Epigenetic Regulation of DNA Repair Pathway Choice by MacroH2A1 Splice Variants Ensures Genome Stability.

The inactive X chromosome (Xi) is inherently susceptible to genomic aberrations. Replication stress (RS) has been proposed as an underlying cause, but the mechanisms that protect from Xi instability remain unknown. Here, we show that macroH2A1.2, an RS-protective histone variant enriched on the Xi, is required for Xi integrity and female survival. Mechanistically, macroH2A1.2 counteracts its structurally distinct and equally Xi-enriched alternative splice variant, macroH2A1.1. Comparative proteomics identified a role for macroH2A1.1 in alternative end joining (alt-EJ), which accounts for Xi anaphase defects in the absence of macroH2A1.2. Genomic instability was rescued by simultaneous depletion of macroH2A1.1 or alt-EJ factors, and mice deficient for both macroH2A1 variants harbor no overt female defects. Notably, macroH2A1 splice variant imbalance affected alt-EJ capacity also in tumor cells. Together, these findings identify macroH2A1 splicing as a modulator of genome maintenance that ensures Xi integrity and may, more broadly, predict DNA repair outcome in malignant cells.

Proteomic Dynamics of Breast Cancer Cell Lines Identifies Potential Therapeutic Protein Targets.

Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We identified 6091 proteins of 76 human BC cell lines using data-independent acquisition (DIA). Integrating our proteomic findings with prior multi-omics datasets, we found that including proteomics data improved drug sensitivity predictions and provided insights into the mechanisms of action. We subsequently profiled the proteomic changes in nine cell lines (five TNBC and four non-TNBC) treated with EGFR/AKT/mTOR inhibitors. In TNBC, metabolism pathways were dysregulated after EGFR/mTOR inhibitor treatment, while RNA modification and cell cycle pathways were affected by AKT inhibitor. This systematic multi-omics and in-depth analysis of the proteome of BC cells can help prioritize potential therapeutic targets and provide insights into adaptive resistance in TNBC.

Metabolism-focused CRISPR screen unveils mitochondrial pyruvate carrier 1 as a critical driver for PARP inhibitor resistance in lung cancer.

Homologous recombination (HR) and poly ADP-ribosylation are partially redundant pathways for the repair of DNA damage in normal and cancer cells. In cell lines that are deficient in HR, inhibition of poly (ADP-ribose) polymerase (poly (ADP-ribose) polymerase [PARP]1/2) is a proven target with several PARP inhibitors (PARPis) currently in clinical use. Resistance to PARPi often develops, usually involving genetic alterations in DNA repair signaling cascades, but also metabolic rewiring particularly in HR-proficient cells. We surmised that alterations in metabolic pathways by cancer drugs such as Olaparib might be involved in the development of resistance to drug therapy. To test this hypothesis, we conducted a metabolism-focused clustered regularly interspaced short palindromic repeats knockout screen to identify genes that undergo alterations during the treatment of tumor cells with PARPis. Of about 3000 genes in the screen, our data revealed that mitochondrial pyruvate carrier 1 (MPC1) is an essential factor in desensitizing nonsmall cell lung cancer (NSCLC) lung cancer lines to PARP inhibition. In contrast to NSCLC lung cancer cells, triple-negative breast cancer cells do not exhibit such desensitization following MPC1 loss and reprogram the tricarboxylic acid cycle and oxidative phosphorylation pathways to overcome PARPi treatment. Our findings unveil a previously unknown synergistic response between MPC1 loss and PARP inhibition in lung cancer cells.

CellMiner Cross-Database (CellMinerCDB) version 1.2: Exploration of patient-derived cancer cell line pharmacogenomics.

CellMiner Cross-Database (CellMinerCDB, discover.nci.nih.gov/cellminercdb) allows integration and analysis of molecular and pharmacological data within and across cancer cell line datasets from the National Cancer Institute (NCI), Broad Institute, Sanger/MGH and MD Anderson Cancer Center (MDACC). We present CellMinerCDB 1.2 with updates to datasets from NCI-60, Broad Cancer Cell Line Encyclopedia and Sanger/MGH, and the addition of new datasets, including NCI-ALMANAC drug combination, MDACC Cell Line Project proteomic, NCI-SCLC DNA copy number and methylation data, and Broad methylation, genetic dependency and metabolomic datasets. CellMinerCDB (v1.2) includes several improvements over the previously published version: (i) new and updated datasets; (ii) support for pattern comparisons and multivariate analyses across data sources; (iii) updated annotations with drug mechanism of action information and biologically relevant multigene signatures; (iv) analysis speedups via caching; (v) a new dataset download feature; (vi) improved visualization of subsets of multiple tissue types; (vii) breakdown of univariate associations by tissue type; and (viii) enhanced help information. The curation and common annotations (e.g. tissues of origin and identifiers) provided here across pharmacogenomic datasets increase the utility of the individual datasets to address multiple researcher question types, including data reproducibility, biomarker discovery and multivariate analysis of drug activity.

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission.

One of the hallmarks of cancer is chromosome instability (CIN), which leads to aneuploidy, translocations, and other chromosome aberrations. However, in the vast majority of human tumors the molecular basis of CIN remains unknown, partly because not all genes controlling chromosome transmission have yet been identified. To address this question, we developed an experimental high-throughput imaging (HTI) siRNA assay that allows the identification of novel CIN genes. Our method uses a human artificial chromosome (HAC) expressing the GFP transgene. When this assay was applied to screen an siRNA library of protein kinases, we identified PINK1, TRIO, IRAK1, PNCK, and TAOK1 as potential novel genes whose knockdown induces various mitotic abnormalities and results in chromosome loss. The HAC-based assay can be applied for screening different siRNA libraries (cell cycle regulation, DNA damage response, epigenetics, and transcription factors) to identify additional genes involved in CIN. Identification of the complete spectrum of CIN genes will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.

Macrophilones from the Marine Hydroid Macrorhynchia philippina Can Inhibit ERK Cascade Signaling.

Six new macrophilone-type pyrroloiminoquines were isolated and identified from an extract of the marine hydroid Macrorhynchia philippina. The proton-deficient and heteroatom-rich structures of macrophilones B-G (2-7) were elucidated by spectroscopic analysis and comparison of their data with those of the previously reported metabolite macrophilone A (1). Compounds 1-7 are the first pyrroloiminoquines to be reported from a hydroid. The macrophilones were shown to inhibit the enzymatic conjugation of SUMO to peptide substrates, and macrophilones A (1) and C (3) exhibit potent and selective cytotoxic properties in the NCI-60 anticancer screen. Bioinformatic analysis revealed a close association of the cytotoxicity profiles of 1 and 3 with two known B-Raf kinase inhibitory drugs. While compounds 1 and 3 showed no kinase inhibitory activity, they resulted in a dramatic decrease in cellular protein levels of selected components of the ERK signal cascade. As such, the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads that target the ERK signal transduction pathway.

rcellminer: exploring molecular profiles and drug response of the NCI-60 cell lines in R.

PURPOSE: The rcellminer R package provides a wide range of functionality to help R users access and explore molecular profiling and drug response data for the NCI-60. The package enables flexible programmatic access to CellMiner's unparalleled breadth of NCI-60 data, including gene and protein expression, copy number, whole exome mutations, as well as activity data for ∼21K compounds, with information on their structure, mechanism of action and repeat screens. Functions are available to easily visualize compound structures, activity patterns and molecular feature profiles. Additionally, embedded R Shiny applications allow interactive data exploration. AVAILABILITY AND IMPLEMENTATION: rcellminer is compatible with R 3.2 and above on Windows, Mac OS X and Linux. The package, documentation, tutorials and Shiny-based applications are available through Bioconductor (http://www.bioconductor.org/packages/rcellminer); ongoing updates will occur according to the Bioconductor release schedule with new CellMiner data. The package is free and open-source (LGPL 3). CONTACT: lunaa@cbio.mskcc.org or vinodh.rajapakse@nih.gov.

Using drug response data to identify molecular effectors, and molecular "omic" data to identify candidate drugs in cancer.

The current convergence of molecular and pharmacological data provides unprecedented opportunities to gain insights into the relationships between the two types of data. Multiple forms of large-scale molecular data, including but not limited to gene and microRNA transcript expression, DNA somatic and germline variations from next-generation DNA and RNA sequencing, and DNA copy number from array comparative genomic hybridization are all potentially informative when one attempts to recognize the panoply of potentially influential events both for cancer progression and therapeutic outcome. Concurrently, there has also been a substantial expansion of the pharmacological data being accrued in a systematic fashion. For cancer cell lines, the National Cancer Institute cell line panel (NCI-60), the Cancer Cell Line Encyclopedia (CCLE), and the collaborative Genomics of Drug Sensitivity in Cancer (GDSC) databases all provide subsets of these forms of data. For the patient-derived data, The Cancer Genome Atlas (TCGA) provides analogous forms of genomic information along with treatment histories. Integration of these data in turn relies on the fields of statistics and statistical learning. Multiple algorithmic approaches may be chosen, depending on the data being considered, and the nature of the question being asked. Combining these algorithms with prior biological knowledge, the results of molecular biological studies, and the consideration of genes as pathways or functional groups provides both the challenge and the potential of the field. The ultimate goal is to provide a paradigm shift in the way that drugs are selected to provide a more targeted and efficacious outcome for the patient.

Putative DNA/RNA helicase Schlafen-11 (SLFN11) sensitizes cancer cells to DNA-damaging agents.

DNA-damaging agents (DDAs) constitute the backbone of treatment for most human tumors. Here we used the National Cancer Institute Antitumor Cell Line Panel (the NCI-60) to identify predictors of cancer cell response to topoisomerase I (Top1) inhibitors, a widely used class of DDAs. We assessed the NCI-60 transcriptome using Affymetrix Human Exon 1.0 ST microarrays and correlated the in vitro activity of four Top1 inhibitors with gene expression in the 60 cell lines. A single gene, Schlafen-11 (SLFN11), showed an extremely significant positive correlation with the response not only to Top1 inhibitors, but also to Top2 inhibitors, alkylating agents, and DNA synthesis inhibitors. Using cells with endogenously high and low SLFN11 expression and siRNA-mediated silencing, we show that SLFN11 is causative in determining cell death and cell cycle arrest in response to DDAs in cancer cells from different tissues of origin. We next analyzed SLFN11 expression in ovarian and colorectal cancers and normal corresponding tissues from The Cancer Genome Atlas database and observed that SLFN11 has a wide expression range. We also observed that high SLFN11 expression independently predicts overall survival in a group of ovarian cancer patients treated with cisplatin-containing regimens. We conclude that SLFN11 expression is causally associated with the activity of DDAs in cancer cells, has a broad expression range in colon and ovarian adenocarcinomas, and may behave as a biomarker for prediction of response to DDAs in the clinical setting.

Identification of benzodiazepine Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput screen against RUNX1-CBFß interaction.

Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for ~24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia (ALL). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-y survival rate of ~50%. We hypothesize that the interaction between RUNX1 and CBFß is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput AlphaScreen and time-resolved fluorescence resonance energy transfer (TR-FRET) methods to quantify the RUNX1-CBFß interaction and screen a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFß directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued preleukemic phenotype in a RUNX1-ETO transgenic zebrafish, and reduced leukemia burden in a mouse CBFB-MYH11 leukemia model. Our data thus confirmed that RUNX1-CBFß interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose.

Genome-wide depletion of replication initiation events in highly transcribed regions.

This report investigates the mechanisms by which mammalian cells coordinate DNA replication with transcription and chromatin assembly. In yeast, DNA replication initiates within nucleosome-free regions, but studies in mammalian cells have not revealed a similar relationship. Here, we have used genome-wide massively parallel sequencing to map replication initiation events, thereby creating a database of all replication initiation sites within nonrepetitive DNA in two human cell lines. Mining this database revealed that genomic regions transcribed at moderate levels were generally associated with high replication initiation frequency. In genomic regions with high rates of transcription, very few replication initiation events were detected. High-resolution mapping of replication initiation sites showed that replication initiation events were absent from transcription start sites but were highly enriched in adjacent, downstream sequences. Methylation of CpG sequences strongly affected the location of replication initiation events, whereas histone modifications had minimal effects. These observations suggest that high levels of transcription interfere with formation of pre-replication protein complexes. Data presented here identify replication initiation sites throughout the genome, providing a foundation for further analyses of DNA-replication dynamics and cell-cycle progression.

Sarcoma_CellminerCDB: A tool to interrogate the genomic and functional characteristics of a comprehensive collection of sarcoma cell lines.

Sarcomas are a diverse group of rare malignancies composed of multiple different clinical and molecular subtypes. Due to their rarity and heterogeneity, basic, translational, and clinical research in sarcoma has trailed behind that of other cancers. Outcomes for patients remain generally poor due to an incomplete understanding of disease biology and a lack of novel therapies. To address some of the limitations impeding preclinical sarcoma research, we have developed Sarcoma_CellMinerCDB, a publicly available interactive tool that merges publicly available sarcoma cell line data and newly generated omics data to create a comprehensive database of genomic, transcriptomic, methylomic, proteomic, metabolic, and pharmacologic data on 133 annotated sarcoma cell lines. The reproducibility, functionality, biological relevance, and therapeutic applications of Sarcoma_CellMinerCDB described herein are powerful tools to address and generate biological questions and test hypotheses for translational research. Sarcoma_CellMinerCDB (https://discover.nci.nih.gov/SarcomaCellMinerCDB) aims to contribute to advancing the preclinical study of sarcoma.

G4 motifs correlate with promoter-proximal transcriptional pausing in human genes.

The RNA Pol II transcription complex pauses just downstream of the promoter in a significant fraction of human genes. The local features of genomic structure that contribute to pausing have not been defined. Here, we show that genes that pause are more G-rich within the region flanking the transcription start site (TSS) than RefSeq genes or non-paused genes. We show that enrichment of binding motifs for common transcription factors, such as SP1, may account for G-richness upstream but not downstream of the TSS. We further show that pausing correlates with the presence of a GrIn1 element, an element bearing one or more G4 motifs at the 5'-end of the first intron, on the non-template DNA strand. These results suggest potential roles for dynamic G4 DNA and G4 RNA structures in cis-regulation of pausing, and thus genome-wide regulation of gene expression, in human cells.

Coordinated regulation of mitochondrial topoisomerase IB with mitochondrial nuclear encoded genes and MYC.

Mitochondrial DNA (mtDNA) is entirely dependent on nuclear genes for its transcription and replication. One of these genes is TOP1MT, which encodes the mitochondrial DNA topoisomerase IB, involved in mtDNA relaxation. To elucidate TOP1MT regulation, we performed genome-wide profiling across the 60-cell line panel (the NCI-60) of the National Cancer Institute Developmental Therapeutics Program. We show that TOP1MT mRNA expression varies widely across these cell lines with the highest levels in leukemia (HL-60, K-562) and melanoma (SK-MEL-28), intermediate levels in breast (MDA-MB-231), ovarian (OVCAR) and colon (HCT-116, HCT-15, KM-12), and lowest levels in renal (ACHN, A498), prostate (PC-3, DU-145) and central nervous system cell lines (SF-539, SF-268, SF-295). Genome-wide analyses show that TOP1MT expression is significantly correlated with the other mitochondrial nuclear-encoded genes including the mitochondrial nucleoid genes, and demonstrate an overall co-regulation of the mitochondrial nuclear-encoded genes. We also find very high correlation between the expression of TOP1MT and the proto-oncogene MYC (c-myc). TOP1MT contains E-boxes (c-myc binding sites) and TOP1MT transcription follows MYC up- and down-regulation by MYC promoter activation and siRNA against MYC. Our finding implicates MYC as a novel regulator of TOP1MT and confirms its role as a master regulator of MNEGs and mitochondrial nucleoids.

Exclusion of the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy in HPCX1-linked families.

Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. The strongest linkage peak of prostate cancer overlies a variable region of ~750 kb at Xq27 enriched by segmental duplications (SDs), suggesting that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. The large size of SDs and their sequence similarity make it difficult to examine this region for possible rearrangements using standard methods. To overcome this problem, direct isolation of a set of genomic segments by in vivo recombination in yeast (a TAR cloning technique) was used to perform a mutational analysis of the 750 kb region in X-linked families. We did not detect disease-specific rearrangements within this region. In addition, transcriptome and computational analyses were performed to search for nonannotated genes within the Xq27 region, which may be associated with genetic predisposition to prostate cancer. Two candidate genes were identified, one of which is a novel gene termed SPANXL that represents a highly diverged member of the SPANX gene family, and the previously described CDR1 gene that is expressed at a high level in both normal and malignant prostate cells, and mapped 210 kb of upstream the SPANX gene cluster. No disease-specific alterations were identified in these genes. Our results exclude the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. Adjacent regions appear to be the most likely candidates to identify the elusive HPCX1 locus.

CellMinerCDB: NCATS Is a Web-Based Portal Integrating Public Cancer Cell Line Databases for Pharmacogenomic Explorations.

Major advances have been made in the field of precision medicine for treating cancer. However, many open questions remain that need to be answered to realize the goal of matching every patient with cancer to the most efficacious therapy. To facilitate these efforts, we have developed CellMinerCDB: National Center for Advancing Translational Sciences (NCATS; https://discover.nci.nih.gov/rsconnect/cellminercdb_ncats/), which makes available activity information for 2,675 drugs and compounds, including multiple nononcology drugs and 1,866 drugs and compounds unique to the NCATS. CellMinerCDB: NCATS comprises 183 cancer cell lines, with 72 unique to NCATS, including some from previously understudied tissues of origin. Multiple forms of data from different institutes are integrated, including single and combination drug activity, DNA copy number, methylation and mutation, transcriptome, protein levels, histone acetylation and methylation, metabolites, CRISPR, and miscellaneous signatures. Curation of cell lines and drug names enables cross-database (CDB) analyses. Comparison of the datasets is made possible by the overlap between cell lines and drugs across databases. Multiple univariate and multivariate analysis tools are built-in, including linear regression and LASSO. Examples have been presented here for the clinical topoisomerase I (TOP1) inhibitors topotecan and irinotecan/SN-38. This web application provides both substantial new data and significant pharmacogenomic integration, allowing exploration of interrelationships. SIGNIFICANCE: CellMinerCDB: NCATS provides activity information for 2,675 drugs in 183 cancer cell lines and analysis tools to facilitate pharmacogenomic research and to identify determinants of response.

Predicting drug sensitivity and resistance: profiling ABC transporter genes in cancer cells.

For analysis of multidrug resistance, a major barrier to effective cancer chemotherapy, we profiled mRNA expression of the 48 known human ABC transporters in 60 diverse cancer cell lines (the NCI-60) used by the National Cancer Institute to screen for anticancer activity. The use of real-time RT-PCR avoided artifacts commonly encountered with microarray technologies. By correlating the results with the growth inhibitory profiles of 1,429 candidate anticancer drugs tested against the cells, we identified which transporters are more likely than others to confer resistance to which agents. Unexpectedly, we also found and validated compounds whose activity is potentiated, rather than antagonized, by the MDR1 multidrug transporter. Such compounds may serve as leads for development.

Integrative epigenomic analyses of small cell lung cancer cells demonstrates the clinical translational relevance of gene body methylation.

DNA methylation is a key regulator of gene expression and a clinical therapeutic predictor. We examined global DNA methylation beyond the generally used promoter areas in human small cell lung cancer (SCLC) and find that gene body methylation is a robust positive predictor of gene expression. Combining promoter and gene body methylation better predicts gene expression than promoter methylation alone including genes involved in the neuroendocrine classification of SCLC and the expression of therapeutically relevant genes including MGMT, SLFN11, and DLL3. Importantly, for super-enhancer (SE) covered genes such as NEUROD1 or MYC, using H3K27ac and NEUROD1, ASCL1, and POU2F3 ChIP-seq data, we show that genic methylation is inversely proportional to expression, thus providing a new approach to identify potential SE regulated genes involved in SCLC pathogenesis. To advance SCLC transitional research, these data are integrated into our web portal (https://discover.nci.nih.gov/SclcCellMinerCDB/) for open and easy access to basic and clinical investigators.

RedundancyMiner: De-replication of redundant GO categories in microarray and proteomics analysis.

BACKGROUND: The Gene Ontology (GO) Consortium organizes genes into hierarchical categories based on biological process, molecular function and subcellular localization. Tools such as GoMiner can leverage GO to perform ontological analysis of microarray and proteomics studies, typically generating a list of significant functional categories. Two or more of the categories are often redundant, in the sense that identical or nearly-identical sets of genes map to the categories. The redundancy might typically inflate the report of significant categories by a factor of three-fold, create an illusion of an overly long list of significant categories, and obscure the relevant biological interpretation. RESULTS: We now introduce a new resource, RedundancyMiner, that de-replicates the redundant and nearly-redundant GO categories that had been determined by first running GoMiner. The main algorithm of RedundancyMiner, MultiClust, performs a novel form of cluster analysis in which a GO category might belong to several category clusters. Each category cluster follows a "complete linkage" paradigm. The metric is a similarity measure that captures the overlap in gene mapping between pairs of categories. CONCLUSIONS: RedundancyMiner effectively eliminated redundancies from a set of GO categories. For illustration, we have applied it to the clarification of the results arising from two current studies: (1) assessment of the gene expression profiles obtained by laser capture microdissection (LCM) of serial cryosections of the retina at the site of final optic fissure closure in the mouse embryos at specific embryonic stages, and (2) analysis of a conceptual data set obtained by examining a list of genes deemed to be "kinetochore" genes.

Novel regulation of nuclear factor-YB by miR-485-3p affects the expression of DNA topoisomerase IIα and drug responsiveness.

Nuclear factor (NF)-YB, a subunit of the transcription factor nuclear factor Y (NF-Y) complex, binds and activates CCAAT-containing promoters. Our previous work suggested that NF-YB may be a mediator of topoisomerase IIα (Top2α), working through the Top2α promoter. DNA topoisomerase II (Top2) is an essential nuclear enzyme and the primary target for several clinically important anticancer drugs. Our teniposide-resistant human lymphoblastic leukemia CEM cells (CEM/VM-1-5) express reduced Top2α protein compared with parental CEM cells. To study the regulation of Top2α during the development of drug resistance, we found that NF-YB protein expression is increased in CEM/VM-1-5 cells compared with parental CEM cells. This further suggests that increased NF-YB may be a negative regulator of Top2α in CEM/VM-1-5 cells. We asked what causes the up-regulation of NF-YB in CEM/VM-1-5 cells. We found by microRNA profiling that hsa-miR-485-3p is lower in CEM/VM-1-5 cells compared with CEM cells. MicroRNA target prediction programs revealed that the 3'-untranslated region (3'-UTR) of NF-YB harbors a putative hsa-miR-485-3p binding site. We thus hypothesized that hsa-miR-485-3p mediates drug responsiveness by decreasing NF-YB expression, which in turn negatively regulates Top2α expression. To test this, we overexpressed miR-485-3p in CEM/VM-1-5 cells and found that this led to reduced expression of NF-YB, a corresponding up-regulation of Top2α, and increased sensitivity to the Top2 inhibitors. Results in CEM cells were replicated in drug-sensitive and -resistant human rhabdomyosarcoma Rh30 cells, suggesting that our findings represent a general phenomenon. Ours is the first study to show that miR-485-3p mediates Top2α down-regulation in part by altered regulation of NF-YB.