced leukemia, investigation into combination strategies to overcome proposed resistance pathways is warranted. Resistance to MEK inhibition is secondary to upstream hyperactivation of RAS/RAF or activation of the PI3K/PTEN/AKT/mTOR pathway. This phase II multi-institution Cancer Therapy Evaluation Program-sponsored study was conducted to determine efficacy and safety of the combination of the ATP-competitive pan-AKT inhibitor GSK2141795, targeting the PI3K/AKT pathway, and the MEK inhibitor trametinib in RAS-mutated relapsed/refractory acute myeloid leukemia (AML). PATIENTS AND METHODS: The primary objective was to determine the proportion of patients achieving a complete remission. Secondary objectives included assessment of toxicity profile and biologic effects of this combination. Twenty-three patients with RAS-mutated AML received the combination. Two dose levels were explored (dose level 1: 2 mg trametinib, 25 mg GSK2141795 and dose level 2: 1.5 mg trametinib, 50 mg GSK2141795). RESULTS: Dose level 1 was identified as the recommended phase II dose. No complete remissions were identified in either cohort. Minor responses were recognized in 5 (22%) patients. The most common drug-related toxicities included rash and diarrhea, with dose-limiting toxicities of mucositis and colitis. Longitudinal correlative assessment of the modulation of MEK and AKT pathways using reverse phase protein array and phospho-flow analysis revealed significant and near significant down-modulation of pERK and pS6, respectively. Combined MEK and AKT inhibition had no clinical activity in patients with RAS-mutated AML. CONCLUSION: Further investigation is required to explore the discrepancy between the activity of this combination on leukemia cells and the lack of clinical efficacy.

Dysregulation of EMT Drives the Progression to Clinically Aggressive Sarcomatoid Bladder Cancer.

Sarcomatoid urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARC and 84 cases of conventional urothelial carcinoma (UC), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs show a distinct mutational landscape, with enrichment of TP53, RB1, and PIK3CA mutations. They are related to the basal molecular subtype of conventional UCs and could be divided into epithelial-basal and more clinically aggressive mesenchymal subsets on the basis of TP63 and its target gene expression levels. Other analyses reveal that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of the EMT network, and nearly half exhibit a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer.

Proteomic Profiling of Signaling Networks Modulated by G-CSF/Plerixafor/Busulfan-Fludarabine Conditioning in Acute Myeloid Leukemia Patients in Remission or with Active Disease prior to Allogeneic Stem Cell Transplantation.

To characterize intracellular signaling in peripheral blood (PB) cells of acute myeloid leukemia (AML) patients undergoing pretransplant conditioning with CXCR4 inhibitor plerixafor, granulocyte colony-stimulating factor (G-CSF), and busulfan plus fludarabine (Bu+Flu) chemotherapy, we profiled 153 proteins in 33 functional groups using reverse phase protein array. CXCR4 inhibition mobilized AML progenitors and clonal AML cells, and this was associated with molecular markers of cell cycle progression. G-CSF/plerixafor and G-CSF/plerixafor/Bu+Flu modulated distinct signaling networks in AML blasts of patients undergoing conditioning with active disease compared to nonleukemic PB cells of patients in remission. We identified AML-specific proteins that remained aberrantly expressed after chemotherapy, representing putative chemoresistance markers in AML.

Pan-cancer genomic analysis links 3'UTR DNA methylation with increased gene expression in T cells.

BACKGROUND: Investigations into the function of non-promoter DNA methylation have yielded new insights into the epigenetic regulation of gene expression. However, integrated genome-wide non-promoter DNA methylation and gene expression analyses across a wide number of tumour types and corresponding normal tissues have not been performed. METHODS: To investigate the impact of non-promoter DNA methylation on cancer pathogenesis, we performed a large-scale analysis of gene expression and DNA methylation profiles, finding enrichment in the 3'UTR DNA methylation positively correlated with gene expression. Filtering for genes in which 3'UTR DNA methylation strongly correlated with gene expression yielded a list of genes enriched for functions involving T cell activation. FINDINGS: The important immune checkpoint gene Havcr2 showed a substantial increase in 3'UTR DNA methylation upon T cell activation and subsequent upregulation of gene expression in mice. Furthermore, this increase in Havcr2 gene expression was abrogated by treatment with decitabine. INTERPRETATION: These findings indicate that the 3'UTR is a functionally relevant DNA methylation site. Additionally, we show a potential novel mechanism of HAVCR2 regulation in T cells, providing new insights for modulating immune checkpoint blockade.

Complementary Longitudinal Serum Biomarkers to CA125 for Early Detection of Ovarian Cancer.

Early detection of ovarian cancer has the potential to impact mortality. A multimodal screening strategy where rising CA125 values over time, analyzed with the risk of ovarian cancer algorithm (ROCA), triggers transvaginal sonography and possible surgery has high sensitivity and specificity, but still fails to detect the 20% of early-stage cases that do not express CA125. Use of multiple biomarkers could detect cases missed by CA125. We have studied the sensitivity and lead time of a multi-marker panel (CA125, HE4, MMP-7, and CA 72-4) compared with CA125 alone. We used PRoBE design principles to select preclinical longitudinal specimens from 75 women (50 screen-positive, 25 screen-negative) who developed invasive epithelial ovarian cancer (3-5 serial specimens each) and 547 corresponding healthy controls (1-10 serial specimens each) from the ovarian cancer screening trial, UKCTOCS, in a blinded fashion. We measured the multi-marker concentrations in ultra-low serum volumes (16 µL) utilizing multiplexed bead-based immunoassays with low detection limits, high inter- and intra-assay precision, negligible cross-reactivity, and good correlation with standard immunoassays. While, at least one of the complementary biomarkers rose with CA125 in 44% (22/50) of screen-positive cases, there was no advantage in lead time over CA125. Therefore, we developed single-marker longitudinal algorithms (ROCA-like) to determine the presence of a change point to distinguish between the cases and controls. Using these algorithms, at 98% specificity, HE4 and CA72-4 identified 16% (4/25) of screen-negative cases, while MMP-7 identified none. Taken together, HE4 and CA72-4 show promise as complementary biomarkers to CA125 for longitudinal screening.

Whole-Organ Genomic Characterization of Mucosal Field Effects Initiating Bladder Carcinogenesis.

We used whole-organ mapping to study the locoregional molecular changes in a human bladder containing multifocal cancer. Widespread DNA methylation changes were identified in the entire mucosa, representing the initial field effect. The field effect was associated with subclonal low-allele frequency mutations and a small number of DNA copy alterations. A founder mutation in the RNA splicing gene, ACIN1, was identified in normal mucosa and expanded clonally with an additional 21 mutations in progression to carcinoma. The patterns of mutations and copy number changes in carcinoma in situ and foci of carcinoma were almost identical, confirming their clonal origins. The pathways affected by the DNA copy alterations and mutations, including the Kras pathway, were preceded by the field changes in DNA methylation, suggesting that they reinforced mechanisms that had already been initiated by methylation. The results demonstrate that DNA methylation can serve as the initiator of bladder carcinogenesis.

Detection of Bladder Cancer in Urine Sediments by a Novel Multicolor Fluorescence In Situ Hybridization (Quartet) Test.

BACKGROUND: Bladder cancer is among the common human malignancies that show a heavy mutational load and copy number variations of numerous chromosomes, which makes them a target for diagnostic explorations. OBJECTIVE: We aimed to design a multicolor fluorescence in situ hybridization (FISH) test referred to as the quartet test for the detection of bladder cancer in urine. DESIGN, SETTING, AND PARTICIPANTS: We performed genome-wide copy number variation analysis on cohorts from the University of Texas MD Anderson Cancer Center (n=40) and The Cancer Genome Atlas (n=129), and identified the most frequently amplified chromosomal regions. These data were used to select four of the amplified regions to design a multicolor FISH test, referred to as the quartet test. Assay validation was performed on urine samples from 98 patients with bladder cancer: 56 with low-grade papillary, 42 with high-grade invasive disease, and 48 benign controls. INTERVENTION: The quartet test can be used in clinical practice for noninvasive detection of bladder cancer. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We initially analyzed samples using a fraction of abnormal cell scores and then by the quantitative score, which included not only the proportion of cells with abnormal copy numbers, but also the proportion of cells with numbers of altered copies and degree of amplification. We used receiver operator characteristic (ROC) curves to identify cutoff values for the scores at which performances of sensitivity and specificity were maximized. RESULTS AND LIMITATIONS: The copy number status assessed by probes detected in voided urine reflected the amplification status of the primary tumor. An ROC curve summarizing the proportion of assayed cells with any abnormal copy numbers gave specificity of 93.8% and sensitivity of 78.6% using the proportion of cells with abnormal copy numbers. The quantitative score giving extra weight to cells with multiple simultaneous amplifications provided 95.8% specificity and 76.8% sensitivity. Both percentage of abnormal cells and quantitative scores were highly effective for assessing the grade of the tumor. The full spectrum of potential clinical applications was not explored in the current study, and further validation studies are needed. CONCLUSIONS: The quartet test shows promising specificity and sensitivity results, but it requires validation on a larger multi-institutional cohort of samples. PATIENT SUMMARY: The quartet test can be used for noninvasive detection of bladder cancer in voided urine. It can also be used to assess the grade of the tumor and tumor recurrence as well as post-treatment effects.

Predicting Novel Therapies and Targets: Regulation of Notch3 by the Bromodomain Protein BRD4.

Systematic approaches for accurate repurposing of targeted therapies are needed. We developed and aimed to biologically validate our therapy predicting tool (TPT) for the repurposing of targeted therapies for specific tumor types by testing the role of Bromodomain and Extra-Terminal motif inhibitors (BETi) in inhibiting BRD4 function and downregulating Notch3 signaling in ovarian cancer.Utilizing established ovarian cancer preclinical models, we carried out in vitro and in vivo studies with clinically relevant BETis to determine their therapeutic effect and impact on Notch3 signaling.Treatment with BETis or siRNA-mediated BRD4 knockdown resulted in decreased cell viability, reduced cell proliferation, and increased cell apoptosis in vitro. In vivo studies with orthotopic mouse models demonstrated that treatment with BETi decreased tumor growth. In addition, knockdown of BRD4 with doxycycline-inducible shRNA increased survival up to 50% (P < 0.001). Treatment with either BETis or BRD4 siRNA decreased Notch3 expression both in vitro and in vivo BRD4 inhibition also decreased the expression of NOTCH3 targets, including HES1 Chromatin immunoprecipitation revealed that BRD4 was present at the NOTCH3 promoter.Our findings provide biological validation for the TPT by demonstrating that BETis can be an effective therapeutic agent for ovarian cancer by downregulating Notch3 expression.The TPT could rapidly identify candidate drugs for ovarian or other cancers along with novel companion biomarkers.

FABP4 as a key determinant of metastatic potential of ovarian cancer.

The standard treatment for high-grade serous ovarian cancer is primary debulking surgery followed by chemotherapy. The extent of metastasis and invasive potential of lesions can influence the outcome of these primary surgeries. Here, we explored the underlying mechanisms that could increase metastatic potential in ovarian cancer. We discovered that FABP4 (fatty acid binding protein) can substantially increase the metastatic potential of cancer cells. We also found that miR-409-3p regulates FABP4 in ovarian cancer cells and that hypoxia decreases miR-409-3p levels. Treatment with DOPC nanoliposomes containing either miR-409-3p mimic or FABP4 siRNA inhibited tumor progression in mouse models. With RPPA and metabolite arrays, we found that FABP4 regulates pathways associated with metastasis and affects metabolic pathways in ovarian cancer cells. Collectively, these findings demonstrate that FABP4 is functionally responsible for aggressive patterns of disease that likely contribute to poor prognosis in ovarian cancer.