Array Datasets Research Articles

Transcriptome sequencing study implicates immune-related genes differentially expressed in schizophrenia: new data and a meta-analysis

We undertook an RNA sequencing (RNAseq)-based transcriptomic profiling study on lymphoblastoid cell lines of a European ancestry sample of 529 schizophrenia cases and 660 controls, and found 1058 genes to be differentially expressed by affection status. These differentially expressed genes were enriched for involvement in immunity, especially the 697 genes with higher expression in cases. Comparing the current RNAseq transcriptomic profiling to our previous findings in an array-based study of 268 schizophrenia cases and 446 controls showed a highly significant positive correlation over all genes. Fifteen (18%) of the 84 genes with significant (false discovery rate<0.05) expression differences between cases and controls in the previous study and analyzed here again were differentially expressed by affection status here at a genome-wide significance level (Bonferroni P<0.05 adjusted for 8141 analyzed genes in total, or P<~6.1 × 10−6), all with the same direction of effect, thus providing corroborative evidence despite each sample of fully independent subjects being studied by different technological approaches. Meta-analysis of the RNAseq and array data sets (797 cases and 1106 controls) showed 169 additional genes (besides those found in the primary RNAseq-based analysis) to be differentially expressed, and provided further evidence of immune gene enrichment. In addition to strengthening our previous array-based gene expression differences in schizophrenia cases versus controls and providing transcriptomic support for some genes implicated by other approaches for schizophrenia, our study detected new genes differentially expressed in schizophrenia. We highlight RNAseq-based differential expression of various genes involved in neurodevelopment and/or neuronal function, and discuss caveats of the approach.

Transcriptome analysis reveals differentially expressed lncRNAs between oral squamous cell carcinoma and healthy oral mucosa.

Oral cavity and oropharyngeal squamous cell carcinoma (OSCC) is a major cancer type in the head and neck region. To better understand the roles long non-coding RNA (lncRNA) play in OSCC carcinogenesis, we compared the expression levels of 3,054 probe sets for lncRNAs between 167 OSCCs and 45 healthy oral mucosa using an Affymetrix HG U133 plus 2.0 array dataset. We found 658 lncRNA transcripts (790 probe sets) to be significantly differentially expressed using a criteria of FDR < 0.01, with 36 of them (39 probe sets) showing more than a 2-fold change. We further validated the top differentially expressed lncRNAs in three independent datasets from Gene Expression Omnibus (GEO) repository: GSE42743, GSE9844, and GSE6791. Fourteen lncRNAs (15 probe sets) were validated in all three datasets using the criteria FDR < 0.01: LOC441178, C5orf66-AS1, HCG22, FLG-AS1, CCL14/CCL15-CCL14, LOC100506990, TRIP10, PCBP1-AS1, LINC01315, LINC00478, COX10-AS1/LOC100506974, MLLT4-AS1, MIR31HG, and DUXAP10/LINC01296. Three lncRNAs in the validated list which showed the highest fold change (LOC441178, HCG22 and C5orf66-AS1) were verified by quantitative RT-PCR in a subset of 20 OSCCs and 10 control samples. In silico prediction of their functional role has given us directions for further investigation.

Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tumor tissue-of-origin mapping from plasma DNA.

Adjacent CpG sites in mammalian genomes can be co-methylated due to the processivity of methyltransferases or demethylases. Yet discordant methylation patterns have also been observed, and found related to stochastic or uncoordinated molecular processes. We focused on a systematic search and investigation of regions in the full human genome that exhibit highly coordinated methylation. We defined 147,888 blocks of tightly coupled CpG sites, called methylation haplotype blocks (MHBs) with 61 sets of whole genome bisulfite sequencing (WGBS) data, and further validated with 101 sets of reduced representation bisulfite sequencing (RRBS) data and 637 sets of methylation array data. Using a metric called methylation haplotype load (MHL), we performed tissue-specific methylation analysis at the block level. Subsets of informative blocks were further identified for deconvolution of heterogeneous samples. Finally, we demonstrated quantitative estimation of tumor load and tissue-of-origin mapping in the circulating cell-free DNA of 59 cancer patients using methylation haplotypes.

Detrimental ELAVL-1/HuR-dependent GSK3β mRNA stabilization impairs resolution in acute respiratory distress syndrome

A hallmark of acute respiratory distress syndrome (ARDS) is accumulation of protein-rich edema in the distal airspaces and its removal is critical for patient survival. Previous studies have shown a detrimental role of Glycogen Synthase Kinase (GSK) 3β during ARDS via inhibition of alveolar epithelial protein transport. We hypothesized that post-transcriptional regulation of GSK3β could play a functional role in ARDS resolution. To address this hypothesis, we performed an in silico analysis to identify regulatory genes whose expression correlation to GSK3β messenger RNA utilizing two lung cancer cell line array datasets. Among potential regulatory partners of GSK3β, these studies identified the RNA-binding protein ELAVL-1/HuR (Embryonic Lethal, Abnormal Vision, Drosophila-Like) as a central component in a likely GSK3β signaling network. ELAVL-1/HuR is a RNA-binding protein that selectively binds to AU-rich elements of mRNA and enhances its stability thereby increasing target gene expression. Subsequent studies with siRNA suppression of ELAVL-1/HuR demonstrated deceased GSK3β mRNA and protein expression and improved clearance of FITC-albumin in A549 cells. Conversely, stabilization of ELAVL-1/HuR with the proteasome inhibitor MG-132 resulted in induction of GSK3β at mRNA and protein level and attenuated FITC-albumin clearance. Utilizing ventilator-induced lung injury or intra-tracheal installation of hydrochloric acid to induce ARDS in mice, we observed increased mRNA and protein expression of ELAVL-1/HuR and GSK3β. Together, our findings indicate a previously unknown interaction between GSK3β and ELAV-1 during ARDS, and suggest the inhibition of the ELAV-1- GSK3β pathways as a novel ARDS treatment approach.

All correlations must die: Assessing the significance of a stochastic gravitational-wave background in pulsar timing arrays

We present two methods for determining the significance of a stochastic gravitational-wave (GW) background affecting a pulsar-timing array, where detection is based on evidence for quadrupolar spatial correlations between pulsars. Rather than constructing noise simulations, we eliminate the GWB spatial correlations in the true datasets to assess detection significance with all real data features intact. In our first method, we perform random phase shifts in the signal-model basis functions. This phase shifting eliminates signal phase coherence between pulsars, while keeping the statistical properties of the pulsar timing residuals intact. We then explore a method to null correlations between pulsars by using a "scrambled" overlap-reduction function in the signal model for the array. This scrambled function is orthogonal to what we expect of a real GW background signal. We demonstrate the efficacy of these methods using Bayesian model selection on a set of simulated datasets that contain a stochastic GW signal, timing noise, undiagnosed glitches, and uncertainties in the Solar system ephemeris. Finally, we introduce an overarching formalism under which these two techniques are naturally linked. These methods are immediately applicable to all current pulsar-timing array datasets, and should become standard tools for future analyses.

Genome-wide identification and analysis of biotic and abiotic stress regulation of small heat shock protein (HSP20) family genes in bread wheat

Small Heat Shock Proteins (sHSPs)/HSP20 are molecular chaperones that protect plants by preventing protein aggregation during abiotic stress conditions, especially heat stress. Due to global climate change, high temperature is emerging as a major threat to wheat productivity. Thus, the identification of HSP20 and analysis of HSP transcriptional regulation under different abiotic stresses in wheat would help in understanding the role of these proteins in abiotic stress tolerance. We used sequences of known rice and Arabidopsis HSP20 HMM profiles as queries against publicly available wheat genome and wheat full length cDNA databases (TriFLDB) to identify the respective orthologues from wheat. 163 TaHSP20 (including 109 sHSP and 54 ACD) genes were identified and classified according to the sub-cellular localization and phylogenetic relationship with sequenced grass genomes (Oryza sativa, Sorghum bicolor, Zea mays, Brachypodium distachyon and Setaria italica). Spatio-temporal, biotic and abiotic stress-specific expression patterns in normalized RNA seq and wheat array datasets revealed constitutive as well as inductive responses of HSP20 in different tissues and developmental stages of wheat. Promoter analysis of TaHSP20 genes showed the presence of tissue-specific, biotic, abiotic, light-responsive, circadian and cell cycle-responsive cis-regulatory elements. 14 TaHSP20 family genes were under the regulation of 8 TamiRNA genes. The expression levels of twelve HSP20 genes were studied under abiotic stress conditions in the drought- and heat-tolerant wheat genotype C306. Of the 13 TaHSP20 genes, TaHSP16.9H-CI showed high constitutive expression with upregulation only under salt stress. Both heat and salt stresses upregulated the expression of TaHSP17.4-CI, TaHSP17.7A-CI, TaHSP19.1-CIII, TaACD20.0B-CII and TaACD20.6C-CIV, while TaHSP23.7-MTI was specifically induced only under heat stress. Our results showed that the identified TaHSP20 genes play an important role under different abiotic stress conditions. Thus, the results illustrate the complexity of the TaHSP20 gene family and its stress regulation in wheat, and suggest that sHSPs as attractive breeding targets for improvement of the heat tolerance of wheat.

Genome-wide methylation data mirror ancestry information

BackgroundGenetic data are known to harbor information about human demographics, and genotyping data are commonly used for capturing ancestry information by leveraging genome-wide differences between populations. In contrast, it is not clear to what extent population structure is captured by whole-genome DNA methylation data.ResultsWe demonstrate, using three large-cohort 450K methylation array data sets, that ancestry information signal is mirrored in genome-wide DNA methylation data and that it can be further isolated more effectively by leveraging the correlation structure of CpGs with cis-located SNPs. Based on these insights, we propose a method, EPISTRUCTURE, for the inference of ancestry from methylation data, without the need for genotype data.ConclusionsEPISTRUCTURE can be used to infer ancestry information of individuals based on their methylation data in the absence of corresponding genetic data. Although genetic data are often collected in epigenetic studies of large cohorts, these are typically not made publicly available, making the application of EPISTRUCTURE especially useful for anyone working on public data. Implementation of EPISTRUCTURE is available in GLINT, our recently released toolset for DNA methylation analysis at: http://glint-epigenetics.readthedocs.io.

Genome-Wide Identification and Characterization of Light Harvesting Chlorophyll a/b Binding Protein Genes in Barley (Hordeum vulgare L.)

Light harvesting chlorophyll a/b binding proteins (LHC) play an important role in photosynthesis and development of plant. However, limited data is available for this important gene family in barley, especially for their biological functions. In this study, sequences of rice and Arabidopsis LHCs were used as queries to identify the respective orthologues from available barley genomic database. Total 17 non-reduntant HvLHCs were identified. Genomic sequences of these genes were ranged from 780 bp to 2779 bp in length, and ORF sequence of 738 bp to 933 bp. The phylogenetic relationship of members from barley, rice and Arabidopsis revealed that most of them were common to the three species, while some of them were specific to Arabidopsis or the poaceae family. Cis-element analysis showed that along with the universal transcription initiation and enhancement relevant elements, there were also light as well as biotic and abiotic stresses responsive elements in promoter regions of these HvLHCs. Additionally, these genes exhibited quite similar expression pattern during the development of barley plant according to the public normalized RNA-seq and barley array datasets, but responded distinctively to different treatments including dark, heat, salinity and drought, which were confirmed by quantitative real-time PCR analysis. This is the first report of identification of HvLHCs at the genome level, and results presented here would be useful not only for determining the precise role of HvLHCs during barley development and abiotic stress responses, but also for using them in molecular breeding of barley varieties with high yield or high tolerance to stresses.

Epsin Family Member 3 and Ribosome-Related Genes Are Associated with Late Metastasis in Estrogen Receptor-Positive Breast Cancer and Long-Term Survival in Non-Small Cell Lung Cancer Using a Genome-Wide Identification and Validation Strategy.

BackgroundIn breast cancer, gene signatures that predict the risk of metastasis after surgical tumor resection are mainly indicative of early events. The purpose of this study was to identify genes linked to metastatic recurrence more than three years after surgery.MethodsAffymetrix HG U133A and Plus 2.0 array datasets with information on metastasis-free, disease-free or overall survival were accessed via public repositories. Time restricted Cox regression models were used to identify genes associated with metastasis during or after the first three years post-surgery (early- and late-type genes). A sequential validation study design, with two non-adjuvantly treated discovery cohorts (n = 409) and one validation cohort (n = 169) was applied and identified genes were further evaluated in tamoxifen-treated breast cancer patients (n = 923), as well as in patients with non-small cell lung (n = 1779), colon (n = 893) and ovarian (n = 922) cancer.ResultsTen late- and 243 early-type genes were identified in adjuvantly untreated breast cancer. Adjustment to clinicopathological factors and an established proliferation-related signature markedly reduced the number of early-type genes to 16, whereas nine late-type genes still remained significant. These nine genes were associated with metastasis-free survival (MFS) also in a non-time restricted model, but not in the early period alone, stressing that their prognostic impact was primarily based on MFS more than three years after surgery. Four of the ten late-type genes, the ribosome-related factors EIF4B, RPL5, RPL3, and the tumor angiogenesis modifier EPN3 were significantly associated with MFS in the late period also in a meta-analysis of tamoxifen-treated breast cancer cohorts. In contrast, only one late-type gene (EPN3) showed consistent survival associations in more than one cohort in the other cancer types, being associated with worse outcome in two non-small cell lung cancer cohorts. No late-type gene was validated in ovarian and colon cancer.ConclusionsRibosome-related genes were associated with decreased risk of late metastasis in both adjuvantly untreated and tamoxifen-treated breast cancer patients. In contrast, high expression of epsin (EPN3) was associated with increased risk of late metastasis. This is of clinical relevance considering the well-understood role of epsins in tumor angiogenesis and the ongoing development of epsin antagonizing therapies.

Simultaneous dimension reduction and adjustment for confounding variation

Dimension reduction methods are commonly applied to high-throughput biological datasets. However, the results can be hindered by confounding factors, either biological or technical in origin. In this study, we extend principal component analysis (PCA) to propose AC-PCA for simultaneous dimension reduction and adjustment for confounding (AC) variation. We show that AC-PCA can adjust for (i) variations across individual donors present in a human brain exon array dataset and (ii) variations of different species in a model organism ENCODE RNA sequencing dataset. Our approach is able to recover the anatomical structure of neocortical regions and to capture the shared variation among species during embryonic development. For gene selection purposes, we extend AC-PCA with sparsity constraints and propose and implement an efficient algorithm. The methods developed in this paper can also be applied to more general settings. The R package and MATLAB source code are available at https://github.com/linzx06/AC-PCA.

Cluster Regulation of RUNX Family By "Gene Switch" Triggers a Profound Tumor Regression of Diverse Origins

Although Runt-related transcription factor 1 (RUNX1), a member of RUNX family and a distant relative of p53, has been generally considered to be a tumor suppressor, a growing body of evidence strongly suggests its pro-oncogenic property in acute myeloid leukemia (AML). Here we demonstrate that switching off RUNX cluster utilizing the newly synthesized compound, which specifically bound to a particular base sequence of DNA, was highly effective against leukemia as well as dismal-prognostic solid tumors arising from diverse origins in vivo. Firstly, to assess the RUNX1 loss in AML cells, we performed shRNA-mediated RUNX1 knockdown experiments. Silencing of RUNX1 stimulated cell cycle arrest at G0/G1 phase and simultaneously induced apoptosis in AML cells bearing wild-type p53. RUNX1 depletion induced remarkable induction of p53 as well as its target gene products and additive knockdown of p53 in these cell lines reverted the phenotype of RUNX1-depletion, indicating that RUNX1 is functionally dependent on proficient p53 pathway. In addition, cycloheximide chase assay revealed that RUNX1 negatively regulates p53 protein in AML cells. In silico data analysis of clinical gene expression array data sets and ChIP-seq experiments using anti-RUNX1 antibody identified 32 candidate genes potentially required for RUNX1-dependent degradation of p53. Among them, we focused on BCL11A and TRIM24, both of which are established mediators of p53 degradation. In accordance with these observations, knockdown of RUNX1 resulted in a significant down-regulation of BCL11A and TRIM24 both at mRNA and protein levels. ChIP-qPCR assay further validated the actual binding of RUNX1 at the promoter regions of these genes, and reintroduction of BCL11A or TRIM24 into RUNX1-silenced AML cells restored their proliferation speed to the control levels. These data suggests that RUNX1 depletion-mediated growth inhibitory effect on leukemia cells depends on p53 activation via transcriptional regulation of BCL11A and TRIM24. Though RUNX1 depletion was highly effective on proliferation of AML cells, a small sub-population of leukemia cells retained the proliferation potential even after the silencing of RUNX1. Since it has been shown that RUNX family member has a redundant function, we next examined the other RUNX family members such as RUNX2 and RUNX3 in RUNX1-knocked down AML cells. Under our tetracycline-inducible shRNA expression system, the expression levels of RUNX1-target genes were decreased at 24 h after RUNX1 knockdown, however, their expression levels were reciprocally increased at 48 h accompanied by increment of RUNX2 and RUNX3 expressions, suggesting that RUNX2 and RUNX3 might compensate for the loss of RUNX1 functions. ChIP-qPCR assay and luciferase reporter experiments confirmed that individual RUNX family member consistently suppressed the promoter activity of the other RUNX members. In accordance with these findings, additional knockdown of RUNX2, RUNX3 or both of them in RUNX1-depleted AML cells effectively repressed RUNX1-target gene expressions and completely suppressed their proliferation. Thus the simultaneous targeting of all RUNX family members as a cluster achieves more stringent control of leukemia cells. Since sequencing analysis of the functional gene alterations of RUNX family members revealed the existence of mutations in a mutual-exclusive manner not only in AML cells but also in various cancers, their functional redundancy in the maintenance of AML cells might be generally accepted. To achieve cluster regulations of RUNX, we conducted a synthesized molecule library screening and succeeded in extracting agents that could irreversibly block the RUNX cluster genes expression profiling through dismantling protein-DNA interactions sequence-specifically. These reagents were highly effective against leukemia as well as dismal-prognostic solid tumors arising from diverse origins in vitro. Furthermore, these reagents were exceptionally well-tolerated in mice and exerted excellent efficacy against xenograft mice models of AML, acute lymphoblastc leukemia, lung and gastric cancers, extending their overall survival periods in vivo. Together, this work identifies the crucial role of RUNX cluster in the maintenance and the progression of cancer cells, and the indicated gene switch technology-dependent its modulation would be a novel strategy to control malignancies. DisclosuresNo relevant conflicts of interest to declare.

A link between adipogenesis and innate immunity: RNase-L promotes 3T3-L1 adipogenesis by destabilizing Pref-1 mRNA.

Ribonuclease L (RNase-L) is an endoribonuclease well known for its roles in innate immunity. Recently it has been shown to regulate several cellular functions by modulating the levels of specific mRNAs. In this study, we investigated whether RNase-L may regulate adipocyte functions. We showed that knockdown of RNase-L reduced 3T3-L1 adipocyte differentiation and lipid accumulation. After mRNA profiling, we found that upregulation of Pref-1 mRNA, an inhibitory regulator of adipogenesis, could explain the reduced adipocyte differentiation with RNase-L downregulation. The signaling molecules downstream to Pref-1, including focal adhesion kinase, extracellular signal-regulated kinases and SRY-box 9, were activated by RNase-L suppression. The presence of Pref-1 mRNA was detected in the mRNP complexes precipitated by anti-RNase-L antibody. Moreover, the Pref-1 mRNA decay rate was raised by elevated RNase-L ribonuclease activity. Finally, in stable cell clones with RNase-L silencing, suppression of Pref-1 mRNA by specific siRNA partially recovered the adipocyte differentiation phenotype. Consistent with our findings, meta-analysis of 45 public array datasets from seven independent studies showed a significant negative relationship between RNase-L and Pref-1 mRNA levels in mouse adipose tissues. Higher RNase-L and lower Pref-1 mRNAs were found in the adipose tissues of high-fat diet mice compared to those of ND mice. In line with this, our animal data also showed that the adipose tissues of obese rats contained higher RNase-L and lower Pref-1 expression in comparison to that of lean rats. This study demonstrated that Pref-1 mRNA is a novel substrate of RNase-L. RNase-L is involved in adipocyte differentiation through destabilizing Pref-1 mRNA, thus offering a new link among RNA metabolism, innate immunity and adipogenesis in obesity progression.

Functional redundancy between Apc and Apc2 regulates tissue homeostasis and prevents tumorigenesis in murine mammary epithelium

Aberrant Wnt signaling within breast cancer is associated with poor prognosis, but regulation of this pathway in breast tissue remains poorly understood and the consequences of immediate or long-term dysregulation remain elusive. The exact contribution of the Wnt-regulating proteins adenomatous polyposis coli (APC) and APC2 in the pathogenesis of human breast cancer are ill-defined, but our analysis of publically available array data sets indicates that tumors with concomitant low expression of both proteins occurs more frequently in the ‘triple negative’ phenotype, which is a subtype of breast cancer with particularly poor prognosis. We have used mouse transgenics to delete Apc and/or Apc2 from mouse mammary epithelium to elucidate the significance of these proteins in mammary homeostasis and delineate their influences on Wnt signaling and tumorigenesis. Loss of either protein alone failed to affect Wnt signaling levels or tissue homeostasis. Strikingly, concomitant loss led to local disruption of β-catenin status, disruption in epithelial integrity, cohesion and polarity, increased cell division and a distinctive form of ductal hyperplasia with ‘squamoid’ ghost cell nodules in young animals. Upon aging, the development of Wnt activated mammary carcinomas with squamous differentiation was accompanied by a significantly reduced survival. This novel Wnt-driven mammary tumor model highlights the importance of functional redundancies existing between the Apc proteins both in normal homeostasis and in tumorigenesis.

Altered gene expression by sedaxane increases PSII efficiency, photosynthesis and growth and improves tolerance to drought in wheat seedlings

Succinate dehydrogenase inhibitor (SDHI) fungicides have been shown to increase PSII efficiency and photosynthesis under drought stress in the absence of disease to enhance the biomass and yield of winter wheat. However, the molecular mechanism of improved photosynthetic efficiency observed in SDHI-treated wheat has not been previously elucidated. Here we used a combination of chlorophyll fluorescence, gas exchange and gene expression analysis, to aid our understanding of the basis of the physiological responses of wheat seedlings under drought conditions to sedaxane, a novel SDHI seed treatment. We show that sedaxane increased the efficiency of PSII photochemistry, reduced non-photochemical quenching and improved the photosynthesis and biomass in wheat correlating with systemic changes in the expression of genes involved in defense, chlorophyll synthesis and cell wall modification. We applied a coexpression network-based approach using differentially expressed genes of leaves, roots and pregerminated seeds from our wheat array datasets to identify the most important hub genes, with top ranked correlation (higher gene association value and z-score) involved in cell wall expansion and strengthening, wax and pigment biosynthesis and defense. The results indicate that sedaxane confers tolerant responses of wheat plants grown under drought conditions by redirecting metabolites from defense/stress responses towards growth and adaptive development.

Classifying lower grade glioma cases according to whole genome gene expression.

OBJECTIVETo identify a gene-based signature as a novel prognostic model in lower grade gliomas.RESULTSA gene signature developed from HOXA7, SLC2A4RG and MN1 could segregate patients into low and high risk score groups with different overall survival (OS), and was validated in TCGA RNA-seq and GSE16011 mRNA array datasets. Receiver operating characteristic (ROC) was performed to show that the three-gene signature was more sensitive and specific than histology, grade, age, IDH1 mutation and 1p/19q co-deletion. Gene Set Enrichment Analysis (GSEA) and GO analysis showed high-risk samples were associated with tumor associated macrophages (TAMs) and highly invasive phenotypes. Moreover, HOXA7-siRNA inhibited migration and invasion in vitro, and downregulated MMP9 at the protein level in U251 glioma cells.METHODSA cohort of 164 glioma specimens from the Chinese Glioma Genome Atlas (CGGA) array database were assessed as the training group. TCGA RNA-seq and GSE16011 mRNA array datasets were used for validation. Regression analyses and linear risk score assessment were performed for the identification of the three-gene signature comprising HOXA7, SLC2A4RG and MN1.CONCLUSIONSWe established a three-gene signature for lower grade gliomas, which could independently predict overall survival (OS) of lower grade glioma patients with higher sensitivity and specificity compared with other clinical characteristics. These findings indicate that the three-gene signature is a new prognostic model that could provide improved OS prediction and accurate therapies for lower grade glioma patients.

Two‐dimensional joint inversions of cross‐hole resistivity data and resolution analysis of combined arrays

ABSTRACTIn this study, a new two‐dimensional inversion algorithm was developed for the inversion of cross‐hole direct current resistivity measurements. In the last decades, various array optimisation methods were suggested for resistivity tomography. However, researchers have still collected data by using classical electrode arrays in most cross‐hole applications. Therefore, we investigated the accuracy of both the individual and the joint inversion of the classical cross‐hole arrays by using both synthetic and field data with the developed algorithm. We showed that the joint inversion of bipole–bipole, pole–bipole, bipole–pole, and pole–tripole electrode arrays gives inverse solutions that are closer to the real model than the individual inversions of the electrode array datasets for the synthetic data inversion. The model resolution matrix of the suggested arrays was used to analyse the inversion results. This model resolution analysis also showed the advantage of the joint inversion of bipole–bipole, pole–bipole, bipole–pole, and pole–tripole arrays. We also used sensitivity sections from each of the arrays and their superpositions to explain why joint inversion gives better resolution than the any individual inversion result.

Abstract 3756: PARP inhibitor sensitivity in small cell lung cancer cell lines and patient-derived xenografts correlates with SLFN11 expression but not with structural homologous recombination deficiency

Abstract Poly-(ADP)-ribose polymerase (PARP) was identified as a potential novel therapeutic target for small cell lung cancer (SCLC) in two key proteomic studies. These findings led to the inclusion of a SCLC cohort in a Phase I study of talazoparib (BMN 673) - currently the most potent PARP inhibitor available - with promising efficacy signals. Identifying predictors of response to PARP inhibition would bring a needed therapeutic advance in this recalcitrant disease. PARP inhibition was first demonstrated to have marked efficacy in BRCA1/2 mutant cancers that are deficient in homologous recombination (HR). Large chromosomal structural alterations characteristic of these tumors can be quantitated by three correlated HR deficiency (HRD) metrics. We sought to determine if HRD would be a relevant predictive biomarker in SCLC. Thirty-six SCLC cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) were interrogated for sensitivity to talazoparib, olaparib and cisplatin. We found significant correlation between these three pharmacologic agents and sought to determine response predictors. Three different HRD metrics were computed in these 36 SCLC cell lines using publically available CCLE Affymetrix SNP 6.0 array datasets. Surprisingly, none of these three measures of HRD were discriminatory for response to talazoparib in vitro. However, we found that after controlling for HRD and PARP1 target expression, expression of a single gene, SLFN11, was significantly correlated with talazoparib sensitivity as measured by the CCLE Affymetrix U133+2 arrays (q value = 0.008). After deriving SLFN11 knock-out SCLC cell lines by CRISPR/Cas9 gene editing, we found that loss of SLFN11 conferred resistance to talazoparib (IC50 &amp;gt;log difference) as compared to parental SLFN11 proficient cells. We then treated several SCLC patient-derived xenograft (PDX) models with talazoparib daily and found that SLFN11 expression in vivo was necessary but not sufficient for response. None of the SLFN11 low expressing PDXs responded to therapy, whereas 3/5 (60%) of SLFN11 high expressing PDXs demonstrated significant tumor growth inhibition of 54-75% after 21-28 days of daily treatment with talazoparib 0.3mg/kg. In conclusion, we demonstrate that SLFN11 expression is correlated with response to PARP inhibition in SCLC in vitro and in vivo. HRD structural aberration scores do not predict for in vitro PARP inhibitor sensitivity, however functional HR interrogation (such as RAD51 foci recruitment) may still be a relevant discriminator in addition to ongoing in vivo analysis. SLFN11 may be a pertinent predictive biomarker in SCLC for therapeutic response to PARP inhibition with considerable clinical implications. Citation Format: Benjamin H. Lok, Ying Feng, G. Karen Yu, Eric E. Gardner, Nadeem Riaz, Yuanbin (Kevin) Ru, Elisa de Stanchina, Simon N. Powell, Jerry Shen, John T. Poirier, Charles M. Rudin. PARP inhibitor sensitivity in small cell lung cancer cell lines and patient-derived xenografts correlates with SLFN11 expression but not with structural homologous recombination deficiency. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3756.

Abstract 1124: Expression of the lipocalin 2 oncogene in the development of smoking-associated and Kras mutant lung adenocarcinoma

Abstract There are more than 90 million smokers in the United States who are at elevated risk for lung adenocarcinoma (LUAD), the most common lung cancer subtype. LUADs in smokers frequently (greater than 25%) harbor activating mutations in the Kras oncogene. Kras mutant LUAD exhibits very poor prognosis warranting the need for new strategies to prevent and treat this fatal lung cancer subtype. Limiting these advances is a poor understanding of the earliest events that drive Kras mutant LUAD development in smokers and that would constitute targets for early treatment. The retinoic acid-inducible G-protein coupled receptor Gprc5a is preferentially expressed in normal lung and mice with knockout of the gene (Gprc5a-/-) develop late onset lung tumors spontaneously. In our data aimed at understanding smoking-associated lung cancer pathogenesis, we found that Gprc5a-/- mice, in contrast to wild type (WT) littermates, not only developed LUADs after tobacco carcinogen exposure but also that these lesions harbored somatic Kras mutations, the same variants thought to act as drivers of human LUAD in smokers. In the present study we construed that pinpointing early events preceding lung oncogenesis in Gprc5a-/- mice will help us comprehend how smoke-associated LUADs with Kras mutations evolve. Towards this, we performed RNA-sequencing, using the Ion Proton platform, of visually normal airway epithelia in Gprc5a-/- mice prior to tumor development compared to epithelia in WT mice. We noted distinctively marked up-regulation of the Lcn2 oncogene in normal airways of Gprc5a-/- mice. Functional pathways and gene set enrichment analyses revealed that Lcn2 up-regulation was associated with activation of mitogen activated protein kinase- and interleukin-associated pathways and with gene sets attributable to cigarette smoke. We then analyzed Lcn2 expression in murine cell lines we had derived and found that Lcn2 was significantly elevated in both Gprc5a-/- LUAD and airway cell lines compared to WT airway cells. Moreover, immunohistochemical expression of Lcn2 protein was elevated, compared to normal lung tissue, in early lesions (hyperplasias and adenomas) and in the LUADs of tobacco carcinogen exposed Gprc5a-/- mice. Of note, we also found that alternatively activated M2 macrophages surrounding all lesions were positive for Lcn2. We then analyzed human LCN2 expression in publicly available array datasets and found that the gene was significantly elevated in human KRAS mutant LUADs compared to wild type LUADs or normal lung (all P &amp;lt; 0.05). Our preliminary findings suggest that elevated Lcn2 expression, in lung (airway and tumor) epithelia and in the protumorigenic inflammatory environment, may be implicated in the early development of smoking-associated Kras mutant LUAD. Efforts are underway to further probe this supposition in mice with knockout of Lcn2. Citation Format: Joshua Ochieng, Sayuri Nunomura, Wenhua Lang, Jinsong Wei, Li Xu, Christina McDowell, Edwin Ostrin, Ralph Arlinghaus, Junya Fujimoto, Humam Kadara. Expression of the lipocalin 2 oncogene in the development of smoking-associated and Kras mutant lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1124.

Abstract 781: Modeling the impact of somatic alterations across human cancers

Abstract Large-scale cancer genomics projects like The Cancer Genome Atlas have generated a comprehensive catalog of somatic mutations and copy number aberrations across many tumor types, but the role of most frequently altered genes remains obscure. To better model the impact of these alterations, we developed a novel computational strategy for exploiting parallel phosphoproteomics and mRNA sequencing data for large tumor sets by linking dysregulation of upstream signaling pathways with altered transcriptional response through the transcriptional circuitry. Our modeling allows us to interpret the impact of somatic alterations in terms of functional outcomes such as altered signaling and transcription factor (TF) activity. We used this novel machine learning strategy to train phosphoprotein-TF interaction models across 12 human cancers for which large reverse-phase protein array and RNA-seq data sets are available through TCGA. First, we used this approach to identify shared and cancer-specific roles of TF/signaling regulators across cancer types. Then we performed a statistical analysis to associate frequent somatic aberrations with alterations in inferred TF and signaling protein activities. From our analysis, we gained many novel insights into cancer biology. We identified both known (e.g FOXO1 for breast cancer) and novel TF regulators of cancer (e.g. ELK1 for head and neck cancer). Many of these identified TF regulators were significantly associated with survival outcome in bladder urothelial, renal cell clear and endometrial carcinoma. Next we performed a comprehensive cross-cancer analysis and identified relationships between somatic alterations and downstream transcriptional effects and signaling pathway activation. We observed that specific molecular aberrations have different functional consequences in different cancer types. For example, PIK3CA mutations are associated with altered activities of a diverse set of TFs across cancers that are involved in cell cycle, apoptosis, metabolism and MAPK/ERK signaling. Notably, in cell line models, we validated some of the altered TFs predicted by our model. We showed that PIK3CA mutation leads to ELK1 activation in breast and head and neck cancer models. We further found that different set of TFs are associated with a specific mutation in different cancers due to the different background of genomic aberrations in each cancer. For example, KRAS mutations are associated with a distinct set of TFs depending of cancer-specific co-mutation profiles (e.g. co-mutations of KRAS and TP53 in lung adenocarcinoma and co-mutations of KRAS and APC in colorectal cancer). Our analysis revealed both known and novel interactions of frequently altered genes with signaling pathways and transcriptional programs in a pan-cancer context. Patterns of co-alterations across cancers may provide new insights relevant to targeted therapy and may be crucial to optimizing combination therapies. Citation Format: Hatice U. Osmanbeyoglu, Eneda Toska, José Baselga, Christina Leslie. Modeling the impact of somatic alterations across human cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 781.

Abstract 2387: Pan-cancer analysis of clonal evolution reveals the costs and adaptive benefits of genomic instability

Abstract Cancers are a mosaic of clones of varying population sizes. Any single cancer sample encodes a tumor-metagenome, since it represents the aggregate genomes of diverse clones that coexist within the sample. We quantified genomic instability as the fraction of the tumor-metagenome affected by copy number variations (CNVs) and leveraged two tumor mixture separation algorithms, EXPANDS and PyClone, to quantify genetic intra-tumor heterogeneity (ITH) from single cancer samples. We tested the potential of measures of genomic instability and ITH as prognostic biomarkers across 1,165 exome sequenced primary tumors from 12 cancer types at TCGA. Our results suggest that a tradeoff between the costs and adaptive benefits of genomic instability governs differential radiotherapy sensitivity. Between 1 and 18 clones were estimated to coexist per tumor sample at &amp;gt;10% cell frequency (median = 4). Clone number varied considerably within and between cancer types, with melanomas representing the most heterogeneous group. 86% all analyzed tumor samples contained at least 2 clones. Across cancer types, the presence of &amp;gt;2 clones was associated with worse overall survival as compared to tumors in which &amp;lt; = 2 clones were detected (Log-rank test: P = 8.6E-4, HR = 1.49). An exceptionally favorable outcome was observed when &amp;gt;75% genomic instability was shared among &amp;lt; = 2 clones. The highest risk was observed among individuals with an intermediate number of 4 clones - additional diversification beyond 4 clones did not impart further risk. The highest risk was also observed among individuals with 50-75% genomic instability, in both the original exome sequencing dataset and an independent SNP array dataset. Genomic instability &amp;lt;25% or &amp;gt;75% predicted reduced risk (HR = 0.15, 95% CI: 0.08-0.29). We analyzed the relation between radiotherapy intensity and overall survival among 242 individuals (21%) treated with radiotherapy and found that not all individuals did benefit equally from therapy. In order to achieve the same benefit from therapy, individuals with 25-50% genomic instability required higher therapy intensity (regression slope = 1.83; P = 0.009) than individuals with 50-75% genomic instability (slope = 2.09; P = 0.005). In contrast, individuals with &amp;lt;25% genomic instability did not benefit from increasing therapy intensity (slope = 0.71; P = 0.311). Radiotherapy may be particularly effective against tumors with intermediate CNV burden, by pushing them past the limit of ‘tolerable’ genomic instability. Our results from two independent pan-cancer cohorts suggest that this limit is exceeded when &amp;gt;75% of a tumor's metagenome is affected by CNVs. This upper limit of tolerable genomic instability may be responsible for the non-linear association we observed between genetic ITH and survival. Leveraging a clone's distance to the upper limit of tolerable genomic instability may represent a new strategy to optimize therapy intensity. Citation Format: Noemi Andor, Trevor A. Graham, Marnix Jansen, Li C. Xia, Athena Aktipis, Claudia Petritsch, Hanlee P. Ji, Carlo C. Maley. Pan-cancer analysis of clonal evolution reveals the costs and adaptive benefits of genomic instability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2387.