An integrative model of pathway convergence in genetically heterogeneous blast crisis chronic myeloid leukemia.

Targeted therapies against the BCR-ABL1 kinase have revolutionized treatment of chronic phase (CP) chronic myeloid leukemia (CML). In contrast, management of blast crisis (BC) CML remains challenging because BC cells acquire complex molecular alterations that confer stemness features to progenitor populations and resistance to BCR-ABL1 tyrosine kinase inhibitors. Comprehensive models of BC transformation have proved elusive because of the rarity and genetic heterogeneity of BC, but are important for developing biomarkers predicting BC progression and effective therapies. To better understand BC, we performed an integrated multiomics analysis of 74 CP and BC samples using whole-genome and exome sequencing, transcriptome and methylome profiling, and chromatin immunoprecipitation followed by high-throughput sequencing. Employing pathway-based analysis, we found the BC genome was significantly enriched for mutations affecting components of the polycomb repressive complex (PRC) pathway. While transcriptomically, BC progenitors were enriched and depleted for PRC1- and PRC2-related gene sets respectively. By integrating our data sets, we determined that BC progenitors undergo PRC-driven epigenetic reprogramming toward a convergent transcriptomic state. Specifically, PRC2 directs BC DNA hypermethylation, which in turn silences key genes involved in myeloid differentiation and tumor suppressor function via so-called epigenetic switching, whereas PRC1 represses an overlapping and distinct set of genes, including novel BC tumor suppressors. On the basis of these observations, we developed an integrated model of BC that facilitated the identification of combinatorial therapies capable of reversing BC reprogramming (decitabine+PRC1 inhibitors), novel PRC-silenced tumor suppressor genes (NR4A2), and gene expression signatures predictive of disease progression and drug resistance in CP.

Genome-scale analysis of aberrant DNA methylation in colorectal cancer.

Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation-based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally high frequency of cancer-specific DNA hypermethylation, is strongly associated with MLH1 DNA hypermethylation and the BRAF(V600E) mutation. A CIMP-low (CIMP-L) subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-H-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 112 genes that were down-regulated more than twofold in CIMP-H tumors together with promoter DNA hypermethylation. These represent ∼7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/112 genes were also transcriptionally down-regulated in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing.

Estrogen and insulin-like growth factor-I (IGF-I) independently down-regulate critical repressors of breast cancer growth.

Although estrogen receptor alpha (ERα) and insulin-like growth factor (IGF) signaling are important for normal mammary development and breast cancer, cross-talk between these pathways, particularly at the level of transcription, remains poorly understood. We performed microarray analysis on MCF-7 breast cancer cells treated with estradiol (E2) or IGF-I for 3 or 24 h. IGF-I regulated mRNA of five to tenfold more genes than E2, and many genes were co-regulated by both ligands. Importantly, expression of these co-regulated genes correlated with poor prognosis of human breast cancer. Closer examination revealed enrichment of repressed transcripts. Interestingly, a number of potential tumor suppressors, for example, B-cell linker (BLNK), were down-regulated by IGF-I and E2. Analysis of three down-regulated genes showed that E2-mediated repression occurred independently of IGF-IR, and IGF-I-mediated repression occurred independently of ERα. However, repression by IGF-I or E2 required common kinases, such as PI3K and MEK, suggesting downstream convergence of the two pathways. In conclusion, E2 and IGF-I co-regulate a set of genes that affect breast cancer outcome. There is enrichment of repressed transcripts, and, for some genes, the down-regulation is independent at the receptor level. This may be important clinically, as tumors with active ERα and IGF-IR signaling may require co-targeting of both pathways.

Estrogen-mediated downregulation of CD24 in breast cancer cells.

We have previously reported on the relevance of the prevalence of CD44(+)/CD24(-/low) cells in primary breast tumors. To study regulation of CD24, we queried a number of publicly available expression array studies in breast cancer cells and found that CD24 was downregulated upon estrogen treatment. We confirmed this estrogen-mediated repression of CD24 mRNA by quantitative real-time PCR in MCF7, T47D and ZR75-1 cells. Repression was also seen at the protein level as measured by flow cytometry. CD24 was not downregulated in the ER alpha negative MDA-MB-231 cells suggesting that ER alpha was necessary. This was further confirmed by ER alpha silencing in MCF7 cells resulting in increased CD24 levels and by reintroduction of ER alpha into C4-12 cells resulting in decreased CD24 levels. Estrogen treatment did not alter half-life of CD24 mRNA and new protein synthesis was not essential for repression, suggesting a primary transcriptional effect. Histone deacetylase inhibition by Trichostatin A completely abolished the repression, but decrease of the ER alpha corepressors NCoR, LCoR, RIP140, silencing mediator of retinoid and thyroid hormone receptors, SAFB1 and SAFB2 by siRNA or overexpression of SAFB2, NCoR and silencing mediator of retinoid and thyroid hormone receptors had no effect. In silico promoter analyses led to the identification of two estrogen responsive elements in the CD24 promoter, one of which was able to bind ER alpha as shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Together, our results show that CD24 is repressed by estrogen and that this repression is a direct transcriptional effect depending on ER alpha and histone deacetylases.

Germline Mutations in Cancer Predisposition Genes are Frequent in Sporadic Sarcomas.

Associations of sarcoma with inherited cancer syndromes implicate genetic predisposition in sarcoma development. However, due to the apparently sporadic nature of sarcomas, little attention has been paid to the role genetic susceptibility in sporadic sarcoma. To address this, we performed targeted-genomic sequencing to investigate the prevalence of germline mutations in known cancer-associated genes within an Asian cohort of sporadic sarcoma patients younger than 50 years old. We observed 13.6% (n = 9) amongst 66 patients harbour at least one predicted pathogenic germline mutation in 10 cancer-associated genes including ATM, BRCA2, ERCC4, FANCC, FANCE, FANCI, MSH6, POLE, SDHA and TP53. The most frequently affected genes are involved in the DNA damage repair pathway, with a germline mutation prevalence of 10.6%. Our findings suggests that genetic predisposition plays a larger role than expected in our Asian cohort of sporadic sarcoma, therefore clinicians should be aware of the possibility that young sarcoma patients may be carriers of inherited mutations in cancer genes and should be considered for genetic testing, regardless of family history. The prevalence of germline mutations in DNA damage repair genes imply that therapeutic strategies exploiting the vulnerabilities resulting from impaired DNA repair may be promising areas for translational research.

Technical Validation of a Next-Generation Sequencing Assay for Detecting Actionable Mutations in Patients with Gastrointestinal Cancer.

Targeted next-generation sequencing is becoming increasingly common as a clinical diagnostic and prognostic test for patient- and tumor-specific genetic profiles as well as to optimally select targeted therapies. Here, we describe a custom-developed, next-generation sequencing test for detecting single-nucleotide variants (SNVs) and short insertions and deletions (indels) in 93 genes related to gastrointestinal cancer from routine formalin-fixed, paraffin-embedded clinical specimens. We implemented a validation strategy, based on the College of American Pathologists requirements, using reference DNA mixtures from cell lines with known genetic variants, which model a broad range of allele frequencies. Test sensitivity achieved >99% for both SNVs and indels, with allele frequencies >10%, with high specificity (97.4% for SNVs and 93.6% for indels). We further confirmed test accuracies using primary formalin-fixed, paraffin-embedded colorectal cancer specimens characterized by alternative and conventional clinical diagnostic technologies. Robust performance was observed on the formalin-fixed, paraffin-embedded specimens: sensitivity was 97.2% and specificity was 99.2%. We also observed high intrarun and inter-run reproducibility, as well as a low cross-contamination rate. Overall assessment using cell line samples and formalin-fixed, paraffin-embedded samples showed that our custom next-generation sequencing assay has consistent detection sensitivity down to 10% variant frequency.

High-depth sequencing of over 750 genes supports linear progression of primary tumors and metastases in most patients with liver-limited metastatic colorectal cancer.

BACKGROUND: Colorectal cancer with metastases limited to the liver (liver-limited mCRC) is a distinct clinical subset characterized by possible cure with surgery. We performed high-depth sequencing of over 750 cancer-associated genes and copy number profiling in matched primary, metastasis and normal tissues to characterize genomic progression in 18 patients with liver-limited mCRC. RESULTS: High depth Illumina sequencing and use of three different variant callers enable comprehensive and accurate identification of somatic variants down to 2.5% variant allele frequency. We identify a median of 11 somatic single nucleotide variants (SNVs) per tumor. Across patients, a median of 79.3% of somatic SNVs present in the primary are present in the metastasis and 81.7% of all alterations present in the metastasis are present in the primary. Private alterations are found at lower allele frequencies; a different mutational signature characterized shared and private variants, suggesting distinct mutational processes. Using B-allele frequencies of heterozygous germline SNPs and copy number profiling, we find that broad regions of allelic imbalance and focal copy number changes, respectively, are generally shared between the primary tumor and metastasis. CONCLUSIONS: Our analyses point to high genomic concordance of primary tumor and metastasis, with a thick common trunk and smaller genomic branches in general support of the linear progression model in most patients with liver-limited mCRC. More extensive studies are warranted to further characterize genomic progression in this important clinical population.

Nanoscale chromatin profiling of gastric adenocarcinoma reveals cancer-associated cryptic promoters and somatically acquired regulatory elements.

Chromatin alterations are fundamental hallmarks of cancer. To study chromatin alterations in primary gastric adenocarcinomas, we perform nanoscale chromatin immunoprecipitation sequencing of multiple histone modifications in five gastric cancers and matched normal tissues. We identify hundreds of somatically altered promoters and predicted enhancers. Many cancer-associated promoters localize to genomic sites lacking previously annotated transcription start sites (cryptic promoters), driving expression of nearby genes involved in gastrointestinal cancer, embryonic development and tissue specification. Cancer-associated promoters overlap with embryonic stem cell regions targeted by polycomb repressive complex 2, exhibiting promoter bivalency and DNA methylation loss. We identify somatically acquired elements exhibiting germline allelic biases and non-coding somatic mutations creating new promoters. Our findings demonstrate the feasibility of profiling chromatin from solid tumours with limited tissue to identify regulatory elements, transcriptional patterns and regulatory genetic variants associated with cancer.

Histone deacetylase 7 and FoxA1 in estrogen-mediated repression of RPRM.

Activation of estrogen receptor alpha (ERalpha) results in both induction and repression of gene transcription; while mechanistic details of estrogen induction are well described, details of repression remain largely unknown. We characterized several ERalpha-repressed targets and examined in detail the mechanism for estrogen repression of Reprimo (RPRM), a cell cycle inhibitor. Estrogen repression of RPRM is rapid and robust and requires a tripartite interaction between ERalpha, histone deacetylase 7 (HDAC7), and FoxA1. HDAC7 is the critical HDAC needed for repression of RPRM; it can bind to ERalpha and represses ERalpha's transcriptional activity--this repression does not require HDAC7's deacetylase activity. We further show that the chromatin pioneer factor FoxA1, well known for its role in estrogen induction of genes, is recruited to the RPRM promoter, is necessary for repression of RPRM, and interacts with HDAC7. Like other FoxA1 recruitment sites, the RPRM promoter is characterized by H3K4me1/me2. Estrogen treatment causes decreases in H3K4me1/me2 and release of RNA polymerase II (Pol II) from the RPRM proximal promoter. Overall, these data implicate a novel role for HDAC7 and FoxA1 in estrogen repression of RPRM, a mechanism which could potentially be generalized to many more estrogen-repressed genes and hence be important in both normal physiology and pathological processes.