DNA Copy Number Gain Research Articles

Abstract 760: Concordance of DNA copy number profiles between primary and metastatic colorectal carcinoma

Abstract The most common form of genomic instability in sporadic colorectal cancer (CRC) is chromosomal instability, a phenotype characterized by a high frequency of DNA copy number gain (CNG) and loss (CNL). Given the fundamental role of chromosomal instability in the pathogenesis of CRC, this study aimed to investigate the concordance of DNA copy number variants (CNV) between primary and metastatic CRC to better understand how the genomic structure differs during tumor evolution. In this study, the SurePrint G3 Human Genome CGH+SNP 2x400K Microarray (Agilent, SC, California, USA) was used for genome-wide screening of CNV in 9 pairs of primary tumors and metachronous liver metastases obtained during surgical resection from patients diagnosed with advanced CRC. Data from the Microarray-based Comparative Genomic Hybridization was analyzed by COSMIC Cancer Gene Census (CGC) and an independent non-CGC panel to map hotspot regions harboring genes causally implicated in cancer in the course of the metastatic cascade. Also, unique CNV in metastases were further investigated to identify loci advantageous for tumor progression and those presumably associated with therapy resistance. Overall, CNG observed in primary tumors were highly concordant with those in liver metastases. The CNG hotspot regions for both tissues represented chr7 (p22.3-p13), chr8 (q21.13-q24.3), chr13 (q12.11-q13.3), chr20 (q11.1-q13.33) and chrX (q11.1-q28) loci, while the most frequent CNL was at chr8 (p21.3-p12) locus. All identified loci underwent CNV events with a higher frequency in metastases (67-100%) than in primary tumors (55%). The identified genomic loci with CNG encode DNA repair genes (such as PMS2, NBN, BRCA2, RAD54B, RAD21, RPA3,4, and TREX2), homeobox genes (HOXA9,11,13), genes encoding centrosome-associated proteins (CETN2, MPLKIP), proto-oncogenes (MYC), and also tumor suppressor (ATRX, ASXL1). Additionally, an analysis of unique CNV within distinct tissues revealed CNG at Chr6 (p25.1-p22.3) in 89% of metastatic samples and absent in all the primary tumor lesions. This particular genomic region contains proto-oncogene DEK. Although the analysis is still underway, we have identified several shared and unique regions of altered copy number in both, primary tumors and metastases. We conclude that despite the high inter- and intra-tumoral genomic heterogeneity, metachronous metastases “inherit” a similar profile of DNA CNV from their respective primary tumor tissues. We intend to further extend our studied group of patients and perform integrative analyses of CNV with gene expression. This study was supported by the Charles University (UNCE/MED/006) and the Grant Agency of the Czech Republic (21-04607X, 21-27902S) Citation Format: Michal Kroupa, Kristyna Tomasova, Katerina Saskova, Jana Drabova, Josef Horak, Ludmila Vodickova, Jachym Rosendorf, Vaclav Liska, Pavel Vodicka. Concordance of DNA copy number profiles between primary and metastatic colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 760.

N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer

N6-methyladenosine (m6A) governs the fate of RNAs through m6A readers. Colorectal cancer (CRC) exhibits aberrant m6A modifications and expression of m6A regulators. However, how m6A readers interpret oncogenic m6A methylome to promote malignant transformation remains to be illustrated. YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis invivo. Multiomic analysis of RNA-sequencing, m6A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m6A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP). DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRCcell lines and primary organoids and lung and liver metastasis invivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m6A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both invitro and invivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis invivo. We identify a novel oncogenic epitranscriptome axis of YTHDF1-m6A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.

Systematic Characterization of Genes Representing Preferential Molecular Vulnerabilities for Myeloma Cells Compared to Other Neoplasias - Implications for the Biology and Therapeutic Targeting of Myeloma

In the last 2 decades, the improved clinical outcomes for multiple myeloma (MM) patients have been driven predominantly by therapeutics which exhibit limited activity outside plasma cell (PC) dyscrasias; do not target specific oncogenic mutations in MM cells, but rather pathways which are critical for PCs and dispensable for normal or malignant cells of most other lineages. We reasoned that identification of genes that are more potently / recurrently essential for MM cells, but less so for other neoplasias, would allow us to "re-identify" targets of currently used "PC-selective" anti-MM therapies. We also reasoned that systematic identification of MM-preferential dependencies could also uncover additional, previously underappreciated, genes which can serve as targets for potential future therapies and hopefully contribute to additional improvements in the therapeutic outcome for MM. To this end, we performed genome-scale CRISPR gene-editing studies to systematically characterize the molecular vulnerabilities of 20 MM cell lines and define which of these genes are more pronounced and/or recurrent dependencies for MM vs. cell lines (n=679) from other blood cancers and solid tumors. We identified 90+ genes whose function was significantly more essential for MM lines than other neoplasias. These MM-preferential dependencies included a large collection of transcription factors (e.g. IRF4, PRDM1, MAF, NFKB1, RELB, IKZF3, IKZF1, TCF3, CCND2, CBFB, MEF2C); transcriptional cofactors (e.g. POU2AF1); epigenetic regulators (e.g. EP300, DOT1L, HDAC1,ARID1A, CARM1); kinases such as IKBKB and CHUK/IKKa (both upstream of NF-κB), PIM2, IGF1R, SIK3,STK11; genes related to endoplasmic reticulum (ER) or Golgi function (e.g. HERPUD1, SYVN1,UBE2J1, SEC23B); as well as BCL2 and SMAD7. Results for several of these genes were further supported by in vitro studies with individual sgRNAs for CRISPR-based gene editing or activation of the respective genes; "addback" experiments with CRISPR-resistant cDNAs; shRNA studies in MM lines; use of small molecule inhibitors (e.g. against PIM kinases, CBFB, CARM1); and a focused in vivo CRISPR screen with MM.1S cells implanted in mice with BM-like scaffolds harboring a "humanized" stromal compartment: this latter in vivo study examined 46 MM-preferential dependencies which are also essential for MM.1S cells in vitro and observed that 41 of these genes were also essential for MM.1Scells in the "humanized" BM-like in vivo system. Some MM-preferential dependencies are essential for subsets of leukemia or lymphoma lines, but most have more pronounced/recurrent essentiality in MM vs. other blood cancers. In terms of overexpression (in high- vs. standard-risk MM; MM vs. normal PCs; or MM vs other cancers); frequency of mutations, DNA copy number gain or proximity to superenhancers, most of the MM-preferential dependencies do not exhibit such alterations or are not ranked in the top-100 genes in terms of the magnitude or frequency of these alterations. Notably, among the MM-preferential dependencies identified in our study, the majority are universally expressed in MM patient samples, while >80% and >75% of these genes have detectable transcript levels (RPKM>1) in CD138+ cells from at least 50% or 80%, respectively, of newly-diagnosed MM patients (MMRF CoMMpass study), suggesting broad expression of these dependencies across MM patients, including individuals with high-risk disease. It was reassuring to observe that MM-preferential dependencies identified in our study include prominent known targets for therapeutics with relatively MM-selective clinical activity (e.g. thalidomide derivatives [IKZF1/IKZF3], proteasome inhibitors [NF-kappaB genes and ER function] or panobinostat [HDAC1]). The identification of these known genes as preferential MM dependencies provides a mechanistic explanation for the relatively selective clinical activity of the respective therapies in MM/PC dyscrasias and also underscores the promising therapeutic implications of the large number of additional and previously underappreciated / understudied MM-preferential dependencies identified in our CRISPR-based functional studies. Disclosures Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

Overexpression of TWA1 predicts poor prognosis in patients with gastric cancer

TWA1 is associated with microtubule dynamics, cell migration, nucleokinesis and chromosome segregation. However, the role of TWA1 in gastric cancer (GC) remains unclear. In this study, Cosmic database revealed that the expression level of TWA1 ranks in the top 20 of overexpressed genes in GC. Further bioinformatic analysis revealed that the expression level of TWA1 was not in connection with the infection status of HP or EB. IHC and IF showed that TWA1 protein was present in both the cytoplasm and nucleus, but mainly in the cytoplasm. The high expression level of TWA1 was also related to tumor size, depth of invasion, lymph node metastasis, TNM stage, cancerous node and vascular invasion. Furthermore, higher TWA1 expression was also associated with shorter PFS and OS in GC. The univariate and multivariate analysis suggested the expression of TWA1 was an independent poor prognostic factor in GC. DNA copy number gain contributes to TWA1 overexpression and promoter methylation of TWA1 predicts profitable prognosis. Co-expression showed that TAF4 may function as a transcription factor (TF) regulates TWA1 expression, which further to mediate tumor invasion and metastasis. These findings revealed that TWA1 plays an important role in the development of GC and is expected to become an important biomarker and therapeutic target of tumors.

Functional Genomic Landscape of Genes with Recurrent Mutations in Multiple Myeloma

During the last two decades, cell lines and patient-derived samples from multiple myeloma (MM) have been extensively profiled for alterations in their genome with the anticipation that those genes with the most recurrent lesions could represent attractive novel therapeutic targets or markers for aggressive disease. Yet for many of these genes, their functional significance for MM cells has not been formally evaluated. With the advent of new CRISPR/Cas9-based functional genomics platforms, it is possible to generate in genome- or subgenome-scale direct quantitative information on the impact that perturbation of these genes exerts on tumor cell survival, proliferation or other phenotypes. We therefore examined the landscape of our CRISPR-based functional genomic data for these recurrently dysregulated genes We specifically curated information from the MMRF CoMMpass study and multiple other publicly available studies, to identify genes which are recurrently identified to harbor nonsynonymous mutation (SNV or indel), DNA copy number loss or gain, or participation in chromosomal translocations. We then examined the patterns of results for these genes in our genome scale CRISPR-based gene-editing studies for loss-of-function in n=18 MM cell lines.We identified a subset of genes (e.g. FAM46C, CDKN2C, RASA2) which are considered targets for recurrent loss-of-function events and indeed exhibit, for large fractions of the cell lines tested enrichment, of their sgRNAs in CRISPR knock-out studies, consistent with a role of these genes as suppressors of tumor cell survival or proliferation. CRISPR KO of TP53 leads to increased survival/proliferation of only a small minority (2/18 of cell lines tested thus far), which reflects the fact that the overwhelming majority of MM cell lines already harbor LOF events for this gene. Interestingly, a substantial number of genes which have been considered to harbor recurrent LOF events in MM patient samples (e.g. NF1, NF2, CYLD) do not exhibit sgRNA enrichment in CRISPR KO screens in the MM cell lines tested so far. In addition, several other recurrently mutated genes for which their loss- or gain-of-function status had not been previously evaluated with extensive functional studies in MM (e.g. SP140, LTB, EGR1, ATM, PARK2, PRKD2, RAPGEF5, DOCK5, TGDS, TNFAIP8) exhibit in the majority of cell lines tested in in CRISPR knockout studies no significant enrichment or depletion of their sgRNAs. In contrast, PTPN11, CREBBP, EP300, KMT2B, KMT2C, SETD2, SF3B1 and UBR5, are notable examples of recurrently mutated genes which represent dependencies for large fractions of MM cell lines in vitro. These results highlight the value of interpreting results from next generation sequencing studies in the context of information provided by the genome scale by use of functional genomic characterization of available cell line models. We envision that, similar sub-genome scale assays were performed at the level of patient derived samples will also provide direct information about the relevance of some of these genes. In addition, functional studies conducted with context of tumor-microenvironemtn compartment interactions and tumor interface will be needed to evaluate several genes identified in the study. DisclosuresLicht:Celgene: Research Funding. Mitsiades:Takeda: Other: employment of a relative; Janssen/ Johnson & Johnson: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding; TEVA: Research Funding.

Appraising the relevance of DNA copy number loss and gain in prostate cancer using whole genome DNA sequence data.

A variety of models have been proposed to explain regions of recurrent somatic copy number alteration (SCNA) in human cancer. Our study employs Whole Genome DNA Sequence (WGS) data from tumor samples (n = 103) to comprehensively assess the role of the Knudson two hit genetic model in SCNA generation in prostate cancer. 64 recurrent regions of loss and gain were detected, of which 28 were novel, including regions of loss with more than 15% frequency at Chr4p15.2-p15.1 (15.53%), Chr6q27 (16.50%) and Chr18q12.3 (17.48%). Comprehensive mutation screens of genes, lincRNA encoding sequences, control regions and conserved domains within SCNAs demonstrated that a two-hit genetic model was supported in only a minor proportion of recurrent SCNA losses examined (15/40). We found that recurrent breakpoints and regions of inversion often occur within Knudson model SCNAs, leading to the identification of ZNF292 as a target gene for the deletion at 6q14.3-q15 and NKX3.1 as a two-hit target at 8p21.3-p21.2. The importance of alterations of lincRNA sequences was illustrated by the identification of a novel mutational hotspot at the KCCAT42, FENDRR, CAT1886 and STCAT2 loci at the 16q23.1-q24.3 loss. Our data confirm that the burden of SCNAs is predictive of biochemical recurrence, define nine individual regions that are associated with relapse, and highlight the possible importance of ion channel and G-protein coupled-receptor (GPCR) pathways in cancer development. We concluded that a two-hit genetic model accounts for about one third of SCNA indicating that mechanisms, such haploinsufficiency and epigenetic inactivation, account for the remaining SCNA losses.

Abstract 685: GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma

Abstract Background: GD2-directed immunotherapeutic strategies have improved outcomes in neuroblastoma; however, the majority of patients treated suffer relapse and GD2 expression on pain fibers causes dose-limiting toxicities. Methods: To identify alternative cell surface immunotherapeutic targets, we compared high-risk neuroblastoma (n=126 tumors) and normal tissue RNA sequencing data (GTEx; n=7859 samples from 31 normal tissues) and prioritized genes by differential and absolute expression and cell surface prediction. Genes were further surveyed for somatic copy number gain and correlative expression with MYCN amplification. Differential protein expression and localization were confirmed in neuroblastoma primary tumors (n=98), patient-derived xenografts (n=32; PDXs), cell lines (n=23), and normal pediatric tissues (n=36). Cell lines were subjected to candidate gene gain and loss of function studies (n=11). Additional pediatric tumor RNA sequencing data was surveyed followed by confirmatory immunohistochemistry (IHC). Finally, candidate specific antibodies were isolated from a human Fab phage library and utilized for antibody-drug conjugate (ADC) engineering followed by cytotoxicity studies. Results: We identified 33 differentially expressed cell surface molecules from which we prioritized glypican-2 (GPC2) for validation given GPC2’s robust differential expression (log-fold change tumor vs. normal tissue = 1.71-9.22; p=1.99 x 10-9-1.88 x10-300), high-level absolute RNA expression (median FPKM=60), and frequent DNA copy number gain associated with higher GPC2 expression (35%, n=182 tumors; p<0.005). GPC2 expression was also higher in MYCN amplified neuroblastomas (p<0.05), MYCN binds the GPC2 promoter shown by chromatin immunoprecipitation (ChIP) sequencing and reporter assays, and MYCN depletion resulted in decreased GPC2 expression. Immunoblot, flow cytometry, immunofluorescence, and IHC analysis of primary tumors, PDXs, and cell lines confirmed dense cell surface GPC2 expression. Medulloblastomas (n=62) were also found to have high GPC2 expression that positively correlated with MYC, MYCN, and GPC2 loci gain (p<0.0001). Pediatric normal tissues had very restricted cell surface GPC2 expression, with only low levels found in the esophagus and skin. GPC2 depletion in neuroblastoma cell lines resulted in apoptosis and growth inhibition and GPC2 forced over-expression increased neuroblastoma cell proliferation (p<0.001 for all assays). Finally, a human GPC2 antibody, D3-GPC2-Fab, was developed and shown to bind GPC2 with high affinity and specificity. D3-GPC2-IgG1 induced internalization of GPC2 and was conjugated to pyrrolobenzodiazepine (PBD) dimers to form an ADC which induced potent and specific cytotoxicity to GPC2 expressing neuroblastoma cells (IC50 = 1.7-11 pM). Conclusions: GPC2 is an oncogene and immunotherapeutic target in neuroblastoma and potentially other cancers. Citation Format: Kristopher R. Bosse, Pichai Raman, Maria Lane, Robyn T. Sussman, Jo Lynne Harenza, Daniel Martinez, Sabine Heitzeneder, Zhongyu Zhu, Komal Rathi, Michael Randall, Laura Donovan, Sorana Morrissy, Doncho V. Zhelev, Yang Feng, Jennifer Hwang, Yanping Wang, Bruce Pawel, Tricia Bhatti, Mariarita Santi, Javed Khan, Michael Taylor, Dimiter S. Dimitrov, Crystal Mackall, John M. Maris. GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma [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 685. doi:10.1158/1538-7445.AM2017-685

MicroRNA-645 is an oncogenic regulator in colon cancer

Despite advances in early diagnosis and the development of molecularly targeted therapy, curative treatment of colon cancer once it has metastasized is yet to be accomplished. This is closely associated with deregulated CRC cell proliferation and resistance to apoptosis. Here we reveal that upregulation of microRNA-645 (miR-645) through DNA copy number gain is responsible for enhanced proliferation and resistance to apoptosis in colon cancer. MiR-645 was upregulated in most colon cancer tissues related to adjacent normal mucosa. This appeared to be associated with amplification of a section of chromosome 20q13.13, where miR-645 is located. Inhibition of miR-645 reduced proliferation and enhanced sensitivity to apoptosis triggered by the chemotherapeutic drugs 5-fluorouracil and cisplatin in CRC cells, and retarded colon cancer xenograft growth. Conversely, overexpression of miR-645 in normal colon epithelial cells enhanced proliferation and triggered anchorage-independent cell growth. Although SRY-related HMG-box 30 (SOX30) was identified as a miR-645 target, its expression was only partially affected by miR-645, suggesting that miR-645 is a fine-tuning mechanism of SOX30 expression. Moreover, overexpression of SOX30 only moderately inhibited promotion of CRC cell proliferation by miR-645, indicating that miR-645 may have more targets that contribute to its pro-proliferation effect in colon cancer. Together, this study reveals that miR-645 can regulate oncogenesis in colon cancer with SOX30 being one of its targets.

The significance of over-expression of NUAK2 on the survival of patients with acral melanomas and its relevance with DNA copy number gain

The significance of over-expression of NUAK2 on the survival of patients with acral melanomas and its relevance with DNA copy number gain

DNA Copy Number Gains of TCF4 (E2-2) Are Associated with Poor Outcome in Diffuse Large B-Cell Lymphoma

The development of B-cells is a complex process that proceeds through multiple stages and is regulated by a hierarchy of transcription factors and other physiologic signals. Each unique B-cell malignancy can be aligned with a 'normal counterpart' at one or more of these discrete developmental stages. However, with the exception of translocations of transcription factor genes, the genetic basis for this is not well defined.We performed an analysis of high-resolution single nucleotide polymorphism (SNP) microarrays from 694 diffuse large B-cell (DLBCL) tumors to identify significant somatic copy number alterations (SCNA). Through integrative analysis of 249 tumors with matched gene expression profiling (GEP) data, we identified the likely targets of these alterations and found that genes that were targeted by DNA copy number gain were significantly enriched for DNA binding activity and transcription factor function. We extended upon this observation by analyzing SNP microarray data of a further 2,716 tumors from 7 additional subtypes of B-cell malignancy. Through this analysis, we identified patterns of transcription factor alterations that aligned with the differentiation state of the 'normal B-cell counterpart' of each malignancy. This provides evidence that SCNA of B-cell transcription factors may underlie the differentiation state of B-cell malignancies.DLBCL can be divided into two subtypes based upon gene expression profiles that align with either the germinal center B-cell differentiation state (GCB-like) or a post-GCB activated B-cell state (ABC-like). Having observed an enrichment for transcription factor SCNAs in DLBCL, and an alignment between transcription factor alterations and differentiation states in other B-cell malignancies, we hypothesized that SCNAs of transcription factors may also underlie the etiology of these molecular subtypes. By testing for associations between SCNAs and cell of origin subtype, we identified three co-segregating DNA copy number gains that were significantly enriched in the ABC-like subtype. These included gains of the BCL6 and SPIB genes that have been previously observed to be associated with the ABC-like subtype. In addition, we found gains of the TCF4 (E2-2) gene to be significantly enriched in ABC-like tumors. In line with this, TCF4 alterations were significantly associated with reduced overall survival in cohorts of patients treated with either CHOP (n=232, P=0.009) or R-CHOP (n=197, P=0.041).B-cell receptor (BCR) signaling is a key survival pathway in ABC-like DLBCL, and the TCF4 gene has a defined role in promoting the expression of immunoglobulin (Ig) genes that encode the B-cell receptor (BCR). The analysis of paired SCNA and GEP data revealed a significantly higher expression of Ig genes in tumors with TCF4 DNA copy number gain compared to those without, suggesting that normal BCR expression may be deregulated by this genetic alteration. In addition, chromatin-immunoprecipitation sequencing (ChIP-seq) for TCF4 in ABC-like DLBCL cell lines also revealed binding of TCF4 to an Ig gene enhancer region. As BCR signaling can be altered by somatic mutations in the CARD11, CD79B and MYD88 genes, we evaluated the relative representation of these mutations and TCF4 DNA copy number gains using targeted deep sequencing of 124 DLBCL tumors. This revealed that TCF4 DNA copy number gains largely mutually excluded CARD11 mutations, but significantly co-segregated with both MYD88 (FDR=0.005) and CD79B (FDR=0.053) mutations. In addition, we observed significant co-segregation between CD79B and MYD88 mutations (FDR<0.001). This is particularly notable due to the preliminary associations between combined CD79B and MYD88 mutation status and response to an inhibitor of BCR signaling, Ibrutinib.Together these data highlight an association between SCNA of B-cell transcription factors and the differentiation state of the 'normal counterpart' of the respective malignant B-cell. In line with this, we show that DNA copy number gains of the TCF4 transcription factor are associated with the ABC-like subtype of DLBCL, significantly worse overall survival, and increased Ig expression. These characteristics, in addition to the co-association between TCF4 DNA copy number gains and somatic mutations of CD79B and MYD88, suggest that TCF4 may be an important modifier of BCR signaling and contribute to the etiology of ABC-like DLBCL. DisclosuresRosenquist:Gilead Sciences: Speakers Bureau. Lunning:TG Therapeutics: Consultancy; AbbVie: Consultancy; Gilead: Consultancy; Bristol-Myer-Squibb: Consultancy; Juno: Consultancy; Genentech: Consultancy; Spectrum: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

Genomic Landscape Survey Identifies SRSF1 as a Key Oncodriver in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.

Abstract PR02: GPC2 is a candidate immunotherapeutic target and putative oncogene in high-risk neuroblastoma and other pediatric cancers

Abstract Background: Children with high-risk neuroblastoma have a poor prognosis despite intensive multimodal chemoradiotherapy. While monoclonal antibodies targeting the disialoganglioside GD2 improve outcomes in neuroblastoma, this therapy is associated with significant “on target-off tumor” toxicities. Thus, a major challenge remains in identifying novel cell surface molecules that meet the stringent criteria for modern immunotherapeutics, including unique tumor expression compared to normal childhood tissues, and preferably that these cell surface molecules be required for tumor sustenance and thus may be less susceptible to immune escape mechanisms. Methods: Differentially expressed genes that represent putative cell surface immunotherapeutic targets were initially identified by comparing high-risk neuroblastoma RNA sequencing data (N=126 primary tumors) to paired normal tissue data (GTEx; N=25 unique normal tissues; N=1-313 replicate samples/tissue type). For further prioritization, gene sets were filtered by in silico cell surface prediction and by absolute RNA expression, and then by assessing primary tumor DNA copy number via SNP genotyping data (N=177). For prioritized genes, protein expression and cellular localization was confirmed by Western blot, immunohistiochemistry (IHC), immunofluroescence (IF) and membrane extraction techniques in neuroblastoma cell lines and primary tumors. For functional characterization, neuroblastoma cell lines (N=12) were subjected to both gain and loss of function studies for each candidate gene. Results: The initial transcriptome-based discovery effort identified 649 significantly differentially expressed genes (log-fold change tumor vs. normal &amp;gt;1 for each tissue; adjusted p&amp;lt;0.05), 86 (13%) of which were predicted to be potential cell surface molecules. Through our analytic pipeline, we prioritized the extracellular glycosylphosphatidylinositol (GPI) anchored, signaling co-receptor Glypican-2 (GPC2) for validation given robust differential RNA expression (log-fold change tumor vs. normal tissue = 2.1-8.2; p&amp;lt;3 x 10-10), high-level absolute RNA expression (median FPKM=57; 85% of tumors with FPKM &amp;gt;25) and consistent DNA copy number gain (31% of primary neuroblastomas; N=177) associated with significantly higher GPC2 expression (p&amp;lt;0.0001). Immunoblot analysis confirmed ubiquitous GPC2 protein expression (N=8 high-risk neuroblastomas and 23 cell lines) and membrane extraction, IF, and IHC confirmed dense plasma membrane associated GPC2 protein expression in neuroblastoma cell lines. IHC analysis of primary neuroblastoma tumors (N=165) compared to a parallel array of pediatric normal tissues (N=41) further confirmed GPC2 protein expression to be membrane associated and tumor specific with very limited normal tissue expression. Lentiviral mediated RNAi induced GPC2 depletion in a panel of 12 neuroblastoma cell lines resulted in significant apoptosis and growth inhibition both in transient CellTiter-Glo and Caspase-Glo assays (20-87% decreased growth and 1.5-18.4-fold increased caspase 3/7 level vs. control) and with longer term real-time monitoring of cell growth (RT-CES). GPC2 overexpression resulted in significantly increased cellular proliferation (2.7-fold growth increase vs. control, p&amp;lt;0.0001). Finally, GPC2 was also found to be significantly differentially overexpressed in other embryonal cancers, most notably medulloblastoma. Conclusions: GPC2 is a candidate cell surface immunotherapeutic target and putative oncogene in high-risk neuroblastoma. More globally, these data show that genome-wide transcriptome analysis integrated with genomic and functional validation can identify differentially expressed cell surface oncogenes that may be attractive immunotherapeutic targets. Development of a GPC2 directed chimeric antigen receptor is ongoing, and progress will be reported. This abstract is also presented as Poster A13. Citation Format: Kristopher R. Bosse, Pichai Raman, Robyn T. Sussman, Michael Randall, Dan Martinez, Zhongyu Zhu, Bruce Pawel, Tricia Bhatti, Javed Khan, Dimiter S. Dimitrov, Crystal Mackall, John M. Maris. GPC2 is a candidate immunotherapeutic target and putative oncogene in high-risk neuroblastoma and other pediatric cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR02.

DNA Copy Number Gain in Lung Cancer and NonInvolved Tissue

DNA Copy Number Gain in Lung Cancer and NonInvolved Tissue

Deep Sequencing in Conjunction with Expression and Functional Analyses Reveals Activation of FGFR1 in Ewing Sarcoma.

A low mutation rate seems to be a general feature of pediatric cancers, in particular in oncofusion gene-driven tumors. Genetically, Ewing sarcoma is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Other contributing somatic mutations involved in disease development have only been observed at low frequency. Tumor samples of 116 Ewing sarcoma patients were analyzed here. Whole-genome sequencing was performed on two patients with normal, primary, and relapsed tissue. Whole-exome sequencing was performed on 50 Ewing sarcoma and 22 matched normal tissues. A discovery dataset of 14 of these tumor/normal pairs identified 232 somatic mutations. Recurrent nonsynonymous mutations were validated in the 36 remaining exomes. Transcriptome analysis was performed in a subset of 14 of 50 Ewing sarcomas and DNA copy number gain and expression of FGFR1 in 63 of 116 Ewing sarcomas. Relapsed tumors consistently showed a 2- to 3-fold increased number of mutations. We identified several recurrently mutated genes at low frequency (ANKRD30A, CCDC19, KIAA0319, KIAA1522, LAMB4, SLFN11, STAG2, TP53, UNC80, ZNF98). An oncogenic fibroblast growth factor receptor 1 (FGFR1) mutation (N546K) was detected, and the FGFR1 locus frequently showed copy number gain (31.7%) in primary tumors. Furthermore, high-level FGFR1 expression was noted as a characteristic feature of Ewing sarcoma. RNA interference of FGFR1 expression in Ewing sarcoma lines blocked proliferation and completely suppressed xenograft tumor growth. FGFR1 tyrosine kinase inhibitor (TKI) therapy in a patient with Ewing sarcoma relapse significantly reduced 18-FDG-PET activity. FGFR1 may constitute a promising target for novel therapeutic approaches in Ewing sarcoma.

Abstract 56: Receptor-Interacting protein kinase 1 functions as an oncogenic regulator in human melanoma

Abstract Although many studies in recent years have uncovered an important role of receptor-interacting protein kinase 1 (RIP1) in mediating cell survival and death signaling, its potential part in the pathogenesis of cancer remains less understood. Here we report that RIP1 functions as an oncogenic regulator in human melanoma. While the expression of RIP1 was commonly upregulated in melanoma, knockdown of RIP1 inhibited melanoma cell proliferation in vitro, and retarded melanoma growth in a xenograft model. Conversely, despite induction of apoptosis in a small proportion of melanoma cells, overexpression of RIP1 enhanced proliferation in the remaining cells. The promoting effect of RIP1 on melanoma cell proliferation was mediated by activation of NF-κB, as blockade of NF-κB activation eliminated RIP1 overexpression-triggered increase in cell proliferation, whereas hyperactivation of NF-κB abolished inhibition of cell proliferation caused by RIP1 knockdown. In support, RIP1 knockdown led to reduction, whereas its overexpression caused an increase, in NF-κB activation. Strikingly, ectopic expression of RIP1 enhanced melanocyte proliferation and triggered anchorage-independent growth of the cells similarly in a NF-κB-dependent manner. While upregulation of RIP1 was associated with DNA copy number gain in a subset of melanomas, constitutive ubiquitination and subsequent stabilization of the RIP1 protein driven by TNFα autocrine appeared to be another mechanism commonly responsible for upregulation of RIP1 in melanoma cells. Collectively, these results identify RIP1 as an oncogenic regulator in melanoma, and points to the possibility of targeting the NF-κB activating mechanism of RIP1 as a novel approach in the treatment of the disease. Citation Format: Lei Jin, Xiao Ying Liu, Fritz Lai, Xu Guang Yan, Chen Chen Jiang, Su Tang Guo, Chun Yan Wang, Amanda Croft, Hsin-Yi Tseng, James S. Wilmott, Richard A. Scolyer, Xu Dong Zhang. Receptor-Interacting protein kinase 1 functions as an oncogenic regulator in human melanoma. [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 56. doi:10.1158/1538-7445.AM2015-56

Abstract 4954: Serine/arginine splicing factor 1 (SRSF1) mediates DNA repair and chemo-sensitivity and drives growth in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Despite a high response rate to chemotherapy, more than 95% of patients eventually die from SCLC. We have identified that Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression in tumor is strongly associated with poor survival based on whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Here, SRSF1 is evaluated as a tumor driver in SCLC. Treatment of SCLC cell lines in vitro with a low dose of cisplatin or topotecan (two of the most common standard of care in SCLC) only induced a modest decrease of cell growth. However, knockdown of SRSF1 with siRNA along with a sub-lethal dose of cisplatin or topotecan enhanced the overall growth inhibition effect compared to the chemotherapy alone. SRSF1 siRNA alone induced modest but significant caspase-3 activation, similar to cisplatin or topotecan treatment alone. The combination of SRSF1 siRNA with chemotherapy treatments produced a significantly higher caspase induction. DNA-damage induction as a potential mechanism of SRSF1 knockdown was assessed. Phosphorylation of H2AX and Chk2, established markers of DNA-strand breaks and DNA-repair response, was consistently induced upon SRSF1 abrogation in cells, and further increased the phosphorylation of these proteins when combined with cisplatin or topotecan treatment. The knockdown of SRSF1 in SCLC cells also resulted in significant growth inhibition when cells were grown as 3D spheroids. Cells transfected with non-targeting siRNA produced large and well-organized spheroids; in contrast, cells transfected with SRSF1 siRNA did not form well-organized structures but mainly existed as single cells with poor viability. Results were confirmed by colony formation assays and could be rescued with a siRNA-resistant SRSF1 expression construct. Finally, we investigated the impact of SRSF1 loss on kinase signaling pathways in SCLC cells through phospho-kinase array profiling. Strong phospho-AKT and ERK signals were observed in control siRNA-transfected cells, and were abrogated by SRSF1 siRNA. Western blot confirmed this in several SCLC cell lines. These targeting studies demonstrate that SRSF1 plays a key role in DNA repair, chemo-sensitivity and cell proliferation. Together, these data reveal SRSF1 as a therapeutic target in SCLC and provide a rationale for personalized therapy in SCLC. Citation Format: Sarah J. Conley, Xin Yao, Jiaqi Huang, Brandon Higgs, Zhibin Hu, Zhan Xiao, Haihong Zhong, Zheng Liu, Philip Brohawn, Xiaoxiao Ge, Meggan Czapiga, Vaheh Oganesyan, Haihua Fu, David Tice, Ronald Herbst, Xinying Su, Yi Gu, Jianren Gu, Baohui Han, Laura Richman, Bahija Jallal, Liyan Jiang, Hongbing Shen, Yihong Yao. Serine/arginine splicing factor 1 (SRSF1) mediates DNA repair and chemo-sensitivity and drives growth in small cell lung 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 4954. doi:10.1158/1538-7445.AM2015-4954

CLIP2 as radiation biomarker in papillary thyroid carcinoma

A substantial increase in papillary thyroid carcinoma (PTC) among children exposed to the radioiodine fallout has been one of the main consequences of the Chernobyl reactor accident. Recently, the investigation of PTCs from a cohort of young patients exposed to the post-Chernobyl radioiodine fallout at very young age and a matched nonexposed control group revealed a radiation-specific DNA copy number gain on chromosomal band 7q11.23 and the radiation-associated mRNA overexpression of CLIP2. In this study, we investigated the potential role of CLIP2 as a radiation marker to be used for the individual classification of PTCs into CLIP2-positive and -negative cases-a prerequisite for the integration of CLIP2 into epidemiological modelling of the risk of radiation-induced PTC. We were able to validate the radiation-associated CLIP2 overexpression at the protein level by immunohistochemistry (IHC) followed by relative quantification using digital image analysis software (P=0.0149). Furthermore, we developed a standardized workflow for the determination of CLIP2-positive and -negative cases that combines visual CLIP2 IHC scoring and CLIP2 genomic copy number status. In addition to the discovery cohort (n=33), two independent validation cohorts of PTCs (n=115) were investigated. High sensitivity and specificity rates for all three investigated cohorts were obtained, demonstrating robustness of the developed workflow. To analyse the function of CLIP2 in radiation-associated PTC, the CLIP2 gene regulatory network was reconstructed using global mRNA expression data from PTC patient samples. The genes comprising the first neighbourhood of CLIP2 (BAG2, CHST3, KIF3C, NEURL1, PPIL3 and RGS4) suggest the involvement of CLIP2 in the fundamental carcinogenic processes including apoptosis, mitogen-activated protein kinase signalling and genomic instability. In our study, we successfully developed and independently validated a workflow for the typing of PTC clinical samples into CLIP2-positive and CLIP2-negative and provided first insights into the CLIP2 interactome in the context of radiation-associated PTC.

Abstract 3427: Uncovering vulvar cancer: Integrated analysis of genomic and transcriptomic data

Abstract Vulvar squamous cell carcinoma (VSCC) is a rare gynecological cancer, representing approximately 3-5% of genital tumors. However, its incidence has risen considerably and no consistent profile of this disease was established so far. Also, little is known about the genomic abnormalities of VSCC and how they correlate with gene expression. In order to verify which are the major genomic alterations that lead to gene expression abnormalities we used integrative analysis in VSCC samples. Seventeen frozen samples of VSCC were carried out in genome-wide expression (GWE) profiling using the Agilent Whole Human Genome Microarray 60K (Agilent Technologies, Santa Clara, CA, USA) and in Array-CGH (aCGH) using the Agilent Human Genome CGH Microarray 60K (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer's protocol. Data were extracted and flagged with Feature Extraction version 10.5 (Agilent Technologies, Santa Clara, CA) and the aCGH data was processed using NEXUS 6.0 (BioDiscovery, Hawthorne, CA, USA). JISTIC was used to classify genes mapped at gains and losses in genomic regions. Differentially expressed genes were identified by SAM statistical test. The CONEXIC algorithm was applied to integrate the data and in silico functional analysis were performed using Ingenuity Pathways Analysis (IPA) (Ingenuity® Systems). The aCGH analysis revealed 216 significant genomic alterations including 196 gains and 20 losses. GWE profile identified 3799 differentially expressed genes in comparison with normal tissue including 1352 up-regulated and 2447 down-regulated. The integrated analysis showed that genomic and transcriptomic results were concordant in 47 genes, in which 46 were up regulated and involved in gain of DNA copy number and only 1 gene (PLXDC2) was down-regulated and associated to genomic loss. According to IPA analysis of the top biological functions associated with the 47 genes, 3 of them were related with “tumor morphology”. Also, “cell death and survival” and “cellular function and maintenance” were included in the list of pathways disrupted by the genes selected as possible drivers of VSCC. Three significant networks could be defined comprising these genes. The first network comprised 18 of the concordant genes and was associated to functions as carbohydrate metabolism, small molecule biochemistry, cellular function and maintenance. The second network (18 genes) was related to RNA post-transcriptional modification, developmental disorder and hereditary disorder. The third network (15 genes) was associated to RNA post-transcriptional modification, gene expression and organ development. Thereby, important signaling networks were found disrupted by these genes which could influence tumor development or progression in VSCC. Therefore, these genes might be useful as a first step to identify molecular markers to improve diagnosis and therapeutic approaches for this tumor. Citation Format: André M. Lavorato-Rocha, Beatriz de Melo Maia, Iara S. Rodrigues, Fabio A. Marchi, Gabriel R. Fernandes, Glauco Baiocchi, Fernando A. Soares, Silvia R. Rogatto, Yukie Sato-Kuwabara, Rafael M. Rocha. Uncovering vulvar cancer: Integrated analysis of genomic and transcriptomic data. [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 3427. doi:10.1158/1538-7445.AM2014-3427

Genome resilience and prevalence of segmental duplications following fast neutron irradiation of soybean.

Fast neutron radiation has been used as a mutagen to develop extensive mutant collections. However, the genome-wide structural consequences of fast neutron radiation are not well understood. Here, we examine the genome-wide structural variants observed among 264 soybean [Glycine max (L.) Merrill] plants sampled from a large fast neutron-mutagenized population. While deletion rates were similar to previous reports, surprisingly high rates of segmental duplication were also found throughout the genome. Duplication coverage extended across entire chromosomes and often prevailed at chromosome ends. High-throughput resequencing analysis of selected mutants resolved specific chromosomal events, including the rearrangement junctions for a large deletion, a tandem duplication, and a translocation. Genetic mapping associated a large deletion on chromosome 10 with a quantitative change in seed composition for one mutant. A tandem duplication event, located on chromosome 17 in a second mutant, was found to cosegregate with a short petiole mutant phenotype, and thus may serve as an example of a morphological change attributable to a DNA copy number gain. Overall, this study provides insight into the resilience of the soybean genome, the patterns of structural variation resulting from fast neutron mutagenesis, and the utility of fast neutron-irradiated mutants as a source of novel genetic losses and gains.

Recurring DNA copy number gain at chromosome 9p13 plays a role in the activation of multiple candidate oncogenes in progressing oral premalignant lesions.

Genomic alteration at chromosome 9p has been previously reported as a frequent and critical event in oral premalignancy. While this alteration is typically reported as a loss driven by selection for CDKN2A deactivation (at 9p21.3), we detect a recurrent DNA copy number gain of ∼2.49 Mbp at chromosome 9p13 in oral premalignant lesions (OPLs) that later progressed to invasive lesions. This recurrent alteration event has been validated using fluorescence in situ hybridization in an independent set of OPLs. Analysis of publicly available gene expression datasets aided in identifying three oncogene candidates that may have driven selection for DNA copy number increases in this region (VCP, DCTN3, and STOML2). We performed in vitro silencing and activation experiments for each of these genes in oral cancer cell lines and found that each gene is independently capable of upregulating proliferation and anchorage-independent growth. We next analyzed the activity of each of these genes in biopsies of varying histological grades that were obtained from a diseased oral tissue field in a single patient, finding further molecular evidence of parallel activation of VCP, DCTN3, and STOML2 during progression from normal healthy tissue to invasive oral carcinoma. Our results support the conclusion that DNA gain at 9p13 is important to the earliest stages of oral tumorigenesis and that this alteration event likely contributes to the activation of multiple oncogene candidates capable of governing oral cancer phenotypes.