Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Dose, schedule, safety, and efficacy of guadecitabine in relapsed or refractory acute myeloid leukemia.

BACKGROUND: Outcomes for patients with relapsed or refractory acute myeloid leukemia (AML) are poor. Guadecitabine, a next-generation hypomethylating agent, could be useful in treating such patients. METHODS: In this multicenter, open-label, phase 2 dose-expansion study, AML patients from 10 North American medical centers were first randomized (1:1) to receive subcutaneous guadecitabine at 60 or 90 mg/m2 on 5 consecutive days in each 28-day cycle (5-day regimen). Subsequently, another cohort was treated for 10 days with 60 mg/m2 (10-day regimen). RESULTS: Between June 15, 2012, and August 19, 2013, 108 patients with previously treated AML consented to enroll in the study, and 103 of these patients were treated; 5 patients did not receive the study treatment. A total of 103 patients were included in the safety and efficacy analyses (24 and 26 patients who were randomly assigned to 60 and 90 mg/m2 /d, respectively [5-day regimen] and 53 patients who were assigned to 60 mg/m2 /d [10-day regimen]). The 90 mg/m2 dose showed no benefit in clinical outcomes in comparison with 60 mg/m2 in the randomized cohort. Composite complete response (CRc) and complete response (CR) rates were higher with the 10-day regimen versus the 5-day regimen (CRc, 30.2% vs 16.0%; P = .1061; CR, 18.9% vs 8%; P = .15). Adverse events (grade ≥ 3) were mainly hematologic, with a higher incidence on the 10-day regimen. Early all-cause mortality was low and similar between regimens. Twenty patients (8 on the 5-day regimen and 12 on the 10-day regimen) were bridged to hematopoietic cell transplantation. CONCLUSIONS: Guadecitabine has promising clinical activity and an acceptable safety profile and thus warrants further development in this population. Cancer 2018;124:325-34. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Safety and tolerability of guadecitabine (SGI-110) in patients with myelodysplastic syndrome and acute myeloid leukaemia: a multicentre, randomised, dose-escalation phase 1 study.

BACKGROUND: Hypomethylating agents are used to treat cancers driven by aberrant DNA methylation, but their short half-life might limit their activity, particularly in patients with less proliferative diseases. Guadecitabine (SGI-110) is a novel hypomethylating dinucleotide of decitabine and deoxyguanosine resistant to degradation by cytidine deaminase. We aimed to assess the safety and clinical activity of subcutaneously given guadecitabine in patients with acute myeloid leukaemia or myelodysplastic syndrome. METHODS: In this multicentre, open-label, phase 1 study, patients from nine North American medical centres with myelodysplastic syndrome or acute myeloid leukaemia that was refractory to or had relapsed after standard treatment were randomly assigned (1:1) to receive subcutaneous guadecitabine, either once-daily for 5 consecutive days (daily × 5), or once-weekly for 3 weeks, in a 28-day treatment cycle. Patients were stratified by disease. A 3 + 3 dose-escalation design was used in which we treated patients with guadecitabine doses of 3-125 mg/m(2) in separate dose-escalation cohorts. A twice-weekly treatment schedule was added to the study after a protocol amendment. The primary objective was to assess safety and tolerability of guadecitabine, determine the maximum tolerated and biologically effective dose, and identify the recommended phase 2 dose of guadecitabine. Safety analyses included all patients who received at least one dose of guadecitabine. Pharmacokinetic and pharmacodynamic analyses to determine the biologically effective dose included all patients for whom samples were available. This study is registered with ClinicalTrials.gov, number NCT01261312. FINDINGS: Between Jan 4, 2011, and April 11, 2014, we enrolled and treated 93 patients: 35 patients with acute myeloid leukaemia and nine patients with myelodysplastic syndrome in the daily × 5 dose-escalation cohorts, 28 patients with acute myeloid leukaemia and six patients with myelodysplastic syndrome in the once-weekly dose-escalation cohorts, and 11 patients with acute myeloid leukaemia and four patients with myelodysplastic syndrome in the twice-weekly dose-escalation cohorts. The most common grade 3 or higher adverse events were febrile neutropenia (38 [41%] of 93 patients), pneumonia (27 [29%] of 93 patients), thrombocytopenia (23 [25%] of 93 patients), anaemia (23 [25%] of 93 patients), and sepsis (16 [17%] of 93 patients). The most common serious adverse events were febrile neutropenia (29 [31%] of 93 patients), pneumonia (26 [28%] of 93 patients), and sepsis (16 [17%] of 93 patients). Six of the 74 patients with acute myeloid leukaemia and six of the 19 patients with myelodysplastic syndrome had a clinical response to treatment. Two dose-limiting toxicities were noted in patients with myelodysplastic syndrome at 125 mg/m(2) daily × 5, thus the maximum tolerated dose in patients with myelodysplastic syndrome was 90 mg/m(2) daily × 5. The maximum tolerated dose was not reached in patients with acute myeloid leukaemia. Potent dose-related DNA demethylation occurred on the daily × 5 regimen, reaching a plateau at 60 mg/m(2) (designated as the biologically effective dose). INTERPRETATION: Guadecitabine given subcutaneously at 60 mg/m(2) daily × 5 is well tolerated and is clinically and biologically active in patients with myelodysplastic syndrome and acute myeloid leukaemia. Guadecitabine 60 mg/m(2) daily × 5 is the recommended phase 2 dose, and these findings warrant further phase 2 studies. FUNDING: Astex Pharmaceuticals, Stand Up To Cancer.

Colorectal carcinomas with CpG island methylator phenotype 1 frequently contain mutations in chromatin regulators.

BACKGROUND & AIMS: Subgroups of colorectal carcinomas (CRCs) characterized by DNA methylation anomalies are termed CpG island methylator phenotype (CIMP)1, CIMP2, or CIMP-negative. The pathogenesis of CIMP1 colorectal carcinomas, and their effects on patients' prognoses and responses to treatment, differ from those of other CRCs. We sought to identify genetic somatic alterations associated with CIMP1 CRCs. METHODS: We examined genomic DNA samples from 100 primary CRCs, 10 adenomas, and adjacent normal-appearing mucosae from patients undergoing surgery or colonoscopy at 3 tertiary medical centers. We performed exome sequencing of 16 colorectal tumors and their adjacent normal tissues. Extensive comparison with known somatic alterations in CRCs allowed segregation of CIMP1-exclusive alterations. The prevalence of mutations in selected genes was determined from an independent cohort. RESULTS: We found that genes that regulate chromatin were mutated in CIMP1 CRCs; the highest rates of mutation were observed in CHD7 and CHD8, which encode members of the chromodomain helicase/adenosine triphosphate-dependent chromatin remodeling family. Somatic mutations in these 2 genes were detected in 5 of 9 CIMP1 CRCs. A prevalence screen showed that nonsilencing mutations in CHD7 and CHD8 occurred significantly more frequently in CIMP1 tumors (18 of 42 [43%]) than in CIMP2 (3 of 34 [9%]; P < .01) or CIMP-negative tumors (2 of 34 [6%]; P < .001). CIMP1 markers had increased binding by CHD7, compared with all genes. Genes altered in patients with CHARGE syndrome (congenital malformations involving the central nervous system, eye, ear, nose, and mediastinal organs) who had CHD7 mutations were also altered in CRCs with mutations in CHD7. CONCLUSIONS: Aberrations in chromatin remodeling could contribute to the development of CIMP1 CRCs. A better understanding of the biological determinants of CRCs can be achieved when these tumors are categorized according to their epigenetic status.

Genome architecture marked by retrotransposons modulates predisposition to DNA methylation in cancer.

Epigenetic silencing plays an important role in cancer development. An attractive hypothesis is that local DNA features may participate in differential predisposition to gene hypermethylation. We found that, compared with methylation-resistant genes, methylation-prone genes have a lower frequency of SINE and LINE retrotransposons near their transcription start site. In several large testing sets, this distribution was highly predictive of promoter methylation. Genome-wide analysis showed that 22% of human genes were predicted to be methylation-prone in cancer; these tended to be genes that are down-regulated in cancer and that function in developmental processes. Moreover, retrotransposon distribution marks a larger fraction of methylation-prone genes compared to Polycomb group protein (PcG) marking in embryonic stem cells; indeed, PcG marking and our predictive model based on retrotransposon frequency appear to be correlated but also complementary. In summary, our data indicate that retrotransposon elements, which are widespread in our genome, are strongly associated with gene promoter DNA methylation in cancer and may in fact play a role in influencing epigenetic regulation in normal and abnormal physiological states.

A Phase II Trial of Guadecitabine plus Atezolizumab in Metastatic Urothelial Carcinoma Progressing after Initial Immune Checkpoint Inhibitor Therapy.

PURPOSE: On the basis of preclinical evidence of epigenetic contribution to sensitivity and resistance to immune checkpoint inhibitors (ICI), we hypothesized that guadecitabine (hypomethylating agent) and atezolizumab [anti-programmed cell death ligand 1 (PD-L1)] together would potentiate a clinical response in patients with metastatic urothelial carcinoma (UC) unresponsive to initial immune checkpoint blockade therapy. PATIENTS AND METHODS: We designed a single arm phase II study (NCT03179943) with a safety run-in to identify the recommended phase II dose of the combination therapy of guadecitabine and atezolizumab. Patients with recurrent/advanced UC who had previously progressed on ICI therapy with programmed cell death protein 1 or PD-L1 targeting agents were eligible. Preplanned correlative analysis was performed to characterize peripheral immune dynamics and global DNA methylation, transcriptome, and immune infiltration dynamics of patient tumors. RESULTS: Safety run-in enrolled 6 patients and phase II enrolled 15 patients before the trial was closed for futility. No dose-limiting toxicity was observed. Four patients, with best response of stable disease (SD), exhibited extended tumor control (8-11 months) and survival (>14 months). Correlative analysis revealed lack of DNA demethylation in tumors after 2 cycles of treatment. Increased peripheral immune activation and immune infiltration in tumors after treatment correlated with progression-free survival and SD. Furthermore, high IL6 and IL8 levels in the patients' plasma was associated with short survival. CONCLUSIONS: No RECIST responses were observed after combination therapy in this trial. Although we could not detect the anticipated tumor-intrinsic effects of guadecitabine, the addition of hypomethylating agent to ICI therapy induced immune activation in a few patients, which associated with longer patient survival.

Tumor-associated methylation of the putative tumor suppressor AJAP1 gene and association between decreased AJAP1 expression and shorter survival in patients with glioma.

Allelic loss of the short arm of chromosome 1 has been observed frequently in a wide spectrum of cancers, most frequently in oligodendroglioma. In our previous studies, we evaluated 177 oligodendroglial tumor samples and identified the AJAP1 gene (formerly Shrew1) in the consensus region of deletion. AJAP1 is a transmembrane protein found in adheren junctions and functions to inhibit glioma cell adhesion and migration. Whereas a putative tumor suppressor gene, we did not detect AJAP1 gene mutations. In subsequent studies, we found that AJAP1 was underexpressed in oligodendrogliomas relative to normal brain tissues. Bioinformatic analysis revealed the presence of CpG islands in the promoter of AJAP1. Methylation analysis of the AJAP1 promoter identified hypermethylation in 21% of oligodendrogliomas (n =27), and the degree of methylation correlated with low levels of AJAP1 expression (P = 0.045). The AJAP1 promoter was also highly methylated in a wide spectrum of cell lines (n = 22), including cell lines of glioblastoma. Analysis of the National Cancer Institute's REMBRANDT dataset, which contains 343 glioma samples, indicated that low AJAP1 gene expression was associated with decreased survival. Thus, both genetic (gene deletion) and epigenetic alterations (promoter methylation) are likely mechanisms that inactivate the putative tumor suppressor AJAP1 in gliomas, which contributes to poor prognosis.

Sensitive and specific detection of early gastric cancer with DNA methylation analysis of gastric washes.

BACKGROUND & AIMS: Aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for detection of gastric neoplasia. We hypothesized that methylation analysis of DNA recovered from gastric washes could be used to detect gastric cancer. METHODS: We studied 51 candidate genes in 7 gastric cancer cell lines and 24 samples (training set) and identified 6 for further studies. We examined the methylation status of these genes in a test set consisting of 131 gastric neoplasias at various stages. Finally, we validated the 6 candidate genes in a different population of 40 primary gastric cancer samples and 113 nonneoplastic gastric mucosa samples. RESULTS: Six genes (MINT25, RORA, GDNF, ADAM23, PRDM5, MLF1) showed frequent differential methylation between gastric cancer and normal mucosa in the training, test, and validation sets. GDNF and MINT25 were most sensitive molecular markers of early stage gastric cancer, whereas PRDM5 and MLF1 were markers of a field defect. There was a close correlation (r = 0.5-0.9, P = .03-.001) between methylation levels in tumor biopsy and gastric washes. MINT25 methylation had the best sensitivity (90%), specificity (96%), and area under the receiver operating characteristic curve (0.961) in terms of tumor detection in gastric washes. CONCLUSIONS: These findings suggest MINT25 is a sensitive and specific marker for screening in gastric cancer. Additionally, we have developed a new method for gastric cancer detection by DNA methylation in gastric washes.

Aberrant CpG island methylation in acute myeloid leukemia is accentuated at relapse.

DNA methylation of CpG islands around gene transcription start sites results in gene silencing and plays a role in leukemia pathophysiology. Its impact in leukemia progression is not fully understood. We performed genomewide screening for methylated CpG islands and identified 8 genes frequently methylated in leukemia cell lines and in patients with acute myeloid leukemia (AML): NOR1, CDH13, p15, NPM2, OLIG2, PGR, HIN1, and SLC26A4. We assessed the methylation status of these genes and of the repetitive element LINE-1 in 30 patients with AML, both at diagnosis and relapse. Abnormal methylation was found in 23% to 83% of patients at diagnosis and in 47% to 93% at relapse, with CDH13 being the most frequently methylated. We observed concordance in methylation of several genes, confirming the presence of a hypermethylator pathway in AML. DNA methylation levels increased at relapse in 25 of 30 (83%) patients with AML. These changes represent much larger epigenetic dysregulation, since methylation microarray analysis of 9008 autosomal genes in 4 patients showed hypermethylation ranging from 5.9% to 13.6% (median 8.3%) genes at diagnosis and 8.0% to 15.2% (median 10.6%) genes in relapse (P < .001). Our data suggest that DNA methylation is involved in AML progression and provide a rationale for the use of epigenetic agents in remission maintenance.

Genomic and epigenomic predictors of response to guadecitabine in relapsed/refractory acute myelogenous leukemia.

BACKGROUND: Guadecitabine is a novel DNA methyltransferase (DNMT) inhibitor with improved pharmacokinetics and clinical activity in a subset of patients with relapsed/refractory acute myeloid leukemia (r/r AML), but identification of this subset remains difficult. METHODS: To search for biomarkers of response, we measured genome-wide DNA methylation, mutations of 54 genes, and expression of a panel of 7 genes in pre-treatment samples from 128 patients treated at therapeutic doses in a phase I/II study. RESULTS: Response rate to guadecitabine was 17% (2 complete remission (CR), 3 CR with incomplete blood count recovery (CRi), or CR with incomplete platelets recovery (CRp)) in the phase I component and 23% (14 CR, 9 CRi/CRp) in phase II. There were no strong mutation or methylation predictors of response. Gene expression clustering defined a subset of patients (~ 20%) that had (i) high DNMT3B and low CDKN2B, CTCF, and CDA expression; (ii) enrichment for KRAS/NRAS mutations; (iii) frequent CpG island hypermethylation; (iv) low long interspersed nuclear element 1 (LINE-1) hypomethylation after treatment; and (v) resistance to guadecitabine in both phase I (response rate 0% vs. 33%, p = 0.07) and phase II components of the study (response rate 5% vs. 30%, p = 0.02). Multivariate analysis identified peripheral blood (PB) blasts and hemoglobin as predictors of response and cytogenetics, gene expression, RAS mutations, and hemoglobin as predictors of survival. CONCLUSIONS: A subset of patients (~ 20%) with r/r AML is unlikely to benefit from guadecitabine as a single agent. In the remaining 80%, guadecitabine is a viable option with a median survival of 8 months and a 2-year survival rate of 21%. TRIAL REGISTRATION: NCT01261312 .

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.

Understanding the development of human bladder cancer by using a whole-organ genomic mapping strategy.

The search for the genomic sequences involved in human cancers can be greatly facilitated by maps of genomic imbalances identifying the involved chromosomal regions, particularly those that participate in the development of occult preneoplastic conditions that progress to clinically aggressive invasive cancer. The integration of such regions with human genome sequence variation may provide valuable clues about their overall structure and gene content. By extension, such knowledge may help us understand the underlying genetic components involved in the initiation and progression of these cancers. We describe the development of a genome-wide map of human bladder cancer that tracks its progression from in situ precursor conditions to invasive disease. Testing for allelic losses using a genome-wide panel of 787 microsatellite markers was performed on multiple DNA samples, extracted from the entire mucosal surface of the bladder and corresponding to normal urothelium, in situ preneoplastic lesions, and invasive carcinoma. Using this approach, we matched the clonal allelic losses in distinct chromosomal regions to specific phases of bladder neoplasia and produced a detailed genetic map of bladder cancer development. These analyses revealed three major waves of genetic changes associated with growth advantages of successive clones and reflecting a stepwise conversion of normal urothelial cells into cancer cells. The genetic changes map to six regions at 3q22-q24, 5q22-q31, 9q21-q22, 10q26, 13q14, and 17p13, which may represent critical hits driving the development of bladder cancer. Finally, we performed high-resolution mapping using single nucleotide polymorphism markers within one region on chromosome 13q14, containing the model tumor suppressor gene RB1, and defined a minimal deleted region associated with clonal expansion of in situ neoplasia. These analyses provided new insights on the involvement of several non-coding sequences mapping to the region and identified novel target genes, termed forerunner (FR) genes, involved in early phases of cancer development.

Age-related DNA methylation changes in normal human prostate tissues.

PURPOSE: Prostate cancer is a leading cause of cancer death among the aging male population but the mechanism underlying this association is unclear. Aberrant methylation of promoter CpG islands is associated with silencing of genes and age-dependent methylation of several genes has been proposed as a risk factor for sporadic cancer. We examined the extent of gene methylation in pathologically normal human prostate as a function of age. EXPERIMENTAL DESIGN: We used pyrosequencing to quantitatively analyze the methylation status of nine CpG islands in normal prostate tissue DNA from 45 organ donors and 45 patients who had undergone cystoprostatectomy for bladder cancer. We also analyzed 12 pairs of matched benign and prostate cancer tissue DNA from patients with prostate cancer. RESULTS: Linear regression analysis revealed a significant increase in promoter methylation levels correlating with age for CpG islands at RARbeta2 (r = 0.4; P < 0.0001), RASSF1A (r = 0.27; P = 0.01), GSTP1 (r = 0.59; P < 0.0001), NKX2-5 (r = 0.27; P = 0.008), and ESR1 (r = 0.244; P = 0.023) in the normal prostate tissue samples studied. A calculated average methylation (z score) at all nine CpG loci analyzed in the normal prostate tissues showed a strong correlation with age (r = 0.6; P < 0.001). Comparison of the methylation level for the matched benign and prostate cancer tissues from individual patients with prostate cancer showed significantly higher methylation in the prostate cancer tissue samples for RARbeta2 (P < 0.001), RASSF1A (P = 0.005), GSTP1 (P < 0.001), NKX2-5 (P = 0.003), ESR1 (P = 0.016), and CLSTN1 (P = 0.01). CONCLUSIONS: Our findings show aberrant hypermethylation as a function of age in the normal prostate tissues. Such age-related methylation may precede and predispose to full-blown malignancy.

Silencing of bidirectional promoters by DNA methylation in tumorigenesis.

CpG island methylation within promoters is known to silence individual genes in cancer. The involvement of this process in silencing gene pairs controlled by bidirectional promoters is unclear. In a screen for hypermethylated CpG islands in cancer, bidirectional promoters constituted 25.2% of all identified promoters, which matches with the genomic representation of bidirectional promoters. From the screen, we selected three bidirectional gene pairs for detailed analysis, WNT9A/CD558500, CTDSPL/BC040563, and KCNK15/BF195580. Levels of mRNA of all three pairs of genes were inversely correlated with the degree of promoter methylation in multiple cancer cell lines. Hypomethylation of these promoters induced by 5-aza-2'-deoxycytidine treatment reactivated or enhanced gene expression bidirectionally. The bidirectional nature of the WNT9A/CD558500 promoter was confirmed by luciferase assays, and hypermethylation down-regulated expression of both genes in the pair. Methylation of WNT9A/CD558500 and CTDSPL/BC040563 promoters occurs frequently in primary colon cancers and acute lymphoid leukemias (ALL), respectively, and methylation was correlated with decreased gene expression in ALL patient samples. Our study shows that hypermethylation of bidirectional promoter-associated CpG island silences two genes simultaneously, a property that should be taken into account when studying the functional consequences of hypermethylation in cancer.

Transcriptional Selectivity of Epigenetic Therapy in Cancer.

A central challenge in the development of epigenetic cancer therapy is the ability to direct selectivity in modulating gene expression for disease-selective efficacy. To address this issue, we characterized by RNA-seq, DNA methylation, and ChIP-seq analyses the epigenetic response of a set of colon, breast, and leukemia cancer cell lines to small-molecule inhibitors against DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343). We also characterized the effects of DAC as combined with the other compounds. Averaged over the cancer cell models used, we found that DAC affected 8.6% of the transcriptome and that 95.4% of the genes affected were upregulated. DAC preferentially regulated genes that were silenced in cancer and that were methylated at their promoters. In contrast, Depsi affected the expression of 30.4% of the transcriptome but showed little selectivity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 affected only 2% of the transcriptome, with UNC0638 and GSK343 preferentially targeting genes marked with H3K9me2 or H3K27me3, respectively. When combined with histone methylase inhibitors, the extent of gene upregulation by DAC was extended while still maintaining selectivity for DNA-methylated genes and silenced genes. However, the genes upregulated by combination treatment exhibited limited overlap, indicating the possibility of targeting distinct sets of genes based on different epigenetic therapy combinations. Overall, our results demonstrated that DNA methyltransferase inhibitors preferentially target cancer-relevant genes and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining selectivity. Cancer Res; 77(2); 470-81. ©2016 AACR.

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas.

Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor-derived xenografts (PDX) revealed that AR-negative SCPC (AR(-)SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR-positive castration-resistant adenocarcinomas (AR(+)ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR(-) and AR(+) PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR(-)SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR(-)SCPC cell lines. We conclude that the epigenome of AR(-) is distinct from that of AR(+) castration-resistant prostate carcinomas, and that the AR(-) phenotype can be reversed with epigenetic drugs.

Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer.

Targeting epigenetic pathways is a promising approach for cancer therapy. Here, we report on the unexpected finding that targeting calcium signaling can reverse epigenetic silencing of tumor suppressor genes (TSG). In a screen for drugs that reactivate silenced gene expression in colon cancer cells, we found three classical epigenetic targeted drugs (DNA methylation and histone deacetylase inhibitors) and 11 other drugs that induced methylated and silenced CpG island promoters driving a reporter gene (GFP) as well as endogenous TSGs in multiple cancer cell lines. These newly identified drugs, most prominently cardiac glycosides, did not change DNA methylation locally or histone modifications globally. Instead, all 11 drugs altered calcium signaling and triggered calcium-calmodulin kinase (CamK) activity, leading to MeCP2 nuclear exclusion. Blocking CamK activity abolished gene reactivation and cancer cell killing by these drugs, showing that triggering calcium fluxes is an essential component of their epigenetic mechanism of action. Our data identify calcium signaling as a new pathway that can be targeted to reactivate TSGs in cancer.

Fusobacterium in colonic flora and molecular features of colorectal carcinoma.

Fusobacterium species are part of the gut microbiome in humans. Recent studies have identified overrepresentation of Fusobacterium in colorectal cancer tissues, but it is not yet clear whether this is pathogenic or simply an epiphenomenon. In this study, we evaluated the relationship between Fusobacterium status and molecular features in colorectal cancers through quantitative real-time PCR in 149 colorectal cancer tissues, 89 adjacent normal appearing mucosae and 72 colonic mucosae from cancer-free individuals. Results were correlated with CpG island methylator phenotype (CIMP) status, microsatellite instability (MSI), and mutations in BRAF, KRAS, TP53, CHD7, and CHD8. Whole-exome capture sequencing data were also available in 11 cases. Fusobacterium was detectable in 111 of 149 (74%) colorectal cancer tissues and heavily enriched in 9% (14/149) of the cases. As expected, Fusobacterium was also detected in normal appearing mucosae from both cancer and cancer-free individuals, but the amount of bacteria was much lower compared with colorectal cancer tissues (a mean of 250-fold lower for Pan-fusobacterium). We found the Fusobacterium-high colorectal cancer group (FB-high) to be associated with CIMP positivity (P = 0.001), TP53 wild-type (P = 0.015), hMLH1 methylation positivity (P = 0.0028), MSI (P = 0.018), and CHD7/8 mutation positivity (P = 0.002). Among the 11 cases where whole-exome sequencing data were available, two that were FB-high cases also had the highest number of somatic mutations (a mean of 736 per case in FB-high vs. 225 per case in all others). Taken together, our findings show that Fusobacterium enrichment is associated with specific molecular subsets of colorectal cancers, offering support for a pathogenic role in colorectal cancer for this gut microbiome component.

Examination of whole blood DNA methylation as a potential risk marker for gastric cancer.

Whole blood DNA methylation analysis has been proposed to be a risk marker for cancer that can be used to target patients for preventive interventions. To test this, we examined whole blood DNA methylation of 16 CpG island promoters and LINE1 repetitive element in patients with gastric cancer and control subjects. Bisulfite pyrosequencing was used to quantify the methylation of 14 CpG island promoters (MINT25, RORA, GDNF, CDH1, RARAB2, ER, CDH13, MYOD1, SFRP1, P2RX7, SLC16A12, IGF2, DPYS, and N33) and LINE1 from 72 patients with gastric cancer, 67 control, and 52 healthy young individuals. Quantitative methylation-specific real-time PCR was also conducted for 3 CpG island promoters (MINT25, MYO3A, and SOX11). Among all sites tested, only a marginal increase in the methylation of the SFRP1 promoter was observed in the blood of patients with gastric cancer when compared with the control group (11.3 % vs 10.5%; age-adjusted P value: P = 0.009), and this association was also seen in a validation set of 91 patients with gastric cancer (11.5% vs 10.5%; age-adjusted P value: P = 0.001). The methylation of 9 sites (GDNF, CDH1, RARAB2, CDH13, MYOD1, SFRP1, SLC16A12, DPYS, N33, and LINE1) and their mean Z score was correlated with higher age (R = 0.41, P < 0.0001) and marginally with telomere shortening (R = -0.18, P = 0.01) but not with gastric cancer risk (other than SFRP1 methylation). Variability in whole blood DNA methylation of cancer markers is primarily associated with aging, reflecting turnover of white blood cells, and has no direct link to gastric cancer predisposition. SFRP1 methylation in whole blood may be associated with gastric cancer risk.

Integrative genomic characterization of oral squamous cell carcinoma identifies frequent somatic drivers.

The survival of patients with oral squamous cell carcinoma (OSCC) has not changed significantly in several decades, leading clinicians and investigators to search for promising molecular targets. To this end, we conducted comprehensive genomic analysis of gene expression, copy number, methylation, and point mutations in OSCC. Integrated analysis revealed more somatic events than previously reported, identifying four major driver pathways (mitogenic signaling, Notch, cell cycle, and TP53) and two additional key genes (FAT1, CASP8). The Notch pathway was defective in 66% of patients, and in follow-up studies of mechanism, functional NOTCH1 signaling inhibited proliferation of OSCC cell lines. Frequent mutation of caspase-8 (CASP8) defines a new molecular subtype of OSCC with few copy number changes. Although genomic alterations are dominated by loss of tumor suppressor genes, 80% of patients harbored at least one genomic alteration in a targetable gene, suggesting that novel approaches to treatment may be possible for this debilitating subset of head and neck cancers.