Elucidation of the molecular mechanism of the breakage-fusion-bridge (BFB) cycle using a CRISPR-dCas9 cellular model.

Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanisms for the individual steps of the BFB cycle warrants further investigation. Here, we demonstrated that a nuclease-dead Cas9 (dCas9) coupled with a telomere-specific single-guide RNA (sgTelo) can be used to model the BFB cycle. First, we showed that targeting dCas9 to telomeres using sgTelo impeded DNA replication at telomeres and induced a pronounced increase of replication stress and DNA damage. Using Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM), we investigated the genome-wide features of telomeres in the dCas9/sgTelo cells and observed a dramatic increase of chromosome end fusions, including fusion/ITS+ and fusion/ITS-.Consistently, we also observed an increase in the formation of dicentric chromosomes, anaphase bridges, and intercellular telomeric chromosome bridges (ITCBs). Utilizing the dCas9/sgTelo system, we uncovered many novel molecular and structural features of the ITCB and demonstrated that multiple DNA repair pathways are implicated in the formation of ITCBs. Our studies shed new light on the molecular mechanisms of the BFB cycle, which will advance our understanding of tumorigenesis, tumor evolution, and drug resistance.

Singleton mutations in large-scale cancer genome studies: uncovering the tail of cancer genome.

Singleton or low-frequency driver mutations are challenging to identify. We present a domain driver mutation estimator (DOME) to identify rare candidate driver mutations. DOME analyzes positions analogous to known statistical hotspots and resistant mutations in combination with their functional and biochemical residue context as determined by protein structures and somatic mutation propensity within conserved PFAM domains, integrating the CADD scoring scheme. Benchmarked against seven other tools, DOME exhibited superior or comparable accuracy compared to all evaluated tools in the prediction of functional cancer drivers, with the exception of one tool. DOME identified a unique set of 32 917 high-confidence predicted driver mutations from the analysis of whole proteome missense variants within domain boundaries across 1331 genes, including 1192 noncancer gene census genes, emphasizing its unique place in cancer genome analysis. Additionally, analysis of 8799 TCGA (The Cancer Genome Atlas) and in-house tumor samples revealed 847 potential driver mutations, with mutations in tyrosine kinase members forming the dominant burden, underscoring its higher significance in cancer. Overall, DOME complements current approaches for identifying novel, low-frequency drivers and resistant mutations in personalized therapy.

Recurrent UBE3C-LRP5 translocations in head and neck cancer with therapeutic implications.

Head and neck cancer is a major cause of morbidity and mortality worldwide. The identification of genetic alterations in head and neck cancer may improve diagnosis and treatment outcomes. In this study, we report the identification and functional characterization of UBE3C-LRP5 translocation in head and neck cancer. Our whole transcriptome sequencing and RT-PCR analysis of 151 head and neck cancer tumor samples identified the LRP5-UBE3C and UBE3C-LRP5 fusion transcripts in 5.3% of patients of Indian origin (n = 151), and UBE3C-LRP5 fusion transcripts in 1.2% of TCGA-HNSC patients (n = 502). Further, whole genome sequencing identified the breakpoint of UBE3C-LRP5 translocation. We demonstrate that UBE3C-LRP5 fusion is activating in vitro and in vivo, and promotes the proliferation, migration, and invasion of head and neck cancer cells. In contrast, depletion of UBE3C-LRP5 fusion suppresses the clonogenic, migratory, and invasive potential of the cells. The UBE3C-LRP5 fusion activates the Wnt/ß-catenin signaling by promoting nuclear accumulation of ß-catenin, leading to upregulation of Wnt/ß-catenin target genes, MYC, CCND1, TCF4, and LEF1. Consistently, treatment with the FDA-approved drug, pyrvinium pamoate, significantly reduced the transforming ability of cells expressing the fusion protein and improved survival in mice bearing tumors of fusion-overexpressing cells. Interestingly, fusion-expressing cells upon knockdown of CTNNB1, or LEF1 show reduced proliferation, clonogenic abilities, and reduced sensitivity to pyrvinium pamoate. Overall, our study suggests that the UBE3C-LRP5 fusion is a promising therapeutic target for head and neck cancer and that pyrvinium pamoate may be a potential drug candidate for treating head and neck cancer harboring this translocation.

Association of Cutibacterium acnes with human thyroid cancer.

Introduction: The diverse subtypes of thyroid carcinoma have distinct clinical outcomes despite a comparable spectrum of underlying genetic alterations. Beyond genetic alterations, sparse efforts have been made to characterize the microbes associated with thyroid cancer. In this study, we examine the microbial profile of thyroid cancer. Methods: We sequenced the whole transcriptome of 70 thyroid cancers (40 papillary and 30 anaplastic). Using Infectious Pathogen Detector IPD 2.0, we analysed the relative abundance of 1060 microbes across 70 tumours from patients with thyroid cancer against 118 tumour samples from patients with breast, cervical, colorectal, and tongue cancer. Results: Our analysis reveals a significant prevalence of Cutibacterium acnes in 58.6% thyroid cancer samples compared to other cancer types (p=0.00038). Immune cell fraction analysis between thyroid cancer samples with high and low Cutibacterium loads identify enrichment of immunosuppressive cells, including Tregs (p=0.015), and other anti-inflammatory cytokines in the tumour microenvironment, suggesting an immune evasion/immunosuppression milieu is associated with the infection. A higher burden of Cutibacterium acnes was also found to be associated with poor survival defining a distinct sub-group of thyroid cancer. Conclusion: Cutibacterium acnes is associated with immune suppression and poor prognosis in a subpopulation of thyroid cancer. This study may help design novel therapeutic measures involving appropriate antibiotics to manage the disease better.

Influence of pathogens on host genome and epigenome in development of head and neck cancer.

BACKGROUND: Head and neck cancer (HNSCC) is a heterogeneous group of cancers, affecting multiple regions such as oral cavity, pharynx, larynx, and nasal region, each showing a distinct molecular profile. HNSCC accounts for more than 6 million cases worldwide, soaring mainly in the developing countries. RECENT FINDINGS: The aetiology of HNSCC is complex and multifactorial, involving both genetic and environmental factors. The critical role of microbiome, which includes bacteria, viruses, and fungi, is under spotlight due to the recent reports on their contribution in the development and progression of HNSCC. This review focuses on the effect of opportunistic pathogens on the host genome and epigenome, which contributes to the disease progression. Drawing parallels from the host-pathogen interactions observed in other tumour types arising from the epithelial tissue such as colorectal cancer, the review also calls attention to the potential explorations of the role of pathogens in HNSCC biology and discusses the clinical implications of microbiome research in detection and treatment of HNSCC. CONCLUSION: Our understanding of the genomic effects of the microbes on the disease progression and the mechanistic insights of the host-pathogen interaction will pave way to novel treatment and preventive approaches in HNSCC.

The Molecular and Cellular Basis of Hutchinson-Gilford Progeria Syndrome and Potential Treatments.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, autosomal-dominant, and fatal premature aging syndrome. HGPS is most often derived from a de novo point mutation in the LMNA gene, which results in an alternative splicing defect and the generation of the mutant protein, progerin. Progerin behaves in a dominant-negative fashion, leading to a variety of cellular and molecular changes, including nuclear abnormalities, defective DNA damage response (DDR) and DNA repair, and accelerated telomere attrition. Intriguingly, many of the manifestations of the HGPS cells are shared with normal aging cells. However, at a clinical level, HGPS does not fully match normal aging because of the accelerated nature of the phenotypes and its primary effects on connective tissues. Furthermore, the epigenetic changes in HGPS patients are of great interest and may play a crucial role in the pathogenesis of HGPS. Finally, various treatments for the HGPS patients have been developed in recent years with important effects at a cellular level, which translate to symptomatic improvement and increased lifespan.

Role of miR-944/MMP10/AXL- axis in lymph node metastasis in tongue cancer.

Occult lymph-node metastasis is a crucial predictor of tongue cancer mortality, with an unmet need to understand the underlying mechanism. Our immunohistochemical and real-time PCR analysis of 208 tongue tumors show overexpression of Matrix Metalloproteinase, MMP10, in 86% of node-positive tongue tumors (n = 79; p < 0.00001). Additionally, global profiling for non-coding RNAs associated with node-positive tumors reveals that of the 11 significantly de-regulated miRNAs, miR-944 negatively regulates MMP10 by targeting its 3'-UTR. We demonstrate that proliferation, migration, and invasion of tongue cancer cells are suppressed by MMP10 knockdown or miR-944 overexpression. Further, we show that depletion of MMP10 prevents nodal metastases using an orthotopic tongue cancer mice model. In contrast, overexpression of MMP10 leads to opposite effects upregulating epithelial-mesenchymal-transition, mediated by a tyrosine kinase gene, AXL, to promote nodal and distant metastasis in vivo. Strikingly, AXL expression is essential and sufficient to mediate the functional consequence of MMP10 overexpression. Consistent with our findings, TCGA-HNSC data suggests overexpression of MMP10 or AXL positively correlates with poor survival of the patients. In conclusion, our results establish that the miR-944/MMP10/AXL- axis underlies lymph node metastases with potential therapeutic intervention and prediction of nodal metastases in tongue cancer patients.

Progesterone modulates the DSCAM-AS1/miR-130a/ESR1 axis to suppress cell invasion and migration in breast cancer.

BACKGROUND: A preoperative-progesterone intervention increases disease-free survival in patients with breast cancer, with an unknown underlying mechanism. We elucidated the role of non-coding RNAs in response to progesterone in human breast cancer. METHODS: Whole transcriptome sequencing dataset of 30 breast primary tumors (10 tumors exposed to hydroxyprogesterone and 20 tumors as control) were re-analyzed to identify differentially expressed non-coding RNAs followed by real-time PCR analyses to validate the expression of candidates. Functional analyses were performed by genetic knockdown, biochemical, and cell-based assays. RESULTS: We identified a significant downregulation in the expression of a long non-coding RNA, Down syndrome cell adhesion molecule antisense DSCAM-AS1, in response to progesterone treatment in breast cancer. The progesterone-induced expression of DSCAM-AS1 could be effectively blocked by the knockdown of progesterone receptor (PR) or treatment of cells with mifepristone (PR-antagonist). We further show that knockdown of DSCAM-AS1 mimics the effect of progesterone in impeding cell migration and invasion in PR-positive breast cancer cells, while its overexpression shows an opposite effect. Additionally, DSCAM-AS1 sponges the activity of miR-130a that regulates the expression of ESR1 by binding to its 3'-UTR to mediate the effect of progesterone in breast cancer cells. Consistent with our findings, TCGA analysis suggests that high levels of miR-130a correlate with a tendency toward better overall survival in patients with breast cancer. CONCLUSION: This study presents a mechanism involving the DSCAM-AS1/miR-130a/ESR1 genomic axis through which progesterone impedes breast cancer cell invasion and migration. The findings highlight the utility of progesterone treatment in impeding metastasis and improving survival outcomes in patients with breast cancer.

TMC-SNPdb 2.0: an ethnic-specific database of Indian germline variants.

Cancer is a somatic disease. The lack of Indian-specific reference germline variation resources limits the ability to identify true cancer-associated somatic variants among Indian cancer patients. We integrate two recent studies, the GenomeAsia 100K and the Genomics for Public Health in India (IndiGen) program, describing genome sequence variations across 598 and 1029 healthy individuals of Indian origin, respectively, along with the unique variants generated from our in-house 173 normal germline samples derived from cancer patients to generate the Tata Memorial Centre-SNP database (TMC-SNPdb) 2.0. To show its utility, GATK/Mutect2-based somatic variant calling was performed on 224 in-house tumor samples to demonstrate a reduction in false-positive somatic variants. In addition to the ethnic-specific variants from GenomeAsia 100K and IndiGenomes databases, 305 132 unique variants generated from 173 in-house normal germline samples derived from cancer patients of Indian origin constitute the Indian specific, TMC-SNPdb 2.0. Of 305 132 unique variants, 11.13% were found in the coding region with missense variants (31.3%) as the most predominant category. Among the non-coding variations, intronic variants (49%) were the highest contributors. The non-synonymous to synonymous SNP ratio was observed to be 1.9, consistent with the previous version of TMC-SNPdb and literature. Using TMC SNPdb 2.0, we analyzed a whole-exome sequence from 224 in-house tumor samples (180 paired and 44 orphans). We show an average depletion of 3.44% variants per paired tumor and significantly higher depletion (P-value < 0.001) for orphan tumors (4.21%), demonstrating the utility of the rare, unique variants found in the ethnic-specific variant datasets in reducing the false-positive somatic mutations. TMC-SNPdb 2.0 is the most exhaustive open-source reference database of germline variants occurring across 1800 Indian individuals to analyze cancer genomes and other genetic disorders. The database and toolkit package is available for download at the following: Database URL  http://www.actrec.gov.in/pi-webpages/AmitDutt/TMCSNPdb2/TMCSNPdb2.html.

ADAM10 facilitates rapid neural stem cell cycling and proper positioning within the subventricular zone niche via JAMC/RAP1Gap signaling.

The mechanisms that regulate neural stem cell (NSC) lineage progression and maintain NSCs within different domains of the adult neural stem cell niche, the subventricular zone are not well defined. Quiescent NSCs are arranged at the apical ventricular wall, while mitotically activated NSCs are found in the basal, vascular region of the subventricular zone. Here, we found that ADAM10 (a disintegrin and metalloproteinase 10) is essential in NSC association with the ventricular wall, and via this adhesion to the apical domain, ADAM10 regulates the switch from quiescent and undifferentiated NSC to an actively proliferative and differentiating cell state. Processing of JAMC (junctional adhesion molecule C) by ADAM10 increases Rap1GAP activity. This molecular machinery promotes NSC transit from the apical to the basal compartment and subsequent lineage progression. Understanding the molecular mechanisms responsible for regulating the proper positioning of NSCs within the subventricular zone niche and lineage progression of NSCs could provide new targets for drug development to enhance the regenerative properties of neural tissue.

Fusobacterium nucleatum is associated with inflammation and poor survival in early-stage HPV-negative tongue cancer.

Persistent pathogen infection is a known cause of malignancy, although with sparse systematic evaluation across tumor types. We present a comprehensive landscape of 1060 infectious pathogens across 239 whole exomes and 1168 transcriptomes of breast, lung, gallbladder, cervical, colorectal, and head and neck tumors. We identify known cancer-associated pathogens consistent with the literature. In addition, we identify a significant prevalence of Fusobacterium in head and neck tumors, comparable to colorectal tumors. The Fusobacterium-high subgroup of head and neck tumors occurs mutually exclusive to human papillomavirus, and is characterized by overexpression of miRNAs associated with inflammation, elevated innate immune cell fraction and nodal metastases. We validate the association of Fusobacterium with the inflammatory markers IL1B, IL6 and IL8, miRNAs hsa-mir-451a, hsa-mir-675 and hsa-mir-486-1, and MMP10 in the tongue tumor samples. A higher burden of Fusobacterium is also associated with poor survival, nodal metastases and extracapsular spread in tongue tumors defining a distinct subgroup of head and neck cancer.

Mitigating consumer privacy breach in smart grid using obfuscation-based generative adversarial network.

Smart meters allow real-time monitoring and collection of power consumption data of a consumer's premise. With the worldwide integration of smart meters, there has been a substantial rise in concerns regarding threats to consumer privacy. The exposed fine-grained power consumption data results in behaviour leakage by revealing the end-user's home appliance usage information. Previously, researchers have proposed approaches to alter data using perturbation, aggregation or hide identifiers using anonymization. Unfortunately, these techniques suffer from various limitations. In this paper, we propose a privacy preserving architecture for fine-grained power data in a smart grid. The proposed architecture uses generative adversarial network (GAN) and an obfuscator to generate a synthetic timeseries. The proposed architecture enables to replace the existing appliance signature with appliances that are not active during that period while ensuring minimum energy difference between the ground truth and the synthetic timeseries. We use real-world dataset containing power consumption readings for our experiment and use non-intrusive load monitoring (NILM) algorithms to show that our approach is more effective in preserving the privacy level of a consumer's power consumption data.

IPD 2.0: To derive insights from an evolving SARS-CoV-2 genome.

BACKGROUND: Rapid analysis of SARS-CoV-2 genomic data plays a crucial role in surveillance and adoption of measures in controlling spread of Covid-19. Fast, inclusive and adaptive methods are required for the heterogenous SARS-CoV-2 sequence data generated at an unprecedented rate. RESULTS: We present an updated version of the SARS-CoV-2 analysis module of our automated computational pipeline, Infectious Pathogen Detector (IPD) 2.0, to perform genomic analysis to understand the variability and dynamics of the virus. It adopts the recent clade nomenclature and demonstrates the clade prediction accuracy of 92.8%. IPD 2.0 also contains a SARS-CoV-2 updater module, allowing automatic upgrading of the variant database using genome sequences from GISAID. As a proof of principle, analyzing 208,911 SARS-CoV-2 genome sequences, we generate an extensive database of 2.58 million sample-wise variants. A comparative account of lineage-specific mutations in the newer SARS-CoV-2 strains emerging in the UK, South Africa and Brazil and data reported from India identify overlapping and lineages specific acquired mutations suggesting a repetitive convergent and adaptive evolution. CONCLUSIONS: A novel and dynamic feature of the SARS-CoV-2 module of IPD 2.0 makes it a contemporary tool to analyze the diverse and growing genomic strains of the virus and serve as a vital tool to help facilitate rapid genomic surveillance in a population to identify variants involved in breakthrough infections. IPD 2.0 is freely available from http://www.actrec.gov.in/pi-webpages/AmitDutt/IPD/IPD.html and the web-application is available at http://ipd.actrec.gov.in/ipdweb/ .

Molecular characterization of lung squamous cell carcinoma tumors reveals therapeutically relevant alterations.

INTRODUCTION: Unlike lung adenocarcinoma patients, there is no FDA-approved targeted-therapy likely to benefit lung squamous cell carcinoma patients. MATERIALS AND METHODS: We performed survival analyses of lung squamous cell carcinoma patients harboring therapeutically relevant alterations identified by whole exome sequencing and mass spectrometry-based validation across 430 lung squamous tumors. RESULTS: We report a mean of 11.6 mutations/Mb with a characteristic smoking signature along with mutations in TP53 (65%), CDKN2A (20%), NFE2L2 (20%), FAT1 (15%), KMT2C (15%), LRP1B (15%), FGFR1 (14%), PTEN (10%) and PREX2 (5%) among lung squamous cell carcinoma patients of Indian descent. In addition, therapeutically relevant EGFR mutations occur in 5.8% patients, significantly higher than as reported among Caucasians. In overall, our data suggests 13.5% lung squamous patients harboring druggable mutations have lower median overall survival, and 19% patients with a mutation in at least one gene, known to be associated with cancer, result in significantly shorter median overall survival compared to those without mutations. CONCLUSIONS: We present the first comprehensive landscape of genetic alterations underlying Indian lung squamous cell carcinoma patients and identify EGFR, PIK3CA, KRAS and FGFR1 as potentially important therapeutic and prognostic target.

An integrated approach to determine the abundance, mutation rate and phylogeny of the SARS-CoV-2 genome.

The analysis of the SARS-CoV-2 genome datasets has significantly advanced our understanding of the biology and genomic adaptability of the virus. However, the plurality of advanced sequencing datasets-such as short and long reads-presents a formidable computational challenge to uniformly perform quantitative, variant or phylogenetic analysis, thus limiting its application in public health laboratories engaged in studying epidemic outbreaks. We present a computational tool, Infectious Pathogen Detector (IPD), to perform integrated analysis of diverse genomic datasets, with a customized analytical module for the SARS-CoV-2 virus. The IPD pipeline quantitates individual occurrences of 1060 pathogens and performs mutation and phylogenetic analysis from heterogeneous sequencing datasets. Using IPD, we demonstrate a varying burden (5.055-999655.7 fragments per million) of SARS-CoV-2 transcripts across 1500 short- and long-read sequencing SARS-CoV-2 datasets and identify 4634 SARS-CoV-2 variants (~3.05 variants per sample), including 449 novel variants, across the genome with distinct hotspot mutations in the ORF1ab and S genes along with their phylogenetic relationships establishing the utility of IPD in tracing the genome isolates from the genomic data (as accessed on 11 June 2020). The IPD predicts the occurrence and dynamics of variability among infectious pathogens-with a potential for direct utility in the COVID-19 pandemic and beyond to help automate the sequencing-based pathogen analysis and in responding to public health threats, efficaciously. A graphical user interface (GUI)-enabled desktop application is freely available for download for the academic users at http://www.actrec.gov.in/pi-webpages/AmitDutt/IPD/IPD.html and for web-based processing at http://ipd.actrec.gov.in/ipdweb/ to generate an automated report without any prior computational know-how.

A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2.

Early diagnosis of SARS-CoV-2 infected patients is essential to control the dynamics of the COVID-19 pandemic. We develop a rapid and accurate one-step multiplex TaqMan probe-based real-time RT-PCR assay, along with a computational tool to systematically analyse the data. Our assay could detect to a limit of 15 copies of SARS-CoV-2 transcripts-based on experiments performed by spiking total human RNA with in vitro synthesized viral transcripts. The assay was evaluated by performing 184 validations for the SARS-CoV-2 Nucleocapsid gene and human RNase P as an internal control reference gene with dilutions ranging from 1-100 ng for human RNA on a cohort of 26 clinical samples. 5 of 26 patients were confirmed to be infected with SARS-CoV-2, while 21 tested negative, consistent with the standards. The accuracy of the assay was found to be 100% sensitive and 100% specific based on the 26 clinical samples that need to be further verified using a large number of clinical samples. In summary, we present a rapid, easy to implement real-time PCR based assay with automated analysis using a novel COVID qPCR Analyzer tool with graphical user interface (GUI) to analyze the raw qRT-PCR data in an unbiased manner at a cost of under $3 per reaction and turnaround time of less than 2h, to enable in-house SARS-CoV-2 testing across laboratories.

Raman spectroscopy-based detection of RNA viruses in saliva: A preliminary report.

Several non-invasive Raman spectroscopy-based assays have been reported for rapid and sensitive detection of pathogens. We developed a novel statistical model for the detection of RNA viruses in saliva, based on an unbiased selection of a set of 65 Raman spectral features that mostly attribute to the RNA moieties, with a prediction accuracy of 91.6% (92.5% sensitivity and 88.8% specificity). Furthermore, to minimize variability and automate the downstream analysis of the Raman spectra, we developed a GUI-based analytical tool "RNA Virus Detector (RVD)." This conceptual framework to detect RNA viruses in saliva could form the basis for field application of Raman Spectroscopy in managing viral outbreaks, such as the ongoing COVID-19 pandemic. (http://www.actrec.gov.in/pi-webpages/AmitDutt/RVD/RVD.html).

Inhibition of SETMAR-H3K36me2-NHEJ repair axis in residual disease cells prevents glioblastoma recurrence.

BACKGROUND: Residual disease of glioblastoma (GBM) causes recurrence. However, targeting residual cells has failed, due to their inaccessibility and our lack of understanding of their survival mechanisms to radiation therapy. Here we deciphered a residual cell-specific survival mechanism essential for GBM relapse. METHODS: Therapy resistant residual (RR) cells were captured from primary patient samples and cell line models mimicking clinical scenario of radiation resistance. Molecular signaling of resistance in RR cells was identified using RNA sequencing, genetic and pharmacological perturbations, overexpression systems, and molecular and biochemical assays. Findings were validated in patient samples and an orthotopic mouse model. RESULTS: RR cells form more aggressive tumors than the parental cells in an orthotopic mouse model. Upon radiation-induced damage, RR cells preferentially activated a nonhomologous end joining (NHEJ) repair pathway, upregulating Ku80 and Artemis while downregulating meiotic recombination 11 (Mre11) at protein but not RNA levels. Mechanistically, RR cells upregulate the Su(var)3-9/enhancer-of-zeste/trithorax (SET) domain and mariner transposase fusion gene (SETMAR), mediating high levels of H3K36me2 and global euchromatization. High H3K36me2 leads to efficiently recruiting NHEJ proteins. Conditional knockdown of SETMAR in RR cells induced irreversible senescence partly mediated by reduced H3K36me2. RR cells expressing mutant H3K36A could not retain Ku80 at double-strand breaks, thus compromising NHEJ repair, leading to apoptosis and abrogation of tumorigenicity in vitro and in vivo. Pharmacological inhibition of the NHEJ pathway phenocopied H3K36 mutation effect, confirming dependency of RR cells on the NHEJ pathway for their survival. CONCLUSIONS: We demonstrate that the SETMAR-NHEJ regulatory axis is essential for the survival of clinically relevant radiation RR cells, abrogation of which prevents recurrence in GBM.

Identifying cancer driver genes from functional genomics screens.

With the emerging advances made in genomics and functional genomics approaches, there is a critical and growing unmet need to integrate plural datasets in order to identify driver genes in cancer. An integrative approach, with the convergence of multiple types of genetic evidence, can limit false positives through a posterior filtering strategy and reduce the need for multiple hypothesis testing to identify true cancer vulnerabilities. We performed a pooled shRNA screen against 906 human genes in the oral cancer cell line AW13516 in triplicate. The genes that were depleted in the screen were integrated with copy number alteration and gene expression data and ranked based on ROAST analysis, using an integrative scoring system, DepRanker, to compute a Rank Impact Score (RIS) for each gene. The RIS-based ranking of candidate driver genes was used to identify the putative oncogenes AURKB and TK1 as essential for oral cancer cell proliferation. We validated the findings, showing that shRNA mediated genetic knockdown of TK1 or pharmacological inhibition of AURKB by AZD-1152 HQPA in AW13516 cells could significantly impede their proliferation. Next we analysed alterations in AURKB and TK1 genes in head and neck cancer and their association with prognosis using data on 528 patients obtained from TCGA. Patients harbouring alterations in AURKB and TK1 genes were associated with poor survival. To summarise, we present DepRanker as a simple yet robust package with no third-party dependencies for the identification of potential driver genes from a pooled shRNA functional genomic screen by integrating results from RNAi screens with gene expression and copy number data. Using DepRanker, we identify AURKB and TK1 as potential therapeutic targets in oral cancer. DepRanker is in the public domain and available for download at http://www.actrec.gov.in/pi-webpages/AmitDutt/DepRanker/DepRanker.html.