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.

Integrative genomic analyses of European intrahepatic cholangiocarcinoma: Novel ROS1 fusion gene and PBX1 as prognostic marker.

BACKGROUND: Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct with a poor prognosis owing to limited therapeutic options. The incidence of intrahepatic CCA (iCCA) is increasing worldwide, and its molecular basis is emerging. Environmental factors may contribute to regional differences in the mutation spectrum of European patients with iCCA, which are underrepresented in systematic genomic and transcriptomic studies of the disease. METHODS: We describe an integrated whole-exome sequencing and transcriptomic study of 37 iCCAs patients in Germany. RESULTS: We observed as most frequently mutated genes ARID1A (14%), IDH1, BAP1, TP53, KRAS, and ATM in 8% of patients. We identified FGFR2::BICC1 fusions in two tumours, and FGFR2::KCTD1 and TMEM106B::ROS1 as novel fusions with potential therapeutic implications in iCCA and confirmed oncogenic properties of TMEM106B::ROS1 in vitro. Using a data integration framework, we identified PBX1 as a novel central regulatory gene in iCCA. We performed extended screening by targeted sequencing of an additional 40 CCAs. In the joint analysis, IDH1 (13%), BAP1 (10%), TP53 (9%), KRAS (7%), ARID1A (7%), NF1 (5%), and ATM (5%) were the most frequently mutated genes, and we found PBX1 to show copy gain in 20% of the tumours. According to other studies, amplifications of PBX1 tend to occur in European iCCAs in contrast to liver fluke-associated Asian iCCAs. CONCLUSIONS: By analyzing an additional European cohort of iCCA patients, we found that PBX1 protein expression was a marker of poor prognosis. Overall, our findings provide insight into key molecular alterations in iCCA, reveal new targetable fusion genes, and suggest that PBX1 is a novel modulator of this disease.

Epigenetic regulation in estrogen receptor positive breast cancer--role in treatment response.

Recent advances in breast cancer treatment have allowed increasing numbers of patients with estrogen receptor (ER) positive (+) breast cancer to receive various forms of endocrine therapy. Unfortunately, de novo and acquired resistance to endocrine therapy remains a major challenge in the clinic. A number of possible mechanisms for drug resistance have been described, which include activation of growth factor receptor pathways, overexpression of ER coactivators, and metabolic resistance due to polymorphisms in metabolizing enzymes. While many of these changes are caused by genetic alterations, there is also increasing evidence to implicate epigenetic gene regulatory mechanisms in the development of endocrine resistance. Since epigenetic modifications are easier to reverse than genetic mutations, they are appealing therapeutic targets, and thus future improvements in medical care for breast cancer patients will depend upon a better understanding of the roles epigenetic modifications play in endocrine resistance. In this review we will focus on recent advances made in the understanding of epigenetic gene regulation in estrogen response and endocrine resistance in breast cancer. We will also summarize current clinical-translational advances in epigenetic therapy, and discuss potential future clinical use of epigenetic changes as therapeutic targets, especially with respect to endocrine treatment.

A Role for Histone H2B Variants in Endocrine-Resistant Breast Cancer.

Acquired resistance to aromatase inhibitors (AIs) remains a major clinical problem in the treatment of estrogen receptor-positive (ER+) breast cancer. We and others have previously reported widespread changes in DNA methylation using breast cancer cell line models of endocrine resistance. Here, we show that the histone variant HIST1H2BE is hypomethylated in estrogen deprivation-resistant C4-12 and long-term estrogen-deprived (LTED) cells compared with parental MCF-7 cells. As expected, this hypomethylation associates with increased expression of HIST1H2BE in C4-12 and LTED cells. Both overexpression and downregulation of HIST1H2BE caused decreased proliferation in breast cancer cell lines suggesting the need for tightly controlled expression of this histone variant. Gene expression analysis showed varied expression of HIST1H2BE in a large panel of breast cancer cell lines, without restriction to specific molecular subtypes. Analysis of HIST1H2BE messenger RNA (mRNA) expression in ER+ AI-treated breast tumors showed significantly higher expression in resistant (n = 19) compared with sensitive (n = 37) tumors (p = 0.01). Using nanostring analysis, we measured expression of all 61 histone variants in endocrine-resistant and endocrine-sensitive tumors. We found significant overexpression of 22 variant histone genes in tumors resistant to AI therapy. In silico The Cancer Genome Atlas (TCGA) analysis showed frequent amplification of the HIST1 locus. In summary, our studies show, for the first time, that overexpression of histone variants might be important in endocrine response in ER+ breast cancer, and that overexpression is at least in part mediated via epigenetic mechanisms and amplifications. Future studies addressing endocrine response should include a potential role of these currently understudied histone variants.

Targeted DNA Methylation Screen in the Mouse Mammary Genome Reveals a Parity-Induced Hypermethylation of Igf1r That Persists Long after Parturition.

The most effective natural prevention against breast cancer is an early first full-term pregnancy. Understanding how the protective effect is elicited will inform the development of new prevention strategies. To better understand the role of epigenetics in long-term protection, we investigated parity-induced DNA methylation in the mammary gland. FVB mice were bred or remained nulliparous and mammary glands harvested immediately after involution (early) or 6.5 months following involution (late), allowing identification of both transient and persistent changes. Targeted DNA methylation (109 Mb of Ensemble regulatory features) analysis was performed using the SureSelectXT Mouse Methyl-seq assay and massively parallel sequencing. Two hundred sixty-nine genes were hypermethylated and 128 hypomethylated persistently at both the early and late time points. Pathway analysis of the persistently differentially methylated genes revealed Igf1r to be central to one of the top identified signaling networks, and Igf1r itself was one of the most significantly hypermethylated genes. Hypermethylation of Igf1r in the parous mammary gland was associated with a reduction of Igf1r mRNA expression. These data suggest that the IGF pathway is regulated at multiple levels during pregnancy and that its modification might be critical in the protective role of pregnancy. This supports the approach of lowering IGF action for prevention of breast cancer, a concept that is currently being tested clinically.

Recurrent Fusion Genes in Gastric Cancer: CLDN18-ARHGAP26 Induces Loss of Epithelial Integrity.

Genome rearrangements, a hallmark of cancer, can result in gene fusions with oncogenic properties. Using DNA paired-end-tag (DNA-PET) whole-genome sequencing, we analyzed 15 gastric cancers (GCs) from Southeast Asians. Rearrangements were enriched in open chromatin and shaped by chromatin structure. We identified seven rearrangement hot spots and 136 gene fusions. In three out of 100 GC cases, we found recurrent fusions between CLDN18, a tight junction gene, and ARHGAP26, a gene encoding a RHOA inhibitor. Epithelial cell lines expressing CLDN18-ARHGAP26 displayed a dramatic loss of epithelial phenotype and long protrusions indicative of epithelial-mesenchymal transition (EMT). Fusion-positive cell lines showed impaired barrier properties, reduced cell-cell and cell-extracellular matrix adhesion, retarded wound healing, and inhibition of RHOA. Gain of invasion was seen in cancer cell lines expressing the fusion. Thus, CLDN18-ARHGAP26 mediates epithelial disintegration, possibly leading to stomach H(+) leakage, and the fusion might contribute to invasiveness once a cell is transformed.

Epigenetic reprogramming of HOXC10 in endocrine-resistant breast cancer.

Resistance to aromatase inhibitors (AIs) is a major clinical problem in the treatment of estrogen receptor (ER)-positive breast cancer. In two breast cancer cell line models of AI resistance, we identified widespread DNA hyper- and hypomethylation, with enrichment for promoter hypermethylation of developmental genes. For the homeobox gene HOXC10, methylation occurred in a CpG shore, which overlapped with a functional ER binding site, causing repression of HOXC10 expression. Although short-term blockade of ER signaling caused relief of HOXC10 repression in both cell lines and breast tumors, it also resulted in concurrent recruitment of EZH2 and increased H3K27me3, ultimately transitioning to increased DNA methylation and silencing of HOXC10. Reduced HOXC10 in vitro and in xenografts resulted in decreased apoptosis and caused antiestrogen resistance. Supporting this, we used paired primary and metastatic breast cancer specimens to show that HOXC10 was reduced in tumors that recurred during AI treatment. We propose a model in which estrogen represses apoptotic and growth-inhibitory genes such as HOXC10, contributing to tumor survival, whereas AIs induce these genes to cause apoptosis and therapeutic benefit, but long-term AI treatment results in permanent repression of these genes via methylation and confers resistance. Therapies aimed at inhibiting AI-induced histone and DNA methylation may be beneficial in blocking or delaying AI resistance.

Progesterone receptor isoform-specific promoter methylation: association of PRA promoter methylation with worse outcome in breast cancer patients.

PURPOSE: ERα and PR levels are critical determinants for breast cancer prognosis and response to endocrine therapy. Although PR is known to be silenced by methylation of its promoter, few studies have correlated methylation with PR levels and outcome in breast cancer. There is only one previous small study comparing methylation of the two PR isoforms, PRA and PRB, which are expressed from different promoters, and finally, there is no prior knowledge of associations between isoform-specific methylation and outcome. EXPERIMENTAL DESIGN: We conducted a cohort-based study to test for associations between PRA and PRB methylation, expression, and clinical outcome in tamoxifen-treated patients (n = 500), and in patients who underwent surgery only (n = 500). Methylation and PR levels were measured by bisulfite pyrosequencing and ligand-binding assay, respectively. RESULTS: Low PR levels were significantly associated with worse outcome in all patients. PRA and PRB promoters were methylated in 9.6% and 14.1% of the breast tumors, respectively. The majority (74%) of PR-negative tumors were not methylated despite the significant inverse correlation of methylation and PR levels. PRA methylation was significantly associated with PRB methylation, although a subset of tumors had PRA only (3.9%) or PRB only (8.3%) methylated. Methylation of PRA, but not PRB was significantly associated with worse outcome in the tamoxifen-treated group. CONCLUSIONS: Mechanisms other than promoter methylation may be more dominant for loss of PR. Isoform-specific methylation events suggest independent regulation of PRA and PRB. Finally, this article shows for the first time that PRA methylation plays a unique role in tamoxifen-resistant breast cancer.

Parity-induced decrease in systemic growth hormone alters mammary gland signaling: a potential role in pregnancy protection from breast cancer.

Early full-term pregnancy is an effective natural protection against breast cancer in both humans and experimental rodents. The protective effect of an early pregnancy is, in part, linked to changes in circulating hormones that are involved in both normal breast development and breast cancer. For example, a reduction in circulating growth hormone (GH) has been shown to protect rats from carcinogen-induced mammary tumors. We examined the ability of a full-term pregnancy to alter the endocrine GH/insulin-like growth factor-I (IGF-I) axis and how this change affected normal mammary gland function in two commonly used rat models (Sprague-Dawley and Wistar Furth). Circulating GH and IGF-I were measured in blood drawn every 30 minutes from parous and age-matched virgin female rats. Mean serum GH levels were significantly decreased (P < 0.01) in parous compared with age-matched virgin rats for both strains. Changes in GH levels were independent of estrous cycle, indicated by a significant (P < 0.05) reduction in circulating levels of GH during estrus and diestrus in both parous strains. Despite the decrease in circulating GH, pituitary GH mRNA levels were unaltered in parous rats. Circulating IGF-I and hepatic IGF-I mRNA were also unaltered by parity in either rat strain. Immunoblot analysis of mammary glands showed decreases in phosphorylation of signal transducer and activator of transcription 5A and Janus-activated kinase 2, suggesting reduced action of GH in the mammary gland. Therefore, although the parity reduction in circulating GH does not affect circulating IGF-I levels, it is possible that reduced GH acts directly at the mammary gland and may play a role in pregnancy protection from breast cancer.