DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite ...sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors.
The transforming growth factor beta (TGF-β) signaling pathway exerts opposing effects on cancer cells, acting as either a tumor promoter or a tumor suppressor. Here, we show that these opposing ...effects are a result of the synergy between SMAD3, a downstream effector of TGF-β signaling, and the distinct epigenomes of breast-tumor-initiating cells (BTICs). These effects of TGF-β are associated with distinct gene expression programs, but genomic SMAD3 binding patterns are highly similar in the BTIC-promoting and BTIC-suppressing contexts. Our data show cell-type-specific patterns of DNA and histone modifications provide a modulatory layer by determining accessibility of genes to regulation by TGF-β/SMAD3. LBH, one such context-specific target gene, is regulated according to its DNA methylation status and is crucial for TGF-β-dependent promotion of BTICs. Overall, these results reveal that the epigenome plays a central and previously overlooked role in shaping the context-specific effects of TGF-β in cancer.
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•TGF-β has opposing effects in different breast-tumor-initiating cell (BTIC) types•Genomic SMAD3 binding patterns are similar in BTICs with opposing responses to TGF-β•BTIC type-specific epigenomes prime genes for regulation by TGF-β/SMAD3•LBH, a type-specific TGF-β target, is essential for BTIC-promoting effects of TGF-β
The TGF-β pathway uses transcriptional regulation through SMAD transcription factors to modulate cell-context-specific phenotypes. Tufegdzic Vidakovic et al. show that in breast-tumor-initiating cells (BTICs), type-specific DNA and histone modifications help determine whether the response to TGF-β is pro-oncogenic or tumor suppressive. These landscapes act both in synergy and independently of cell-type-specific SMAD3 binding to TGF-β target genes to modulate context-specific transcriptional regulation by TGF-β/SMAD3.
The inter- and intra-tumor heterogeneity of breast cancer needs to be adequately captured in pre-clinical models. We have created a large collection of breast cancer patient-derived tumor xenografts ...(PDTXs), in which the morphological and molecular characteristics of the originating tumor are preserved through passaging in the mouse. An integrated platform combining in vivo maintenance of these PDTXs along with short-term cultures of PDTX-derived tumor cells (PDTCs) was optimized. Remarkably, the intra-tumor genomic clonal architecture present in the originating breast cancers was mostly preserved upon serial passaging in xenografts and in short-term cultured PDTCs. We assessed drug responses in PDTCs on a high-throughput platform and validated several ex vivo responses in vivo. The biobank represents a powerful resource for pre-clinical breast cancer pharmacogenomic studies (http://caldaslab.cruk.cam.ac.uk/bcape), including identification of biomarkers of response or resistance.
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•We developed a biobank of breast cancer patient-derived tumor xenografts (PDTXs)•PDTXs represent diverse molecular subtypes and retain intra-tumor heterogeneity•PDTX-derived tumor cells (PDTCs) were used for high-throughput drug testing•PDTXs/PDTCs are a robust platform for pre-clinical pharmacogenomic studies
Development and analysis of a collection of breast-cancer-patient-derived xenografts indicate that the xenografts and cell cultures derived from them preserve the heterogeneity of the original tumors and can be used for drug screening.
Patient-Derived Tumour Xenografts (PDTXs) have emerged as the pre-clinical models that best represent clinical tumour diversity and intra-tumour heterogeneity. The molecular characterization of PDTXs ...using High-Throughput Sequencing (HTS) is essential; however, the presence of mouse stroma is challenging for HTS data analysis. Indeed, the high homology between the two genomes results in a proportion of mouse reads being mapped as human.
In this study we generated Whole Exome Sequencing (WES), Reduced Representation Bisulfite Sequencing (RRBS) and RNA sequencing (RNA-seq) data from samples with known mixtures of mouse and human DNA or RNA and from a cohort of human breast cancers and their derived PDTXs. We show that using an In silico Combined human-mouse Reference Genome (ICRG) for alignment discriminates between human and mouse reads with up to 99.9% accuracy and decreases the number of false positive somatic mutations caused by misalignment by >99.9%. We also derived a model to estimate the human DNA content in independent PDTX samples. For RNA-seq and RRBS data analysis, the use of the ICRG allows dissecting computationally the transcriptome and methylome of human tumour cells and mouse stroma. In a direct comparison with previously reported approaches, our method showed similar or higher accuracy while requiring significantly less computing time.
The computational pipeline we describe here is a valuable tool for the molecular analysis of PDTXs as well as any other mixture of DNA or RNA species.
Due to compromised homologous recombination (HR) repair, BRCA1‐ and BRCA2‐mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that ...target specifically BRCA2‐deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2‐deficient cells, including olaparib‐resistant and cisplatin‐resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2‐deficient xenografts and inhibits growth of olaparib‐resistant patient‐derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication‐associated DNA double‐strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2‐compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA‐deficient tumours.
Synopsis
BRCA1/2‐deficient tumours accumulate DNA damage and genomic rearrangements conducive for tumour progression, which is exploited in the clinic by targeted therapies against the BRCA1/2‐mutated tumour subset. Chlorambucil is identified as the most effective drug in eliminating BRCA2‐deficient cells.
The bi‐functional alkylator chlorambucil was specifically toxic to BRCA1/2‐deficient cells and tumours, but not to wild type controls.
Chlorambucil effectively eliminated cisplatin‐resistant and olaparib‐resistant BRCA1/2‐deficient cells and tumours.
Mechanistically, chlorambucil toxicity is mediated by accumulation of replication‐associated DNA damage, similarly to cisplatin.
ATR, FANCD2 and SNM1A nuclease are determinants of cellular sensitivity to both drugs.
Chlorambucil is substantially less toxic to normal cells and tissues than cisplatin.
BRCA1/2‐deficient tumours accumulate DNA damage and genomic rearrangements conducive for tumour progression, which is exploited in the clinic by targeted therapies against the BRCA1/2‐mutated tumour subset. Chlorambucil is identified as the most effective drug in eliminating BRCA2‐deficient cells.
BackgroundThe metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The ...immunosuppressive effects of KYN in the tumor microenvironment are predominantly mediated by the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that broadly suppresses immune cell function. Inhibition of AhR thus offers an antitumor therapy opportunity via restoration of immune system functions.MethodsThe expression of AhR was evaluated in tissue microarrays of head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). A structure class of inhibitors that block AhR activation by exogenous and endogenous ligands was identified, and further optimized, using a cellular screening cascade. The antagonistic properties of the selected AhR inhibitor candidate BAY 2416964 were determined using transactivation assays. Nuclear translocation, target engagement and the effect of BAY 2416964 on agonist-induced AhR activation were assessed in human and mouse cancer cells. The immunostimulatory properties on gene and cytokine expression were examined in human immune cell subsets. The in vivo efficacy of BAY 2416964 was tested in the syngeneic ovalbumin-expressing B16F10 melanoma model in mice. Coculture of human H1299 NSCLC cells, primary peripheral blood mononuclear cells and fibroblasts mimicking the human stromal-tumor microenvironment was used to assess the effects of AhR inhibition on human immune cells. Furthermore, tumor spheroids cocultured with tumor antigen-specific MART-1 T cells were used to study the antigen-specific cytotoxic T cell responses. The data were analyzed statistically using linear models.ResultsAhR expression was observed in tumor cells and tumor-infiltrating immune cells in HNSCC, NSCLC and CRC. BAY 2416964 potently and selectively inhibited AhR activation induced by either exogenous or endogenous AhR ligands. In vitro, BAY 2416964 restored immune cell function in human and mouse cells, and furthermore enhanced antigen-specific cytotoxic T cell responses and killing of tumor spheroids. In vivo, oral application with BAY 2416964 was well tolerated, induced a proinflammatory tumor microenvironment, and demonstrated antitumor efficacy in a syngeneic cancer model in mice.ConclusionsThese findings identify AhR inhibition as a novel therapeutic approach to overcome immune resistance in various types of cancers.
Activation of the mitogen-activated protein kinase (MAPK) pathway is frequent in cancer. Drug development efforts have been focused on kinases in this pathway, most notably on RAF and MEK. We show ...here that MEK inhibition activates JNK-JUN signaling through suppression of DUSP4, leading to activation of HER Receptor Tyrosine Kinases. This stimulates the MAPK pathway in the presence of drug, thereby blunting the effect of MEK inhibition. Cancers that have lost MAP3K1 or MAP2K4 fail to activate JNK-JUN. Consequently, loss-of-function mutations in either MAP3K1 or MAP2K4 confer sensitivity to MEK inhibition by disabling JNK-JUN-mediated feedback loop upon MEK inhibition. In a panel of 168 Patient Derived Xenograft (PDX) tumors, MAP3K1 and MAP2K4 mutation status is a strong predictor of response to MEK inhibition. Our findings suggest that cancers having mutations in MAP3K1 or MAP2K4, which are frequent in tumors of breast, prostate and colon, may respond to MEK inhibitors. Our findings also suggest that MAP3K1 and MAP2K4 are potential drug targets in combination with MEK inhibitors, in spite of the fact that they are encoded by tumor suppressor genes.
Lithium, commonly used to treat bipolar disorder, potentiates the ability of the muscarinic agonist pilocarpine to induce seizures in rodents. As this potentiation by lithium is reversed by the ...administration of myo-inositol, the potentiation may be mediated by inhibition of inositol monophosphatase (IMPase), a known target of lithium. Recently, we demonstrated that ebselen is a ‘lithium mimetic’ in regard to behaviours in both mice and man. Ebselen inhibits IMPase in vitro and lowers myo-inositol in vivo in the brains of mice and men, making ebselen the only known inhibitor of IMPase, other than lithium, that penetrates the blood-brain barrier. Our objective was to determine the effects of ebselen on sensitization to pilocarpine-induced seizures and neural activity. We administered ebselen at different doses and time intervals to mice, followed by injection of a sub-seizure dose of pilocarpine. We assessed seizure and neural activity by a subjective seizure rating scale, by monitoring tremors, and by induction of the immediate early gene c-fos. In contrast to lithium, ebselen did not potentiate the ability of pilocarpine to induce seizures. Unexpectedly, ebselen inhibited pilocarpine-induced tremor as well as pilocarpine-induced increases in c-fos mRNA levels. Both lithium and ebselen inhibit a common target, IMPase, but only lithium potentiates pilocarpine-induced seizures, consistent with their polypharmacology at diverse molecular targets. We conclude that ebselen does not potentiate pilocarpine-induced seizures and instead, reduces pilocarpine-mediated neural activation. This lack of potentiation of muscarinic sensitization may be one reason for the lack of side-effects observed with ebselen treatment clinically.
Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular ...metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR‐compromised cells are sensitive to acetaldehyde, similarly to FANCD2‐deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2‐deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR‐deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication‐associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde‐arrested replication forks require BRCA2 and FANCD2 for protection against MRE11‐dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2‐deficient tumors and ex vivo in patient‐derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP‐ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2‐deficient cells and tumors.
Synopsis
Treatment with acetaldehyde or with the alcohol‐deterrent disulfiram, which enhances acetaldehyde levels, selectively eliminates BRCA1/2‐deficient cells and tumors. Increasing cellular acetaldehyde might thus benefit cancer patients with BRCA1/2 mutations.
Acetaldehyde and disulfiram increased the levels of RPA foci and decreased replication fork progression, leading to accumulation of replication‐associated DNA damage specifically in BRCA2‐deficient cells.
The Aldh2 gene encodes an aldehyde dehydrogenase with key roles in endogenous acetaldehyde detoxification.
Aldh2 gene deletion or its point mutation E487K associated with the ethanol‐induced flushing syndrome in humans causes proliferation arrest in cells lacking BRCA1/2 expression.
Growth of BRCA1/2‐defective tumors, including those that have acquired resistance to PARP inhibitors, is suppressed by acetaldehyde treatment.
Treatment with acetaldehyde or with the alcohol‐deterrent disulfiram, which enhances acetaldehyde levels, selectively eliminates BRCA1/2‐deficient cells and tumors. Increasing cellular acetaldehyde might thus benefit cancer patients with BRCA1/2 mutations.
Abstract
Breast cancer is the most common cancer among women showing high clinical and molecular heterogeneity. Current clinical management causes patients overtreatment with implications on both ...patients’ quality of life and healthcare costs. Moreover, intrinsic or acquired tumor resistance to treatment leads to incurable metastatic progression in a significant proportion of patients. Consequently, there is an urgent need for better predictive biomarkers and a better understanding of the mechanisms driving response to treatment. As part of the METABRIC initiative, we fully molecularly characterized 2000 breast primary tumors, measuring gene expression, copy number aberration, somatic mutations and methylation. In addition, a biobank of breast cancer patient-derived tumor xenograft (PDTX) models (n=92) has been generated in our lab and a comprehensive molecular characterization was also obtained. We recently demonstrated that breast cancer PDTXs maintain originating cancers intra-tumor heterogeneity, hence representing a more relevant preclinical model than cell lines. An ex-vivo drug screening was performed in these models generating response data (IC50 and AUC) for 100 different drugs, including "best in class" PI3K, PARP and CDK4/6 inhibitors, novel biological and chemical inhibitors of HER2, ER, IGF1R and HER3, as well as standard of care agents. Here we derived signatures of pathway activation/disruption by integrating different data types in the METABRIC cohort. Their association with previous breast cancer classifications, as well as their prognostic significance was studied. The predictive power of these signatures was investigated in the PDTX cohort to identify novel pharmacogenomics associations. They were tested independently as well as in combination, to derive molecular predictors of response to treatment. We found known and novel associations between genomic/transcriptomic features and drug response. For example, our results confirmed the known association between estrogen receptor-related genes and response to tamoxifen treatment. We also identified markers of response to inhibitors of the PI3K/AKT/mTOR pathway. Selected findings were validated in clinical cohorts as well as in independent PDTX models. In conclusion, by integrating molecular data from large cohorts of clinical samples and PDTX we have generated a computational framework for the systematic identification of pharmacogenomics associations in breast cancer and to generate hypothesis for rational drug-drug combinations.
Citation Format: Maurizio Callari, Rajbir N. Batra, Ankita Sati Batra, Wendy Greenwood, Suet-Feung Chin, Alejandra Bruna, Oscar M. Rueda, Carlos Caldas. Integrative analysis of molecular and drug response data from clinical samples and PDTXs to identify pharmacogenomic associations in breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2991. doi:10.1158/1538-7445.AM2017-2991
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