Degree Of Genomic Instability Research Articles

Abstract 1409: Analysis of 16,172 patient-derived tumor samples indicate TPX2 as being essential for survival of genomically instable cancer cells

Abstract Mutations in homologous recombination (HR) genes, including BRCA1 and BRCA2, compromise DNA repair and lead to genomic instability (GI). GI is lethal to normal cells but is a characteristic of many cancers. Apparently, these cancers are somehow re-wired to survive high levels of GI. Identification of the genetic alterations that allow viability of genomically instable tumor cells may uncover novel therapeutic targets. To elucidate how these tumor cells are rewired, we analyzed publically available mRNA expression data of 16,172 human cancer samples. Functional genomic mRNA profiling (FGmRNA-profiling) was applied on these samples to infer levels of GI and to capture the downstream effects of somatic copy number alterations on gene expression. A genome-wide association analysis was subsequently performed to assess the correlation between FGmRNA signals of individual genes with the degree of GI. From the top 250 genes with strong positive correlation with GI, 11 genes were prioritized based on a co-functionality network in which genes are co-regulated and share similar predicted biological function. The 11 genes that were identified in this cluster were: BIRC5, UBE2C, CENPA, CDCA3, DEK, SKP2, TPX2, KIF2C, RAD21, MYBL2 and WDR67. To validate these findings in genetically-defined models, we engineered a panel of 5 triple negative breast cancer (TNBC) cell lines with doxycycline-inducible shRNAs targeting BRCA2. BRCA2 depletion resulted in a failure of RAD51 foci to localize to DNA double strand breaks which generated isogenic cell line pairs proficient and deficient of HR repair. First, we depleted each of the identified 11 genes using RNA interference in BT-549 cells and observed that depletion of TPX2, a microtubule-associated protein, led to largest differential levels of cell death when comparing the BRCA2-deficient with the BRCA2-proficient context (86.6% vs 32.9% cell death in BRCA2-depleted vs controlled depleted cells respectively). Subsequently, we could replicate this decreased survival with TPX2 depletion in a BRCA2-deficient context in an additional 2 out of 4 other TNBC cell lines. Furthermore, we investigated whether BRCA2-depleted cells were also more sensitive to depletion of Aurora kinase A, a substrate of TPX2. For this purpose, mouse mammary tumor cell lines, derived from Tp53-/- or Brca2-/-;Tp53-/- mice, or a Brca2-reconstituted version thereof were treated with an Aurora A inhibitor, Alisertib. Again, we found that the BRCA2-deficient cell line was more sensitive to Aurora A inhibition than the two BRCA2-proficient cell lines. In conclusion, FGmRNA-profiling of mRNA expression data of human cancer samples identified TPX2 as an essential gene for survival of BRCA2-deficient breast cancer cells, when compared to BRCA2-proficient cells. Thus, targeting the TPX2/AURKA axis could potentially act as a novel therapeutic target in the treatment of genomically instable cancers. Citation Format: Stephanie E. van Gijn, Elles Wierenga, Anne Margriet Heijink, Rudolf S. Fehrmann, Marcel A. van Vugt. Analysis of 16,172 patient-derived tumor samples indicate TPX2 as being essential for survival of genomically instable cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1409. doi:10.1158/1538-7445.AM2017-1409

Abstract 3369: Epigenetic DNA methylation profiling of triple negative breast cancer: a quantitative NGS approach

Abstract Triple negative breast cancer (TNBC) is an aggressive disease with a high degree of genomic instability. TNBC patients have poor prognosis and the risk of metastasis and death is increased for women who relapse within four years of treatment compared to other breast cancer subtypes. Currently, there are no reliable prognostic markers to identify which population is at risk for early relapse and the molecular mechanism for disease recurrence is not well understood. Epigenetic changes, especially DNA methylation, are common in breast cancer and have been found to be associated with increased metastatic capability. Therefore, the methylation states of genes in TNBC tumors compared to non-tumor breast tissue was examined in order to identify potential biomarkers and therapeutic targets in TNBC. Matched tumor and adjacent non-tumor tissue from patients with TNBC were obtained following surgical resection and genomic DNA was extracted. Epigenetic profiling of TNBC tumors and non-tumor tissue was performed using a highly sensitive and quantitative analytics platform, which utilizes methylation sensitive restriction endonucleases to detect changes in methylation of CpG sites. Millions of CpG sites exist within the human genome and many of these are altered with tumor formation and progression, therefore, changes in methylation profiles of CpG sites may be useful as a diagnostic and/or prognostic biomarker in TNBC patients. Non-metric multidimensional scaling ordination analysis of the CpG sites revealed highly distinct methylation patterns between tumor and non-tumor tissue. Approximately 326 sites had a significant methylation score difference (p&lt0.0025) between TNBC tumor and non-tumor tissue, with most of the CpG sites having greater than 2-fold change in methylation status. Analysis of functional gene classes using KEGG classifiers revealed a significant change in methylation patterns of genes involved in response to infections and other immune related functions. Additionally, the top ten genes with hyper- or hypomethylated sites within TNBC tumors when compared to non-tumor tissue were identified. Interestingly, Gli-1 was one of the top hypomethylated genes. Gli-1 has been shown in our lab and others to have significance in chemoresistance and recurrence in TNBC patients. Immunohistochemical analysis of TNBC tumors revealed Gli-1 overexpression in TNBC tumors compared to non-tumor tissue. Expression levels of Gli-1 in TNBC tumors correlated with stage (p&lt0.001) and recurrence free (p&lt0.0213) and overall survival (p&lt0.017). Thus, analysis of CpG methylated sites in TNBC tumors and non-tumor tissue revealed differences in epigenetic profiles allowing for distinction between tumor and non-tumor tissue. Genes in TNBC tumors with significant changes in methylation status may be potential candidate genes that serve as a diagnostic or prognostic biomarker for TNBC. Citation Format: Kimberly M. Arnold, Adam G. Marsh, Jennifer Sims-Mourtada. Epigenetic DNA methylation profiling of triple negative breast cancer: a quantitative NGS approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3369. doi:10.1158/1538-7445.AM2017-3369

Abstract 2256: Establishment and characterization of circulating tumor cell-derived xenografts in non-small cell lung cancer

Abstract Low numbers of circulating tumor cells (CTCs) have so far limited the establishment of CTC-derived xenografts (CDXs) to improve our understanding of tumor progression, drug resistance mechanisms, and their biological properties. We report the establishment and the phenotypic and molecular characterization of one NSCLC CDX. Blood samples (30 ml) were drawn from 49 NSCLC patients with advanced metastatic disease. CTCs were enriched by RosetteSep, embedded in matrigel, and implanted in the interscapular aspect of NSG mouse (Nod/Scid-IL2Rγ-/-). Mice were followed-up for one year according to ethical regulations. CDX tumours and CDX derived cell lines were phenotypically and molecularly characterized by immunofluorescence, immunohistochemistry, CGHarray, exome sequencing and transcriptome gene expression. CTCs from one NSCLC patient with 750 CTCs detected by CellSearch gave rise to a tumor 5 months after initial murine injection. Histological analysis confirmed the human origin of the tumor and the presence of a poorly differentiated adenocarcinoma consistent with the patient's biopsy. Tumor was positive for EpCAM, EMA, CK8;18, and Ki67, and negative for vimentin. A fraction of cells (25%) from freshly dissociated tumors exhibited ALDH activity. CGH from CDX tumors at passage 1 and 2 shows multiple gene rearrangement, revealing a high degree of genomic instability. Transcriptome analysis of ALDH positive and negative cells is ongoing and should help of identifying a cancer stem cell gene expression signature. Whole-exome sequencing of CDX tumor is ongoing and will be compared to data obtained from single CTCs from the patient. A cell line established in vitro from the CDX model grows in 3D clusters and is tumorigenic in mice. Interestingly, this cell line is positive for cytokeratins, EpCAM, E-cadherin, N-cadherin, vimentin, and expresses multiple cancer stem cell markers including CD166, CD24, CD133 and, ALDH activity. The cell line is hypotetraploid (about 70 chromosomes) and its CGH profile was similar to that of the CDX tumour, revealing a high level of genome instability. By investigating DNA replication process in this cell line, we found that it exhibits a spontaneous enhanced DNA damage signaling associated to an accumulation of DNA double strand breaks mainly in S phase strongly suggesting that the CDX-derived cell line displays hallmarks on replication stress that could explain, at least partially, the genomic instability in the cells. We report a low success rate in the establishment of NSCLC CDX (2%). However one NSCLC CDX model harboring cancer stem cell properties and deficiency of DNA replication maintenance was established. Ongoing work to identify a cancer stem cell signature and characteristics replication stress markers in this CDX model will be presented. This NSCLC CDX model will be useful to test drugs targeting these alterations in vivo and improve our knowledge of drug resistance. Citation Format: Vincent Faugeroux, Olivier Deas, Judith Michels, Jean Gabriel Judde, Stefano Cairo, Philippe Vielh, Virginie Marty, Fanny Billiot, Maud Ngocamus, Benjamin Besse, Patricia Kannouche, Françoise Farace. Establishment and characterization of circulating tumor cell-derived xenografts in non-small cell lung cancer. [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 2256.

Abstract C53: Homologous recombination deficiency (HRD) of high grade serous ovarian tumors from the NOVA Phase III clinical study

Abstract Background: Genome-wide analysis was conducted on tumors obtained from patients enrolled in the NOVA study, a Phase 3 clinical trial evaluating the PARP inhibitor niraparib as a maintenance treatment in patients with platinum-sensitive ovarian cancer. Homologous recombination deficiency (HRD) and mutations in DNA damage repair genes were evaluated. Material and methods: DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue and used to create libraries that were hybridized to a custom Agilent SureSelect capture array carrying probes for 54,091 single nucleotide polymorphism sites distributed across the human genome, as well as probes targeting 43 genes involved in DNA repair, including BRCA1 and BRCA2. The captured and enriched DNA was sequenced on an Illumina HiSeq 2500 sequencer. Sequences covering SNP positions were used to generate allelic imbalance profiles. Measures of genomic instability, including determination of an HRD score (integer value of 0-100), were calculated using allelic imbalance profiles and determination of loss of heterozygosity (LOH) by allele-specific copy number (ASCN). A previously identified HRD threshold score of 42 was used to define HRD positivity in the absence of a BRCA mutation. Results: The NOVA study is a Phase 3, multicenter, randomized, double-blind, placebo-controlled study of niraparib as maintenance therapy in ovarian cancer patients who have either gBRCAmut or a tumor with high-grade serous histology and who have responded to their most recent chemotherapy containing a platinum agent. Tumor BRCA mutational status, HRD score and genomic sequencing of 43 DNA repair genes were obtained from tumor samples from both gBRCAmut and non-gBRCAmut cohorts. In the gBRCAmut cohort, HRD analysis of the tumor confirmed the presence of a deleterious or suspected deleterious mutation in all cases. In addition, an HRD score ≥ 42 and the presence of a deleterious mutation in TP53 with loss of heterozygosity (LOH) were observed in nearly all tumors. In the non-gBRCA cohort, somatic BRCA mutations were observed in approximately 13% of tumors, and approximately half of tumors with no evidence of a BRCA mutation had a high HRD score. In both cohorts, the use of three scoring algorithms (LOH, telomeric allelic imbalance [TAI], large-scale state transitions [LST]), was more predictive of BRCA mutational status than LOH alone. Additional genomic sequencing identified deleterious mutations with LOH in DNA repair genes, such as BRIP1, CDK12, RAD51C, PTEN, and RAD51D, with many tumors exhibiting multiple deleterious mutations. Conclusions: High grade serous ovarian cancer is characterized by a high degree of genomic instability. Genomic analysis in the clinical setting is able to identify patients with both germline and somatic BRCA mutations, in addition to BRCAwt tumors with other genetic defects that may be sensitive to agents exploiting deficiencies in HR. Citation Format: Keith Wilcoxen, Christopher Neff, Victor Abkevich, Joshua Timothy Jones, Kathryn Kolquist, Michael Mirza, Jerry Lanchbury, Keith Mikule, Shefali Agarwal, Anne-Renee Hartman, Alexander Gutin, Kirsten Timms. Homologous recombination deficiency (HRD) of high grade serous ovarian tumors from the NOVA Phase III clinical study. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C53.

Abstract IA25: Targeting genetic heterogeneity in multiple myeloma through immune activation.

Abstract IA25: Targeting genetic heterogeneity in multiple myeloma through immune activation.

Abstract AS20: DNA-PKcs is amplified in high grade serous ovarian cancer (HGSC), correlates with poor outcome and drives resistance to platinum therapy via the AKT signaling pathway

Abstract High grade serous ovarian cancer is typified by p53 mutation, high degrees of genomic instability and the development of chemo-resistance. Genomic translocations result from incorrectly repaired DNA double strand breaks (DSBs). DNA-PKcs is a central catalytic component of the error prone non-homologous end joining (NHEJ) DSB repair mechanism. We report here that DNA-PKcs is frequently amplified in copy number in HGSC and that amplification correlates with higher DNA-PKcs gene expression and poorer patient outcome (PFS/OS) using both in-house and publicly available datasets (TCGA). Targeting DNA-PKcs, pharmacologically or by RNAi, enhances apoptosis in response to platinum treatment in platinum resistant cell lines and primary models. Furthermore we report that inhibition of DNA-PK restores response to cisplatin in chemoresistant ovarian cancer cells in vivo. SKOV-3 tumor xenografts were implanted subcutaneously into Balb/c Nu/Nu mice and treated with DNA-PKcs inhibitor NU7441 for 2 weeks alone or in combination with cisplatin. DNA-PK inhibition or cisplatin treatment alone were ineffective however in combination they decreased tumor growth at 14 days by 90%, relative to platinum only treatment. On investigating the mechanism of DNA-PKcs mediated chemoresistance we revealed that, in response to DNA damage, DNA-PKcs phosphorylates AKT on serine residue 473 in the nucleus of platinum resistant ovarian tumor cells, but not sensitive cells from the same patient. Phosphorylation at AKT threonine 308 is unaffected and DNA-PK inhibition does not interfere with insulin-mediated phosphorylation of AKT S473. DNA-PK mediated AKT activation in chemoresistant cells results in inhibitory phosphorylation of the pro-apoptotic protein BAD and stabilisation of the anti-apoptotic Bcl-2 protein resulting in the observed attenuation of apoptotic response to platinum treatment. DNA-PK inhibition is an attractive therapeutic strategy for resensitising resistant tumors to platinum based therapy, directly linking the DNA damage caused by platinum to the pro-survival AKT pathway, without interfering with normal physiological roles of AKT. Citation Format: Curry E, Cheraghchi-Bashi-Astaneh A, Chen M, Cunnea P, De Sousa C, Maginn E, Dai Y, Liu E, Wasan H, Mills G, Bowtell D, Gabra H & Stronach EA. DNA-PKcs is amplified in high grade serous ovarian cancer (HGSC), correlates with poor outcome and drives resistance to platinum therapy via the AKT signaling pathway [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS20.

Abstract 1081: Non-random genomic alterations in BRCA1-related breast cancer

Abstract BRCA1-related breast cancer is characterized by a high degree of genomic instability. However, to which extent this instability is a random result of the homologous recombination defect or the selection of the tumor phenotype, is unknown. The objective of this study is to investigate the genomic aberrations (copy number variation and somatic mutations) of BRCA1-related breast cancer, using a mouse model, to identify likely nonrandom aberrations and their biological functions. We examined genomic aberrations of three independent BRCA1-related tumors from a genetically engineered mouse model, K14-Cre BRCA1f/fp53f/f. Tumors had been propagated in Cre-negative littermates and liver tissue of the recipient mice from the controls were used as a germline control. Fresh frozen tumors and normal samples were used for exome sequencing and RNA-seq using Illumina technology. A commonly used workflow, GATK, was used for the sequence alignment, preprocessing and calling somatic mutations. VarScan2 was used for calling somatic copy number variations. We investigate the prevalence of common aberrations among these mouse in human BRCA1-related breast and ovarian cancer from The Cancer Genome Atlas (TCGA). We used the BioMart software tool to map mouse genes to human genes for the cross species analysis. After identifying common aberrant regions and genes across human and mouse we applied enrichment analysis using GOSeq, MetaCore and MSigDB software tools to identify the biological functions of the genetic alterations observed in BRCA1-related breast cancer across species. The analysis of exome sequencing data from BRCA1-related mouse tumors revealed surprisingly similar patterns of CNVs and rates of non-synonymous mutations. Correspondingly, distinct pattern were identified with regard to CNVs in human BRCA1-related breast and ovarian cancer. The cross species analysis of BRCA1-related TCGA ovarian and breast cancer tumors and BRCA1-related mouse tumors identified commonly aberrant regions that mapped to the long arms of human chromosomes 1, 8, 17 and 20 in both breast and ovarian cancer. Functionally, the blocks of genes that were commonly amplified in BRCA1-related breast or ovarian cancer included genes that transduce mitogenic signaling and ligand independent activation of estrogen receptors ESR1 and ESR2 as well as genes of the renin-angiotensin system. To conclude, copy number aberrations in BRCA1-related breast or ovarian cancers are not random and enrich for genes that enforce mitogenic signaling. The data support the notion that in addition to loss of cell cycle checkpoint control, tumor development requires activation of a mitogenic signaling program that could potentially be targeted for treatment. Citation Format: Sheida Nabavi, Kristina M. Holton, Ashish Juvekar, Nicholas Wang, Olivier Elemento, Lewis C. Cantley, Gerburg M. Wulf. Non-random genomic alterations in BRCA1-related breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1081. doi:10.1158/1538-7445.AM2015-1081

Abstract A47: DNA-PKcs is amplified in high-grade serous ovarian cancer (HGSC), correlates with poor outcome and drives resistance to platinum therapy via the AKT signaling pathway

Abstract High grade serous ovarian cancer is typified by p53 mutation, high degrees of genomic instability and the development of chemo-resistance. Genomic translocations result from incorrectly repaired DNA double strand breaks (DSBs). DNA-PKcs is a central catalytic component of the error prone non-homologous end joining (NHEJ) DSB repair mechanism. We report here that DNA-PKcs is frequently amplified in copy number in HGSC and that amplification correlates with higher DNA-PKcs gene expression and poorer patient outcome using both in-house and publicly available datasets. Targeting DNA-PKcs, pharmacologically or by RNAi, enhances apoptosis in response to platinum treatment in platinum resistant cell lines and primary models and inhibition of DNA-PK restores response to cisplatin in chemoresistant ovarian cancer cells in vivo. SKOV-3 tumor xenografts were implanted subcutaneously into Balb/c Nu/Nu mice and treated with DNA-PKcs inhibitor NU7441 for 2 weeks alone or in combination with cisplatin. DNA-PK inhibition or cisplatin treatment alone were ineffective however in combination they decreased tumor growth at 14 days by 90%, relative to platinum only treatment. On investigating the mechanism of DNA-PKcs mediated chemoresistance we revealed that, in response to DNA damage, DNA-PKcs phosphorylates AKT on serine residue 473 in platinum resistant ovarian tumor cells, but not sensitive cells from the same patient. Phosphorylation at AKT threonine 308 is unaffected and DNA-PK inhibition does not interfere with insulin-mediated phosphorylation of AKT S473. DNA-PK mediated AKT activation in chemoresistant cells results in inhibitory phosphorylation of the pro-apoptotic protein BAD and stabilization of the anti-apoptotic Bcl-2 protein resulting in the observed attenuation of apoptotic response to platinum treatment. DNA-PK inhibition is an attractive therapeutic strategy for resensitizing resistant tumors to platinum based therapy, directly linking the DNA damage caused by platinum to pro-survival AKT pathway signaling. Citation Format: Edward Curry, Azadeh Cheraghchi-Bashi-Astaneh, Michelle Chen, Paula Cunnea, De Sousa Camila, Elaina Maginn, Yan Dai, Edison Liu, Harpreet Wasan, Gordon B. Mills, David Bowtell, Hani Gabra, Euan A. Stronach. DNA-PKcs is amplified in high-grade serous ovarian cancer (HGSC), correlates with poor outcome and drives resistance to platinum therapy via the AKT signaling pathway. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A47.

Characterising phenotypically relevant intratumoural heterogeneity in high grade serous ovarian cancer.

e16569 Background: High-grade serous ovarian cancer is characterized by high degrees of genomic instability and heterogeneity, with the majority of patients eventually acquiring resistance to plati...

Abstract 1227: Preserved histological and molecular phenotypes following serial in vivo propagation of patient-derived xenografts of small cell lung cancer

Abstract Background: Limited availability of tissue samples is a major impediment to small cell lung cancer (SCLC) research but patient-derived xenograft (PDX) offers a potential alternative. The high genomic instability of SCLC necessitates the need to establish whether the molecular characteristics of PDXs are preserved across serial propagation in mice. Method: PDX were raised using biopsy samples from patients with relapsed SCLC under an IRB-approved protocol. Image guided or bronchoscopic biopsies were directly implanted subcutaneously into SCID mice within 60 minutes of collection and without in vitro culture. Tumor growth was monitored and animals were sacrificed while overall tumor size remained <2g. Harvested tumors were immediately re-implanted into a new set of 5 mice for up to 5 generations while freezing remnant tumor samples in liquid nitrogen for future use. Tumor histology was assessed by standard H&E and immunohistochemistry for neuroendocrine markers (NSE, chromogranin and Synaptophysin). Harvested DNA from tumor samples from all generations of PDX using QiAmp DNA Mini Kit was employed for targeted sequencing at greater than 2,000x coverage on the Illumina MiSeq instrument. A 17-gene panel including frequently encountered genetic aberrations in lung cancer (EGFR, KRAS, TP53, STK11, PTEN, NRAS, ERBB2, MET, NF1 and PIK3CA) was used to characterize molecular phenotype of PDX samples. Molecular homogeneity was assessed by comparing the genetic profile of each PDX across all generations. All identified mutations were compared to the COSMIC database.Results: We have successfully raised PDX from 5 of 9 patient biopsies with a lag time from initial implantation to adequate tumor growth ranging between 1.5 and 11 months. Propagation of PDX across generations has been performed for 5 generations (1PDX), 3 generations (2PDXs), 2 generations (2PDXs) and 1 generation (1PDX). There was good preservation of tumor histology assessed by H&E and IHC across generations. Mutations in TP53 gene (c.422G>A[p.Cys141Tyr]; c.913A>T[p.Lys305Ter], c.488A>G[p.Tyr163Cys], c.892G> [p.Glu298Ter]) detected in tumor samples from the first generation mice were preserved across all subsequent generations tested. Additionally, there were no new aberrations detected in this panel of 17 genes when comparing early to later generations of PDX. Conclusions: Despite the high degree of genomic instability expected in SCLC, serially propagated PDX of SCLC maintained histologic and molecular characteristics across many generations. Our finding that original tumor characteristics remain well preserved in PDX supports future use of this resource as reliable platform for translational research in SCLC.Supported through the Georgia Cancer Coalition Distinguished Scholar Award and NIH/NCI 5K23CA164015 grant to TK Owonikoko Citation Format: Taofeek Kunle Owonikoko, Guojing Zhang, Gabriel L. Sica, Hyun S. Kim, Onyinye C. Ogee-Nwankwo, Suresh S. Ramalingam, Michael R. Rossi, Fadlo R. Khuri. Preserved histological and molecular phenotypes following serial in vivo propagation of patient-derived xenografts of small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1227. doi:10.1158/1538-7445.AM2014-1227

Abstract 1959: Lgr5 marks an enriched population of fetal mammary stem cells

Abstract Basal-like breast cancers have unique characteristics that may contribute to their particularly poor prognosis. These include a high degree of genomic instability, intra-tumor heterogeneity, and gene expression profiles similar to those of normal mammary stem cells. These characteristics may arise through the aberrant propagation of normal stem cells. Alternatively, mutations commonly found in this subtype may be the dominant determinant of tumor phenotype. In order to explore these possibilities, pure populations of stem cells need to be isolated and analyzed. Our lab has identified a population of fetal mammary stem cells (fMaSCs) in the mouse whose gene expression profiles show a striking enrichment for those of human basal tumors. When fMaSCs were targeted with a p53 mutation, commonly found in human basal cancer, a tumor arose within 5 weeks- the shortest latency we have observed from any cell type. This suggests that fMaSCs are particularly prone to tumorigenesis and may likely be a source of basal-like tumors in the adult. Mammary stem cell activity becomes detectable at a defined time in late embryonic development, peaks right before birth, diminishes rapidly at birth, and remains low in the adult. We have since identified Lgr5 to be highly expressed in fMaSCs at the peak of their stem cell activity. We have gone on to use it to isolate the most pure population of fMaSCs measured yet by in vitro means. When plated in 3D culture containing 2% matrigel, up to 50% of Lgr5-expressing fetal mammary cells formed correctly polarized mammospheres. Using three independent methods, we will determine how Lgr5 expression tracks with stem cell activity throughout development and use this marker to test for the persistence of fetal-like stem cells in the adult mammary gland. We will develop comprehensive gene expression profiles from Lgr5+ cells throughout development to identify molecular correlates of the stem cell state. The relative contributions of developmental state of cell of origin or mutational status to tumor phenotype are poorly understood. We will test the capacity of stem cells at various stages of development to give rise to tumors when targeted with mutations commonly found in human basal tumors. A thorough analysis of tumors will reveal links between development, tumor initiation and progression, and provide insight into strategies to treat basal breast cancers. Citation Format: Christy L. Trejo, Ben Spike, Geoff Wahl. Lgr5 marks an enriched population of fetal mammary stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1959. doi:10.1158/1538-7445.AM2014-1959

ATR inhibition preferentially targets homologous recombination-deficient tumor cells.

Homologous recombination (HR) is required for faithful repair of double-strand DNA breaks. Defects in HR repair cause severe genomic instability and challenge cellular viability. Paradoxically, various cancers are HR defective and have apparently acquired characteristics to survive genomic instability. We aimed to identify these characteristics to uncover therapeutic targets for HR-deficient cancers. Cytogenetic analysis of 1143 ovarian cancers showed that the degree of genomic instability was correlated to amplification of replication checkpoint genes ataxia telangiectasia and Rad3-related kinase (ATR) and CHEK1. To test whether genomic instability leads to increased reliance on replication checkpoint signaling, we inactivated Rad51 to model HR-related genomic instability. Rad51 inactivation caused defective HR repair and induced aberrant replication dynamics. Notably, inhibition of Rad51 led to increased ATR/checkpoint kinase-1 (Chk1)-mediated replication stress signaling. Importantly, inhibition of ATR or Chk1 preferentially killed HR-deficient cancer cells. Combined, our data show that defective HR caused by Rad51 inhibition results in differential sensitivity for ATR and Chk1 inhibitors, implicating replication checkpoint kinases as potential drug targets for HR-defective cancers.

Minocycline-Mediated Improvement of Cognitive Impairment Induced by Irradiation Involves Preventing Immature Neurons From Apoptosis

Purpose/Objective(s): Cancer cells exhibit high degree of genome instability including frequent micronuclei, extranuclear organelles containing DNA fragments or whole chromosomes that arise due to DNA damage or mitotic errors. Micronuclei exhibit abnormalities in DNA replication, DNA damage response and membrane stability, leading to accumulation of mutations that are subsequently passed on to the main genome. As a consequence, micronuclei may act as a “mutator” organelle in tumorigenesis, and may be responsible for chromothripsis, catastrophic event leading to a large number of clustered rearrangements observed in many malignancies. We performed studies to gain insight into the mechanism of microncuclear-mediated DNA damage and mutagenesis. Materials/Methods: Micronuclei were induced in human RPE-1 and U2OS cell lines by treatment with 100 ng/ml nocodazole for 6 hours, leading to increased rate of chromosome missegregation, followed by synchronization with mitotic shake-off to release cells into subsequent interphase. Cells were fixed at given intervals in G1, S and G2 phases of the cell cycle and indirect immunofluorescence, time-lapse microscopy and flow cytometry were performed per previously established protocols. Antibodies used included anti-RPA70 (Cell Signaling), anti-RPA32 (Abcam), antig-H2AX (Cell Signaling), anti-Mcm2 (Cell Signaling) and anti-Mcm3(Cell Signaling). Results: Sor G2phase micronculei with ruptured nuclear membranes exhibit massive DNA damage as evidenced by positive g-H2AX and TUNEL staining, but G0and G1synchronized micronuclei show no such damage. Loss of nuclear membrane integrity by itself is thus not sufficient to result in DNA damage, and can only lead to damage in the setting of ongoing DNA replication. Strikingly, micronuclei exhibit a dramatic reduction in nucleoplasmic levels of Replication Protein A complex (RPA70 and RPA32) and DNA helicase (Mcm 2 and Mcm3), which may account for the observed delay in DNA replication, replication stress and blunted DNA damage response. Further dilution of these factors in the setting of sudden nuclear membrane rupture leads to replication catastrophe and massive DNA damage. Conclusions: Nucleocytoplasmic transport abnormalities in micronuclei lead to inability to concentrate key replication and DNA damage response factors, including components of RPA and DNA helicase complexes, contributing to error-prone DNA replication and blunted DNA damage response. Sudden loss of micronuclear membrane integrity in the setting of ongoing DNA replication leads to replication catastrophe and accumulation of massive DNA damage that is subsequently passed on to the main tumor genome driving tumor progression. Author Disclosure: A. Spektor: None. S. Liu: None. E. Jackson: None. D. Pellman: None.

Micronuclei-Induced DNA Damage and Mutagenesis in Cancer

Purpose/Objective(s): Cancer cells exhibit high degree of genome instability including frequent micronuclei, extranuclear organelles containing DNA fragments or whole chromosomes that arise due to DNA damage or mitotic errors. Micronuclei exhibit abnormalities in DNA replication, DNA damage response and membrane stability, leading to accumulation of mutations that are subsequently passed on to the main genome. As a consequence, micronuclei may act as a “mutator” organelle in tumorigenesis, and may be responsible for chromothripsis, catastrophic event leading to a large number of clustered rearrangements observed in many malignancies. We performed studies to gain insight into the mechanism of microncuclear-mediated DNA damage and mutagenesis. Materials/Methods: Micronuclei were induced in human RPE-1 and U2OS cell lines by treatment with 100 ng/ml nocodazole for 6 hours, leading to increased rate of chromosome missegregation, followed by synchronization with mitotic shake-off to release cells into subsequent interphase. Cells were fixed at given intervals in G1, S and G2 phases of the cell cycle and indirect immunofluorescence, time-lapse microscopy and flow cytometry were performed per previously established protocols. Antibodies used included anti-RPA70 (Cell Signaling), anti-RPA32 (Abcam), antig-H2AX (Cell Signaling), anti-Mcm2 (Cell Signaling) and anti-Mcm3(Cell Signaling). Results: Sor G2phase micronculei with ruptured nuclear membranes exhibit massive DNA damage as evidenced by positive g-H2AX and TUNEL staining, but G0and G1synchronized micronuclei show no such damage. Loss of nuclear membrane integrity by itself is thus not sufficient to result in DNA damage, and can only lead to damage in the setting of ongoing DNA replication. Strikingly, micronuclei exhibit a dramatic reduction in nucleoplasmic levels of Replication Protein A complex (RPA70 and RPA32) and DNA helicase (Mcm 2 and Mcm3), which may account for the observed delay in DNA replication, replication stress and blunted DNA damage response. Further dilution of these factors in the setting of sudden nuclear membrane rupture leads to replication catastrophe and massive DNA damage. Conclusions: Nucleocytoplasmic transport abnormalities in micronuclei lead to inability to concentrate key replication and DNA damage response factors, including components of RPA and DNA helicase complexes, contributing to error-prone DNA replication and blunted DNA damage response. Sudden loss of micronuclear membrane integrity in the setting of ongoing DNA replication leads to replication catastrophe and accumulation of massive DNA damage that is subsequently passed on to the main tumor genome driving tumor progression. Author Disclosure: A. Spektor: None. S. Liu: None. E. Jackson: None. D. Pellman: None.

Serial exome analysis of disease progression in premalignant gammopathies

Serial exome analysis of disease progression in premalignant gammopathies

Abstract IA08: Studying therapy response and resistance in mouse models of breast cancer

Abstract Women carrying germline mutations in BRCA1 are strongly predisposed to developing triple-negative breast cancers, which frequently contain TP53 mutations. To study the role of BRCA1 loss-of-function in development of triple-negative breast cancer, we have established genetically engineered mouse models (GEMMs) for BRCA1-mutated breast cancer based on cre-mediated tissue-specific deletion of p53 and Brca1. In addition to GEMMs for Brca1-null tumors, we have generated mammary tumor models carrying specific BRCA1 founder mutations (C61G, 185delAG and 5382insC). The mammary tumors that arise in these mouse models show strong similarity to BRCA1-associated breast cancer with respect to expression of basal cell markers, high tumor grade, and lack of expression of hormone receptors and ERBB2. In addition to these GEMMs, we have also established a panel of patient-derived xenograft (PDX) models for BRCA1-proficient and –deficient triple-negative breast cancer. BRCA1 deficiency in these PDX tumors is driven either by genetic mutation or by BRCA1 promoter methylation. The BRCA1-deficient tumors that arise in our various mouse models are in all cases characterized by a high degree of genomic instability and hypersensitivity to DNA-damaging agents due to loss of homology-directed double-strand break (DSB) repair. We have successfully used our GEMMs and PDX models of BRCA1-deficient breast cancer for preclinical evaluation of therapy response and elucidation of mechanisms of acquired drug resistance. BRCA1-deficient mammary tumors are highly sensitive to DNA-damaging agents such as platinum drugs and to PARP inhibitors, which indirectly induce DSBs by inhibiting DNA single-strand break repair. However, none of these drugs are capable of causing tumor eradication and all tumors grow back after drug treatment. By employing in vitro functional genetic screens and in vivo intervention studies, we found that therapy response and resistance is affected by several factors, including type of BRCA1 founder mutation, drug efflux transporter activity, and 53BP1 status. The lack of tumor eradication prompted us to look for additional drugs targeting BRCA-deficient tumors. Using a cell-based screening approach, we found that bifunctional alkylators such as nimustine may cause durable complete remission of BRCA-deficient mouse mammary tumors, suggesting that BRCA-mutated hereditary breast cancers and BRCA-like sporadic tumors may be eradicated by dose-intensive treatment with bifunctional alkylators. In support of this notion, patients with advanced breast cancers that display a BRCA1-like profile of genomic aberrations show a high complete remission rate and long progression-free survival after treatment with high-dose alkylating chemotherapy. Citation Format: Jos Jonkers. Studying therapy response and resistance in mouse models of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA08.

Chromothripsis and Focal Copy Number Alterations Determine Poor Outcome in Malignant Melanoma

Genetic changes during tumorigenesis are usually acquired sequentially. However, a recent study showed that in 2% to 3% of all cancers a single catastrophic event, termed chromothripsis, can lead to massive genomic rearrangements confined to one or a few chromosomes. To explore whether the degree of genomic instability and chromothripsis influences prognosis in cancer, we retrospectively applied array-comparative genomic hybridization (aCGH) to 20 malignant melanomas that showed, despite comparable conventional clinical and pathologic parameters, a profoundly different clinical course. We compared 10 patients who died of malignant melanoma 3.7 years (median, range 0.9-7.6 years) after diagnosis with 10 patients who survived malignant melanoma and had a median disease-free survival of 14.8 years (range 12.5-16.7 years; P = 0.00001). We observed a striking association between the degree of chromosomal instability, both numerical and structural, and outcome. Malignant melanomas associated with good prognosis showed only few chromosomal imbalances (mean 1.6 alterations per case), predominantly whole chromosome or chromosome arm gains and losses, whereas malignant melanomas with poor prognosis harbored significantly more chromosomal aberrations (13.9 per case; P = 0.008). Array-based CGH showed that these aberrations were mostly focal events, culminating in two cases in a pattern consistent with the phenomenon of chromothripsis, which was confirmed by paired-end sequencing. This is the first description of chromothripsis in primary malignant melanomas. Our study therefore links focal copy number alterations and chromothripsis with poor outcome in patients with malignant melanomas (P = 0.0002) and provides a genetic approach to predict outcome in malignant melanomas.

The Synergistic Effect of Chemical Carcinogens Enhances Epstein-Barr Virus Reactivation and Tumor Progression of Nasopharyngeal Carcinoma Cells

Seroepidemiological studies imply a correlation between Epstein-Barr virus (EBV) reactivation and the development of nasopharyngeal carcinoma (NPC). N-nitroso compounds, phorbols, and butyrates are chemicals found in food and herb samples collected from NPC high-risk areas. These chemicals have been reported to be risk factors contributing to the development of NPC, however, the underlying mechanism is not fully understood. We have demonstrated previously that low dose N-methyl-N’-nitro-N-nitrosoguanidine (MNNG, 0.1 µg/ml) had a synergistic effect with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (SB) in enhancing EBV reactivation and genome instability in NPC cells harboring EBV. Considering that residents in NPC high-risk areas may contact regularly with these chemical carcinogens, it is vital to elucidate the relation between chemicals and EBV and their contributions to the carcinogenesis of NPC. In this study, we constructed a cell culture model to show that genome instability, alterations of cancer hallmark gene expression, and tumorigenicity were increased after recurrent EBV reactivation in NPC cells following combined treatment of TPA/SB and MNNG. NPC cells latently infected with EBV, NA, and the corresponding EBV-negative cell, NPC-TW01, were periodically treated with MNNG, TPA/SB, or TPA/SB combined with MNNG. With chemically-induced recurrent reactivation of EBV, the degree of genome instability was significantly enhanced in NA cells treated with a combination of TPA/SB and MNNG than those treated individually. The Matrigel invasiveness, as well as the tumorigenicity in mouse, was also enhanced in NA cells after recurrent EBV reactivation. Expression profile analysis by microarray indicates that many carcinogenesis-related genes were altered after recurrent EBV reactivation, and several aberrations observed in cell lines correspond to alterations in NPC lesions. These results indicate that cooperation between chemical carcinogens can enhance the reactivation of EBV and, over recurrent reactivations, lead to alteration of cancer hallmark gene expression with resultant enhancement of tumorigenesis in NPC.

Copy number alteration and uniparental disomy analysis categorizes Japanese papillary thyroid carcinomas into distinct groups.

The aim of the present study was to investigate chromosomal aberrations in sporadic Japanese papillary thyroid carcinomas (PTCs), concomitant with the analysis of oncogene mutational status. Twenty-five PTCs (11 with BRAFV600E, 4 with RET/PTC1, and 10 without mutation in HRAS, KRAS, NRAS, BRAF, RET/PTC1, or RET/PTC3) were analyzed using Genome-Wide Human SNP Array 6.0 which allows us to detect copy number alteration (CNA) and uniparental disomy (UPD), also referred to as copy neutral loss of heterozygosity, in a single experiment. The Japanese PTCs showed relatively stable karyotypes. Seven cases (28%) showed CNA(s), and 6 (24%) showed UPD(s). Interestingly, CNA and UPD were rarely overlapped in the same tumor; the only one advanced case showed both CNA and UPD with a highly complex karyotype. Thirteen (52%) showed neither CNA nor UPD. Regarding CNA, deletions tended to be more frequent than amplifications. The most frequent and recurrent region was the deletion in chromosome 22; however, it was found in only 4 cases (16%). The degree of genomic instability did not depend on the oncogene status. However, in oncogene-positive cases (BRAFV600E and RET/PTC1), tumors with CNA/UPD were less frequent (5/15, 33%), whereas tumors with CNA/UPD were more frequent in oncogene-negative cases (7/10, 70%), suggesting that chromosomal aberrations may play a role in the development of PTC, especially in oncogene-negative tumors. These data suggest that Japanese PTCs may be classified into three distinct groups: CNA+, UPD+, and no chromosomal aberrations. BRAFV600E mutational status did not correlate with any parameters of chromosomal defects.

Abstract 3641: The predictive and prognostic roles of DNA damage response genes in epithelial ovarian cancer

Abstract Background: Despite platinum-based chemotherapy, 5-year survival of advanced stage ovarian cancer patients is only 15-30%. Exposure to platinum-based chemotherapeutics induces double strand DNA breaks (DSBs) and subsequently leads to activation of the DNA damage response (DDR). Therefore inhibition of components of the DDR may lead to better response to therapy. Aim of this study was to investigate in a large series of ovarian cancer patients the predictive and prognostic role of the activation status of four DDR-proteins- ATM, a key controller in the DDR, and three ATM substrates, including Chk2, 53BP1 and γ-H2AX, of which the latter is a direct readout for DNA damage. Methods: Expression of phospho-ATM, Chk2, phospho-Chk2, 53BP1, phospho-53BP1 and γ-H2AX was immunohistochemically assessed in 309 patients with chemo-naive ovarian cancer. Expression levels were related to clinicopathological characteristics and survival. Response to platinum-based chemotherapy was analyzed by defining two populations with either an optimal therapy response or a very poor therapy response. Both groups consisted of patients with advanced stage ovarian cancer, >2 cm residual disease after primary surgery and were all treated with platinum-based chemotherapy. The responder group had a well-defined PFS of more than 18 months while the non-responder group had a PFS of less than 6 months. Results: In the two well-defined groups with the largest contrast regarding response to treatment, positive γ-H2AX expression (OR=0.193; p=0.036) and positive Chk2 expression (OR=0.149; p=0.011) were related to a good response to platinum-based chemotherapy. In advanced stage patients, phospho-53BP1 expression was independently related to a worse disease-specific survival (HR=1.905; p= 0.044). Conclusion: High DDR protein expression in advanced stage ovarian cancers suggests a high degree of genomic instability, while the negative prognostic impact of phospho-53BP1 may be due to enhanced repair capacity of chemotherapy-induced DSBs on the long term. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3641. doi:1538-7445.AM2012-3641