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Descriptive panel of hot-spot mutations in NFE2L2 and KEAP1 genes across lung and head and neck carcinoma samples available in the TCGA database (Provisional datasets) as obtained from the "Mutations" tool available in the TCGA cBioportal

Supplementary figure legends for figures S1 to S5.

TP53 is the most frequently mutated gene in most types of human cancer, including breast cancer. The triple-negative breast cancer (TNBC) subtype in particular displays TP53 mutation in approximately 80% of patients. Unlike other breast cancer subtypes (e.g., ER/PR-positive or HER2-positive), TNBC patients currently lack an approved highly effective targeted therapy. Notably, most TNBC acquires TP53 mutations, which, in addition to the loss of canonical p53 functions, can result in gain-of-function, activating different cellular mechanisms involved in tumor phenotypes such as proliferation, metastasis, invasiveness, and angiogenesis. Here we evaluate the role of mutant p53 in cancer phenotypes of TNBC cell lines, in particular their inflammatory profile. As expected, loss of p53 protein by small interfering RNA depletion did not show a major impact on the cell viability of MDA-MB231 and Hs578t cells in MTT assay. However, depleting mutant p53 knockdown made MDA-MB231 cells more susceptible to treatment with methyl-methane sulfonate, a genotoxic alkylating agent. Interestingly, cell invasion as measured by the transwell assay demonstrated that depletion of mutant p53 depletion decreased the invasiveness potential of MDA-MB231 and Hs578t cells which was substantiated with decreased migration of MDA-MB231 cells in a scratch assay over 24 h. RNA sequencing of MDA-MB231 and Hs578t cells revealed that mutant p53 knockdown decreased the expression of several constitutively expressed pro-inflammatory cytokines such as IL8, IL6, CXCL2, and CXCL3, but not genes associated with survival to alkylating agents (NRF2 and Endoplasmic reticulum stress markers) or genes typically regulated by wild-type p53 when compared to control-silenced and MMS-treated cells as indicated by Pathway Enrichment Analysis using the Enrich R and DAVID tools. These results were confirmed by ELISA quantification of IL8, IL6, and CXCL2 in MDA-MB231 and Hs578t transfected with two sequences of siRNA targeting mutant p53. On the other hand, RNA sequencing revealed some constitutively expressed genes known to be involved in breast cancer cells malignancy, such as PTGS2 (COX-2 enzyme gene) and MMP1, which were not affected by p53 knockdown. We found that MMP1, and PGE2 (the product of PTGS2/COX-2 enzyme), are upregulated by the ERK1/2 MAPK signaling pathway as determined in MDA-MB231 cells treated with the MEK1/2 inhibitor UO126 and sorafenib. UO126 and sorafenib also decreased IL8 and IL6 production. Furthermore, combined mutant p53 knockdown with MEK/ERK1/2 pathway inhibition caused a more pronounced IL8 and IL6 inhibition when compared to either p53 knockdown or UO126/sorafenib alone, whereas MMP1 and PGE2 levels were only reduced by MEK/ERK1/2 inhibitor treatments. Interestingly, neither mutant p53 knockdown impacted ERK1/2 phosphorylation status nor did UO126/sorafenib alter mutant p53 immunocontent. Reporter assays showed that mutant p53 promotes NFkappaB reporter activation, and MEK/ERK1/2 controls both NFkappaB and AP-1 transcription factors, both associated with the expression of the secretome components evaluated herein. Functional cell assays showed that concomitant inhibition of mutant p53 and MEK/ERK1/2 pathways reduce cell proliferation, invasion, and migration, indicating that mutant p53 protein gain of function cooperates with ERK1/2 MAPK signaling pathway to promote secretome production and malignant phenotypes in TNBC cell models. Citation Format: Raquel Nascimento das Neves, Aparna Gorthi, Alexander James Roy Bishop, Alfeu Zanotto Filho. Mutant p53 and ERK1/2 MAPK cooperate with the production of TNBC inflammatory secretome [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-03.

Supplementary Fig. 1. IB inhibits the growth of breast cancer cells in a dose and time dependent manner. Supplementary Fig. 2. IB inhibits migration and invasion of breast cancer cells. Supplementary Fig. 3. IB treatment inhibits in vivo growth and metastasis of breast tumorigenesis. Supplementary Fig. 4. IB is safe and effective against human breast cancers. Supplementary Fig. 5. (A) Western blot analysis of MCF-7 cells treated with vehicle or IB (5μM) using antibodies against FOXM1, SKP2, PLK1 and XRCC3. Supplementary Fig. 6. (A) Western blot analysis of MCF-7 cells treated with vehicle or IB (5μM) using antibodies against cyclin D 1, CDK4, cyclin E2, CDK2. Supplementary Fig. 7. (A) A549, HeLa and DU145 cells were treated with various doses of IB (1-5 μM) for 24 hours and then cells were re-seeded in 6-well plate and allowed to grow for additional 7 days before colonies were stained with crystal violet.

Purpose: The approaches aimed at inhibiting the ability of cancer cells to repair DNA strand breaks have emerged as promising targets for treating cancers. Here, we assessed the potential of imipramine blue (IB), a novel analogue of antidepressant imipramine, to suppress breast cancer growth and metastasis by inhibiting the ability of breast cancer cells to repair DNA strand breaks by homologous recombination (HR).Experimental Design: The effect of IB on breast cancer growth and metastasis was assessed in vitro as well as in preclinical mouse models. Besides, the therapeutic efficacy and safety of IB was determined in ex vivo explants from breast cancer patients. The mechanism of action of IB was evaluated by performing gene-expression, drug–protein interaction, cell-cycle, and DNA repair studies.Results: We show that the systemic delivery of IB using nanoparticle-based delivery approach suppressed breast cancer growth and metastasis without inducing toxicity in preclinical mouse models. Using ex vivo explants from breast cancer patients, we demonstrated that IB inhibited breast cancer growth without affecting normal mammary epithelial cells. Furthermore, our mechanistic studies revealed that IB may interact and inhibit the activity of proto-oncogene FoxM1 and associated signaling that play critical roles in HR-mediated DNA repair.Conclusions: These findings highlight the potential of IB to be applied as a safe regimen for treating breast cancer patients. Given that FoxM1 is an established therapeutic target for several cancers, the identification of a compound that inhibits FoxM1- and FoxM1-mediated DNA repair has immense translational potential for treating many aggressive cancers. Clin Cancer Res; 22(14); 3524–36. ©2016 AACR.

Supplimentary Methods and Figure Legends

Supplementary Table S1: Primers used in the present investigation. Supplementary Table S 2 : Gene Ontology Enrichment Analysis on differentially expressed genes in IB-treated compared to vehicle-treated breast cancer cells using Database for Annotation, Visualization and Integrated Discovery (DAVID). BP- Biological Processes.

Proteins are manufactured by ribosomes—macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as ‘red laser’). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease. RNA polymerase II has an unexpected function in the nucleolus, helping to drive the expression of ribosomal RNA and to protect nucleolar structure through a mechanism involving triplex R-loop structures.

Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.

The study of high-throughput genomic profiles from a pharmacogenomics viewpoint has provided unprecedented insights into the oncogenic features modulating drug response. A recent screening of ~1,000 cancer cell lines to a collection of anti-cancer drugs illuminated the link between genotypes and vulnerability. However, due to essential differences between cell lines and tumors, the translation into predicting drug response in tumors remains challenging. Here we proposed a DNN model to predict drug response based on mutation and expression profiles of a cancer cell or a tumor. The model contains a mutation and an expression encoders pre-trained using a large pan-cancer dataset to abstract core representations of high-dimension data, followed by a drug response predictor network. Given a pair of mutation and expression profiles, the model predicts IC50 values of 265 drugs. We trained and tested the model on a dataset of 622 cancer cell lines and achieved an overall prediction performance of mean squared error at 1.96 (log-scale IC50 values). The performance was superior in prediction error or stability than two classical methods and four analog DNNs of our model. We then applied the model to predict drug response of 9,059 tumors of 33 cancer types. The model predicted both known, including EGFR inhibitors in non-small cell lung cancer and tamoxifen in ER+ breast cancer, and novel drug targets. The comprehensive analysis further revealed the molecular mechanisms underlying the resistance to a chemotherapeutic drug docetaxel in a pan-cancer setting and the anti-cancer potential of a novel agent, CX-5461, in treating gliomas and hematopoietic malignancies. Overall, our model and findings improve the prediction of drug response and the identification of novel therapeutic options., Accepted for presentation in the International Conference on Intelligent Biology and Medicine (ICIBM 2018) at Los Angeles, CA, USA. Currently under consideration for publication in a Supplement Issue of BMC Genomics

BACKGROUND: Volumetric tissue-engineered constructs are limited in development due to the dependence on well-formed vascular networks. Scaffold pore size and the mechanical properties of the matrix dictates cell attachment, proliferation and successive tissue morphogenesis. We hypothesize scaffold pore architecture also controls stromal-vessel interactions during morphogenesis. METHODS: The interaction between mesenchymal stem cells (MSCs) seeded on hydroxyapatite scaffolds of 450, 340, and 250 μm pores and microvascular fragments (MVFs) seeded within 20 mg/mL fibrin hydrogels that were cast into the cell-seeded scaffolds, was assessed in vitro over 21 days and compared to the fibrin hydrogels without scaffold but containing both MSCs and MVFs. mRNA sequencing was performed across all groups and a computational mechanics model was developed to validate architecture effects on predicting vascularization driven by stiffer matrix behavior at scaffold surfaces compared to the pore interior. RESULTS: Lectin staining of decalcified scaffolds showed continued vessel growth, branching and network formation at 14 days. The fibrin gel provides no resistance to spread-out capillary networks formation, with greater vessel loops within the 450 μm pores and vessels bridging across 250 μm pores. Vessel growth in the scaffolds was observed to be stimulated by hypoxia and successive angiogenic signaling. Fibrin gels showed linear fold increase in VEGF expression and no change in BMP2. Within scaffolds, there was multiple fold increase in VEGF between days 7 and 14 and early multiple fold increases in BMP2 between days 3 and 7, relative to fibrin. There was evidence of yap/taz based hippo signaling and mechanotransduction in the scaffold groups. The vessel growth models determined by computational modeling matched the trends observed experimentally. CONCLUSION: The differing nature of hypoxia signaling between scaffold systems and mechano-transduction sensing matrix mechanics were primarily responsible for differences in osteogenic cell and microvessel growth. The computational model implicated scaffold architecture in dictating branching morphology and strain in the hydrogel within pores in dictating vessel lengths. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-020-00648-7) contains supplementary material, which is available to authorized users.

Ewing sarcoma is an aggressive pediatric cancer of enigmatic cellular origins typically resulting from a single translocation event t (11, 22) (q24, q12). The resulting fusion gene, EWSR1-FLI1, is toxic or unstable in most primary tissues. Consequently, attempts to model Ewing sarcomagenesis have proven unsuccessful thus far, highlighting the need to identify the cellular features which permit stable EWSR1-FLI1 expression. By re-analyzing publicly available RNA-Sequencing data with manifold learning techniques, we uncovered a group of Ewing-like tissues belonging to a developmental trajectory between pluripotent, neuroectodermal, and mesodermal cell states. Furthermore, we demonstrated that EWSR1-FLI1 expression levels control the activation of these developmental trajectories within Ewing sarcoma cells. Subsequent analysis and experimental validation demonstrated that the capability to resolve R-loops and mitigate replication stress are probable prerequisites for stable EWSR1-FLI1 expression in primary tissues. Taken together, our results demonstrate how EWSR1-FLI1 hijacks developmental gene programs and advances our understanding of Ewing sarcomagenesis.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. We observed that SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination-mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways.

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2–4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.

The recent accumulation of cancer genomic data provides an opportunity to understand how a tumor’s genomic characteristics can affect its responses to drugs. This field, called pharmacogenomics, is a key area in the development of precision oncology. Deep learning (DL) methodology has emerged as a powerful technique to characterize and learn from rapidly accumulating pharmacogenomics data. We introduce the fundamentals and typical model architectures of DL. We review the use of DL in classification of cancers and cancer subtypes (diagnosis and treatment stratification of patients), prediction of drug response and drug synergy for individual tumors (treatment prioritization for a patient), drug repositioning and discovery and the study of mechanism/mode of action of treatments. For each topic, we summarize current genomics and pharmacogenomics data resources such as pan-cancer genomics data for cancer cell lines (CCLs) and tumors, and systematic pharmacologic screens of CCLs. By revisiting the published literature, including our in-house analyses, we demonstrate the unprecedented capability of DL enabled by rapid accumulation of data resources to decipher complex drug response patterns, thus potentially improving cancer medicine. Overall, this review provides an in-depth summary of state-of-the-art DL methods and up-to-date pharmacogenomics resources and future opportunities and challenges to realize the goal of precision oncology.

The conserved RNA-binding protein Musashi1 (MSI1) has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation and as a key oncogenic factor in numerous solid tumors, including glioblastoma. To explore the potential use of MSI1 targeting in therapy, we studied MSI1 in the context of radiation sensitivity. Knockdown of MSI1 led to a decrease in cell survival and an increase in DNA damage compared to control in cells treated with ionizing radiation. We subsequently examined mechanisms of double-strand break repair and found that loss of MSI1 reduces the frequency of nonhomologous end-joining. This phenomenon could be attributed to the decreased expression of DNA-protein kinase catalytic subunit, which we have previously identified as a target of MSI1. Collectively, our results suggest a role for MSI1 in double-strand break repair and that its inhibition may enhance the effect of radiotherapy.

Purpose: The approaches aimed at inhibiting the ability of cancer cells to repair DNA strand breaks have emerged as promising targets for treating cancers. Here, we assessed the potential of imipramine blue (IB), a novel analogue of antidepressant imipramine, to suppress breast cancer growth and metastasis by inhibiting the ability of breast cancer cells to repair DNA strand breaks by homologous recombination (HR). Experimental Design: The effect of IB on breast cancer growth and metastasis was assessed in vitro as well as in preclinical mouse models. Besides, the therapeutic efficacy and safety of IB was determined in ex vivo explants from breast cancer patients. The mechanism of action of IB was evaluated by performing gene-expression, drug–protein interaction, cell-cycle, and DNA repair studies. Results: We show that the systemic delivery of IB using nanoparticle-based delivery approach suppressed breast cancer growth and metastasis without inducing toxicity in preclinical mouse models. Using ex vivo explants from breast cancer patients, we demonstrated that IB inhibited breast cancer growth without affecting normal mammary epithelial cells. Furthermore, our mechanistic studies revealed that IB may interact and inhibit the activity of proto-oncogene FoxM1 and associated signaling that play critical roles in HR-mediated DNA repair. Conclusions: These findings highlight the potential of IB to be applied as a safe regimen for treating breast cancer patients. Given that FoxM1 is an established therapeutic target for several cancers, the identification of a compound that inhibits FoxM1- and FoxM1-mediated DNA repair has immense translational potential for treating many aggressive cancers. Clin Cancer Res; 22(14); 3524–36. ©2016 AACR.

Despite improvement in overall survival of breast cancer patients, many women don't survive this disease. Moreover, the quality of life for patients who do survive is often substantially reduced due to the toxicity associated with the chemotherapy. Here, we report that imipramine blue (IB), a novel analogue of anti-depressant imipramine that we recently synthesized, may serve as a safe and potent therapeutic agent for treating breast cancers. We show that IB reduced cell growth, migration and invasion of breast cancer cells. Systemic delivery of IB using nanoparticle-based drug delivery approach suppressed breast cancer growth and metastasis without inducing any toxicity in pre-clinical orthotropic mouse models. Notably, using ex-vivo model of tumor explants from breast cancer patients, we demonstrated that IB inhibited breast cancer growth without affecting normal mammary epithelial cell proliferation. Furthermore, IB improved the sensitivity of breast cancer cells to chemotherapy drugs paclitaxel and doxorubicin. Our results revealed that IB mediated its anti-tumor effect by targeting genes involved in cell cycle progression, microtubule dynamics and DNA damage surveillance pathway including Forkhead Box M1 (FOXM1), stathmin1, S-phase kinase-associated protein 2 (Skp2) and XRCC3, which we show to be highly expressed in breast cancer patients. Importantly, we demonstrated that IB inhibited breast cancer cell's ability to repair DNA strand breaks by impairing homologous recombination events. These findings highlight the potential of IB to be used as a potent therapeutic regimen for treating breast cancer patients. Since IB-1 is derived from a FDA approved drug it has potential to be rapidly translated to the clinic. Citation Format: Rajamanickam S, Subbarayalu P, Timilsina S, Gorthi A, Drake MT, Chen Y, Vadlamudi R, Bishop AJR, Arbiser JL, Rao MK. Imipramine Blue - A safe and potent therapeutic regimen that suppresses breast cancer growth and progression by targeting DNA damage surveillance pathway. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-07-06.

Ewing sarcoma (EwS) is a pediatric and young adult cancer that is driven by the EWSR1-FLI1 translocation. Despite decades of work, this cancer is still an enigma, with poorly understood biology and no targeted treatments. Our recent work published in Nature demonstrated a previously overlooked consequence of EWSR1-FLI1, that this fusion causes hyperphosphorylated RNA polymerase II (pRNAPII) due to loss of EWSR1 inhibition of CDK7 and CDK9. We observed high levels of transcription, with high levels of R-loops present in locations that R-loops normally (physiologically) occur. Based upon these findings, we began to reconsider cellular phenotypes of EwS to identify the molecular basis of these phenotypes and ask whether these changes provide a fundamental defect in all EwS. One phenotype that was previously identified in EwS is that these cells display altered splicing profiles. In our prior work we reported that the normal dephosphorylation of pRNAP in response to damage does not occur, and this results in trapping of BRCA1 protein with the active transcription complex. It was recently shown that the splicing machinery is bound to pRNAPII. This observation raises the interesting possibility that in EwS, like BRCA1 protein, the splicing machinery is also sequestered with pRNAPII. Interestingly, in recent years, there have been several reports linking R-loops to splicing, with splicing defects causing R-loop accumulation and R-loops being associated with sites of alternative splicing. We therefore asked if there is an association between the R-loops and alternative splicing phenotype in EwS cells and whether this results in a defect that can be leveraged to specifically target EwS cells and tumors. Citation Format: L. Lawrence, A. Gorthi, H. Miller, A. J. R. Bishop. Dysregulated Transcription Drives Splicing Defect in Ewing Sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B24.

Introduction: Embryonal tumors with multilayered rosettes (ETMRs) are aggressive brain tumors that occur mainly in infants. Patients face a very poor prognosis with a median overall survival of ~12 months after diagnosis. The tumors harbor in ~90% of all cases amplification of a miRNA cluster on chromosome 19 (C19MC) that is thought to be the driver of the disease. However, current treatment options are lacking as (a) the mechanisms downstream of C19MC are poorly understood and (b) the drivers in cases lacking the C19MC aberration are unknown. To develop better treatment protocols for ETMR patients, more insight is needed in what is driving these tumors and how that can be targeted. Materials and Methods: To investigate the genomic and epigenomic landscape of ETMR in depth, we collected 193 ETMR samples and 23 matched relapses and performed DNA methylation profiling on all and DNA (whole genome, whole exome, and panel) sequencing and mRNA and miRNA transcriptome analysis on a subset of them. The BT183 ETMR cell line was used for drug treatments. Results: Among the 22 tumors without C19MC amplification, we identified 8 cases with truncating DICER1 germline mutations in one allele and somatic missense mutations in the RNASE III domain in the other allele. No DICER1 mutations were identified in C19MC amplified cases. In addition, structural variations (SVs) affecting C19MC were found in 3 other C19MC nonamplified cases and amplification of another miRNA cluster, miR-17-92, in 2 other cases. However, despite the presence of different genetic aberrations, based on DNA methylation and transcriptome profiling no molecular subgrouping was observed within our cohort. Whole-genome sequencing revealed an overall low recurrence and conservation of SNVs but strong conservation of SVs from primary tumors to relapses, especially surrounding C19MC. Moreover, many newly acquired SNVs in the relapses are associated to a new cisplatin treatment-related mutational signature. SVs detected in ETMRs significantly colocalized with R-loops, structures that form upon a collision of replication and transcription and are associated to increased levels of chromosomal instability, which is frequently observed in ETMRs. Using a DICER1 KO model, we found that global deregulation of miRNAs led to increased levels of R-loops and R-loop associated chromosomal instability. Finally, we show that a combination of topoisomerase and PARP inhibitors is highly synergistic and strongly increased the levels of both R-loops and DNA damage in ETMR cells and effectively killed the cells. Conclusions: Our results show that genomically instable ETMR cells are vulnerable to further increases in chromosomal instability, knowledge that may lead to new treatment strategies for ETMR patients and possibly other cancers with high levels of R-loops. Citation Format: Sander Lambo, Susanne Grübner, Tobias Rausch, Sebastian Waszak, Christin Schmidt, Sonja Krausert, Loreen Weichert, Aparna Gorthi, Carolina Romero, Annie Huang, Julia Schueler, Jan Korbel, Alexander Bishop, Stefan Pfister, Andrey Korshunov, Marcel Kool. Molecular characterization of ETMRs reveals role for R-loop mediated genomic instability and new treatment options [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A39.

Ewing sarcoma is an aggressive pediatric cancer that is driven by a fusion oncogene, EWS-FLI1. While the transcriptional targets of EWS-FLI1 have been widely studied, the systemic impact of EWS-FLI...

Molecular targeted compounds are emerging as a strategy to improve classical chemotherapy. Herein, we describe that using low dose of the multikinase inhibitor sorafenib improves cyclophosphamide antitumor activity by inhibiting angiogenesis, metastasis and promoting tumor healing in MDA-MB231 xenografts and the 4T1-12B syngeneic breast cancer metastasis model. Mechanistic studies in MDA-MB231 cells revealed that alkylation upregulates inflammatory genes/proteins such as COX-2, IL8, CXCL2 and MMP1 in a MEK1/2-ERK1/2-dependent manner. These proteins enrich the secretome of cancer cells, stimulating cell invasion and angiogenesis via autocrine and paracrine mechanisms. Sorafenib inhibits MEK1/2-ERK1/2 pathway thereby decreasing inflammatory genes and mitigating cell invasion and angiogenesis at basal and alkylation-induced conditions whereas NRF2 and ER stress pathways involved in alkylation survival are not affected. In non-invasive/non-angiogenic breast cancer cells (SKBR3 and MCF7), alkylation did not elicit inflammatory responses with the only sorafenib effect being ERK1/2-independent ROS-dependent cytotoxicity when using higher drug concentrations. In summary, our data show that alkylating agents may elicit inflammatory responses that seems to contribute to malignant progression in specific breast cancer cells. Identifying and targeting drivers of this phenotype may offer opportunities to optimize combined drug regimens between classical chemotherapeutics and targeted agents.

Ewing sarcoma is an aggressive pediatric bone and soft tissue cancer that is driven by a translocation event, predominantly involving the RNA binding protein EWSR1 and the transcription factor FLI1. The resultant fusion transcription factor EWSR1-FLI1 is a potent oncogene and drives dramatic changes in the transcriptional landscape of Ewing sarcoma. Our previous work demonstrated that EWS-FLI1 drives aberrant accumulation of R-loops, increases replication stress and suppresses homologous recombinational repair in Ewing sarcoma. Similar phenotypes were also observed with the loss of wildtype EWSR1. However, surprisingly, Ewing tumors have highly stable genomes. The most common mutation is in the STAG2 gene, a component of the cohesin ring complex. It was recently reported that STAG2-cohesin complexes not only participate in maintaining chromatin architecture but also play a key role in directing tissue-specific gene expression by interacting with transcription factors. In the present study, we evaluate the STAG2 function in R-loop metabolism, replication and repair and attempt to identify the reason for its selective loss in Ewing sarcoma. Using biochemical and biophysical experimental strategies, we observed that STAG2 and EWSR1 both bind R-loop structures with high affinity and are protective against RNaseH-mediated degradation of R-loops to significantly different degrees. We also examine the effect of STAG2 loss on R-loop accumulation and replication stress in Ewing and non-Ewing cell lines. Finally, we evaluate the correlation between STAG2 binding sites and R-loops. We propose that loss of STAG2 alleviates some of the genomic stresses experienced in Ewing sarcoma. Note: This abstract was not presented at the meeting. Citation Format: Aparna Gorthi. Implications of STAG2 loss in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4401.

ETMRs are aggressive embryonal brain tumors with a very poor prognosis, having a median overall survival of ~12 months after diagnosis. ETMRs harbour in ~90% of all cases amplification of a miRNA cluster on chromosome 19 (C19MC) that is thought to be the driver of the disease. However, current treatment options are lacking as (a) the mechanisms downstream of C19MC are poorly understood and (b) the drivers in cases lacking the C19MC aberration are unknown. To investigate the genomic landscape of ETMR, we collected 186 ETMR samples and 23 matched relapses. Interestingly, among the 18 tumors without C19MC amplification, we identified five cases with truncating DICER1 germline mutations in one allele and somatic missense mutations in the RNASE III domain in the other allele. In addition, SVs affecting C19MC were found in three other cases and amplification of the miR-17–92 cluster in another two cases. However, despite the presence of different genetic aberrations, based on DNA methylation profiling no molecular subgrouping was observed within our cohort. Whole-genome sequencing revealed an overall low recurrence and conservation of SNVs but strong conservation of SVs from primary tumors to relapses especially surrounding C19MC. SVs detected in ETMRs significantly co-localized with R-loops, structures that form upon a collision of replication and transcription and are associated to increased levels of chromosomal instability, which is frequently observed in ETMRs. Using a DICER1 KO model we found that global deregulation of miRNAs led to increased levels of R-loops and R-loop associated chromosomal instability. Finally, we show that treating ETMR cells with topoisomerase and PARP inhibitors strongly increased the levels of both R-loops and DNA damage and effectively killed the cells. Our results show that genomically instable ETMR cells are vulnerable to further increases in chromosomal instability, knowledge that may lead to new treatment strategies in the future.

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis, and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival, an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine or glutathione-ethyl-ester, decreased ER stress and abrogated alkylating agents–induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 target overexpression and poor survival. In KEAP1-mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. Mol Cancer Ther; 15(12); 3000–14. ©2016 AACR.

Embryonal Tumors with Multilayered Rosettes (ETMRs) are pediatric brain tumors mainly occurring in infants. Characteristic to ETMRs is the highly recurrent (~90%) amplification of the C19MC miRNA cluster fused to TTYH1 that drives the expression of this cluster. As the overall survival of these patients is very poor, there is an urgent need for a better understanding of these tumors that may lead to other treatment strategies. Whole genome and panel sequencing data have been generated for 60 ETMRs and matching germline when available. Data have been complemented with DNA methylation profiling and m(i)RNA sequencing data. Our results show that ETMR is a single disease entity without molecular subgroups. ETMRs lacking the C19MC amplification (~10%) are highly similar to tumors with C19MC amplification, based on methylation and m(i)RNA profiling, indicating that they do not represent a distinct subgroup. Germline sequencing revealed mutations in genes involved in DNA repair or miRNA processing, while tumor specific mutations included genes involved in the TP53-, SHH-, WNT-, or miRNA processing pathways. These pathways are also highly upregulated compared to other pediatric brain tumors. Mutations in DNA repair, miRNA processing, structural variations (SVs) and mutations in close proximity of SVs occur at high allele frequencies and are conserved in recurrent tumors while many other SNVs are lost. These data suggest that C19MC amplification/fusion, miRNA processing and DNA repair defects are the early (driving) events in tumor formation while aberrations involving for instance the SHH and WNT pathways are later (passenger) events. Aside from frequent and recurrent copy number changes, ETMRs show pluriploidy, complex rearrangements and strong presence of R-loops suggesting that ETMR genomes are highly unstable. We identified a high number of R-loops in the region forming the C19MC aberration and an enrichment of breakpoints in other R-loop forming regions. This may suggest a role for R-loops in both tumor progression and initiation. Finally, we tested whether further inducing the number of R-loops in these tumors may increase replication stress and cell death. Indeed, topoisomerase inhibition coupled to PARP inhibition increased the amount of R-loops and acted synergistically in killing ETMR cells. These data show that targeting the genomic instability in ETMRs could be a viable treatment option for treating ETMR patients. Citation Format: Sander Lambo, Andrey Korshunov, Christin Schmidt, Carolina Romero, Aparna Gorthi, Sonja Krausert, Tobias Rausch, Susanne Gröbner, Sebastian Brabetz, Sebastian Waszak, Alexander J. Bishop, Stefan Pfister, Marcel Kool. Targeting genomic instability in embryonal tumors with multilayered rosettes (ETMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3172.

Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein1. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

Ewing sarcoma is a rare, yet highly aggressive family of bone and soft tissue tumors that afflicts children and young adults. It is the prototypical example of mesenchymal tumors driven by a fusion oncogene involving the EWSR1 gene, most frequently EWS-FLI1. Ewing sarcoma is exquisitely sensitive to genotoxic agents, and more recently PARP1 inhibitors, predominantly when detected at an early stage. However, the molecular basis for this sensitivity is relatively under-explored. Further, the long term side effects of these drugs pose a critical barrier to effective treatment. In this study, we proposed to delineate the pathways dictating Ewing sarcoma chemosensitivity and identify alternative avenues for targeted therapy. R-loops are three nucleic acid DNA structures consisting of an RNA:DNA hybrid and a displaced complementary DNA loop. The importance of R-loops in both being causative as well as disruptive in DNA damage as well as DNA damage response has emerged through recent studies. Our previous work has demonstrated that Ewing sarcoma cell lines have high levels of endogenous damage as well as replication stress. We observe that Ewing cell lines have elevated levels of persistent R-loops and impaired homologous recombination repair of double strand breaks. In the present study, we use a multimodal, systems biology approach involving high-throughput RNAi based survival data, time-course RNA-seq in response to two chemotherapeutics, Chromatin immunoprecipitation-seq and DNA:RNA hybrid immunoprecipitation-seq datasets across multiple Ewing sarcoma and control cell lines. Our data led to the identification of a subset of genes whose expression patterns in response to DNA damage is dysregulated in Ewing sarcoma. Predominantly, among these genes is a network of RNA binding proteins that highlights the importance of RNA metabolism in DNA damage response. We also observe a concomitant alteration in damage-dependent transcription. Finally, we identified several compensatory mechanisms that are upregulated in Ewing sarcoma (RNASEH2, FEN1, Fanconi Anemia, etc.) in order to manage the exacerbated genomic stress conditions and targeting any of these pathways leads to severe tumor cell cytotoxicity. We therefore propose the use of these pathways as potential candidates for targeted therapy. Citation Format: Aparna Gorthi, Eva Loranc, Cao Lin, Alexander JR Bishop. Systems biology approach to delineating DNA damage response and R-loop dependencies in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A25.

R-loops are physiologically occurring structures in the genome that composed of a DNA/RNA hybrid and a displaced single-stranded DNA. R-loops have been observed in various organisms and shown to play important roles in regulating gene expression, DNA replication, genome stability, and other functions. The recent introduction of the protocol of DNA-RNA Immune-Precipitation (DRIP) followed by next-generation sequencing further propels the data accumulation of R-loop formation in different cellular contexts. In this study, we presented a user-friendly tool, DRIPer, for investigating DRIP-seq data to compare against a collection of publicly available DRIP-seq data and ENCODE ChIP-seq. Such comparisons allow correlation analysis and Kolmogorov-Smirnov tests to study associations via a given gene set, which could be for specific biological pathways, ontological functions, or other co-regulated genes. This powerful method will enable biologists to quickly evaluate the relationship of R-loops to nearby binding protein sites and target gene expression.

Clinical decision support systems augment the quality of medical care by aiding healthcare workers in the evaluation and management of complicated cases. Clinical decision support systems are especially instrumental in quickly assessing the criticality of pregnancy as it involves interpreting multiple maternal and fetal parameters. We propose a machine learning approach for early determination of the risk category of pregnancy based on patterns gleaned from profiles of known clinical parameters. In particular, we demonstrate the usefulness of classification and regression trees in solving multivariate problems in obstetric care since the decision making process and the importance of specific parameters are clearly illustrated in the tree. As proof of concept, an application use case has been presented.

Probe design is the most important step for any microarray based assay. Accurate and efficient probe design and selection for the target sequence is critical in generating reliable and useful results. Several different approaches for probe design are reported in literature and an increasing number of bioinformatics tools are available for the same. However, based on the reported low accuracy, determining the hybridization efficiency of the probes is still a big computational challenge. Present study deals with the extraction of various novel features related to sequence composition, thermodynamics and secondary structure that may be essential for designing good probes. A feature selection method has been used to assess the relative importance of all these features. In this paper, we validate the importance of various features currently used for designing an oligonucleotide probe. Finally, a classification methodology is presented that can be used to predict the hybridization quality of a probe.

Background: Ewing's sarcoma is a rare form of highly aggressive bone tumor of children and young adults. Understanding the molecular mechanisms involved in the development of Ewing's sarcoma forms the key step in the design and development of new therapeutic interventions. Previous studies revealed that genomic alterations play an important role in oncogenesis, disease progression and response of tumors to different therapies. The recent advance of next generation sequencing-based methods provide unprecedented capabilities to scan Ewing's sarcoma genomes for changes such as mutations, insertions/deletions, copy number variations, differential gene expression and discovery of new gene fusions. In this study, we performed detailed investigation to identify the genetic basis of difference between two Ewing cell lines, RDES and SKES1, which show differential sensitivity to natural product extracts, by using RNA-seq, small RNA-seq and exome-capture-seq techniques. Methods: Transcriptomes and genomic DNA of both the Ewing's cell lines, RDES and SKES1 were prepared for paired-end sequencing on the Illumina HiSeq2000 platform using the manufacturer's protocols, for RNA-seq and exome-capture-seq respectively. The reads were aligned to the human hg19 genome assembly and the abundance of mRNAs as well as small RNAs for all annotated genes from the UCSC annotation of human genome was calculated. The count data from abundance estimation was used to compute differentially expressed genes and small RNAs between these two cell lines. Furthermore, we used deFuse for the gene fusion identification and GATK pipeline for the DNA variant analysis. Results: We studied the gene expression profiles from RNA-seq and small RNA-seq reads and identified approximately 270 and 114 differentially expressed genes and microRNAs, respectively between drug-sensitive and insensitive cell lines. Interestingly, we found that two of ABC transporters are significantly overexpressed in the resistant cell line. Moreover, many apoptotic genes show altered expression between these two cell lines. Gene fusion products were also identified from the RNA-seq analysis. Both the cell lines were found to harbor the well-known Ewing's sarcoma gene fusion EWS-FLI1. In addition to EWS-FLI1, we also discovered some new and unique gene fusion candidates in both cell lines. Furthermore, we used exome-capture sequence analysis to identify sequence variation and mutations in these two cell lines. Known mutations reported in COSMIC database and SNPs that are not included in normal populations from the 1000 genome project were carefully studied. Interestingly, some of the non-synonymous changes identified map to kinases and other known cancer-related genes. Conclusions: Our studies suggest that the difference in drug sensitivity of two Ewing's sarcoma cell-lines is probably due to the expression of ATP-dependent efflux pumps as well as alterations in the apoptotic machinery. Further studies are warranted to understand the exact molecular mechanism of drug sensitivity. Citation Format: TABREZ A. MOHAMMAD, Aparna Gorthi, Andrew Robles, Xavier Bernard, Tzu-hung Hsiao, Dawn Garcia, Zhao Lai, Susan L. Mooberry, Gail E. Tomlinson, Alexander JR Bishop, Yidong Chen. Integrative genomic analysis of two Ewing's sarcoma cell lines that show differential drug sensitivity. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A30.

DNA methylation has been associated with several key events of gene regulation and to human cancer. It is not yet known how the epigenome of various populations is associated with clinical manifestations during the course of the disease. In this poster we describe a method for stratifying breast cancer patients from Indian origin using CpG island microarray from the University Healthcare Network (UHN) Toronto (human CpG island 12k microarray chip, HCGI12K). DNA samples were obtained from a prospective study cohort which consisted of 51 female primary breast cancers. All patients had been undergoing treatment in a tertiary care hospital and its associated centers in the southern part of India between 2007 and 2009. We have identified and classified the DNA methylation in CpG islands of patient samples using various clinical parameters such as age of disease onset, menopausal status, hormone receptor status and Her2 status. We present the methods to analyze the data from UHN CpG island arrays used in a high throughput methylation study in order to derive decision rules of stratifying the data into basic classes such as normal and benign conditions, and infiltrating ductal carcinoma. Results will be presented pertaining to differential methylation status of patients in different categories such as age of disease onset, menopausal status, hormone receptor status and Her2 status. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-08.