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T-cells in the tumor microenvironment can often become exhausted and dysfunctional due to chronic and prolonged exposure to antigen. The resulting condition of T cell exhaustion represents an important mechanism of clinical resistance to immune checkpoint blockade. The transcription factor NR4A1 is known to be upregulated in tumor-specific T-cells and is a mediator of T cell dysfunction including driving exhaustion during chronic antigen stimulation of T-cells. Pro-oncogenic activities of NR4A1 have been observed in various solid tumors, including colorectal and breast cancers, and co-expression of NR4A1 with known immune checkpoint markers such as PD-L1 and B7 family members has also been observed in various cancer cell lines. These findings have identified NR4A1 as an important target for overcoming resistance to cancer immunotherapy. We hypothesize that targeted silencing of the NR4A1 and other immunomodulatory genes can reverse T-cell exhaustion and expand the clinical benefit of immune checkpoint blockade in solid tumors resistant to immune therapy. To reverse T-cell exhaustion with a targeted approach, we developed T cell targeting SeekRsTM, which are chimeric RNA therapeutics containing dual-flanking aptamer binders connected by a double-stranded bridge containing siRNA silencers that can target multiple immunomodulatory genes. First-generation aptamer-siRNA chimeras designed with a CTLA-4 targeting aptamer containing a STAT3 siRNA demonstrated effectiveness in silencing its target. Modifications to the structure included the addition of chemically modified nucleotides to improve serum stability. Additionally, dimerization of the structure to form a SeekR dimer molecule greatly improved internalization and target silencing compared to the monomeric chimera. Newly developed SeekRs targeting T cells and designed to silence NR4A1 have effectively downregulated NR4A1 in model cell lines and activated T cells. These results demonstrate that dual targeting SeekRs can be used to specifically deliver siRNA to T cells for targeted silencing that can reverse exhaustion and potentially overcome resistance to cancer immunotherapy in the clinic. Citation Format: Marvin O'Ketch, Jeffrey A. Kiefer, Dnyanesh Rasale, Warren S. Weiner, Lizette A. Castaño, Nathalie A. Azorsa, David W. Lee, Kerry M. Barnhart, Spyro Mousses, David O. Azorsa. Activity of novel RNA therapeutics to overcome resistance to immune checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4417.

SeekR™ chimeric molecules consist of dual flanking RNA aptamers designed to bind specific proteins (BINDERS), joined by a double stranded RNA bridge that encodes multiple siRNAs designed to silence select disease target genes (SILENCERS). Aptamers are folded nucleic acid chains that act much like chemical antibodies in that they can bind proteins in a sequence-specific manner. By identifying RNA aptamers capable of seeking and binding to specific cell surface protein receptors, SeekR™ RNA therapeutics can be engineered to self-deliver to a particular receptor defined tissue type. For example, a SeekR™ can be directed to prostate tumors that selectively express PSMA. The combination of two RNA aptamer binders with two siRNA silencers thus enables the selective delivery of the siRNAs to tumor cells expressing the binder target, wherein it directs sequence-specific silencing of cancer target genes. To identify and prioritize the right combination of SeekR targets, we have taken advantage of single-cell sequencing (scRNA-seq) data sets. By analyzing scRNA-seq data, we can identify specific cell populations within tumors to co-target with an aptamer and siRNA. We leveraged several publicly available colon and non-small cell lung cancer scRNA data sets for this analysis. A custom analysis pipeline was applied to these data to identify specific cell types within tumor samples. We then profiled specific cancer targets and determined percentages of target co-expression within a single tumor compared to normal cells. This provides information for cell-type-specific targeting to reduce potential toxicity within non-tumor tissue. In addition to targeting aptamers, we performed ligand-receptor analyses to prioritize specific siRNA targets in particular cell types within the tumor environment. This analysis revealed novel combinations of binders and silencers that will more precisely deliver the right multi-targeted gene silencing to the correct cell types. In summary, scRNA-seq data provide a rich resource of data to direct SeekR™ development and optimization. Citation Format: Susan Massey, Richard Hsiao, Shweta Yadav, David Azorsa, Spyro Mousses, Jeffrey A. Kiefer. Leveraging single-cell RNA sequencing data to design multi-targeting SeekRRNA therapeutics [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 4081.

Ubiquitination is a metabolic process in eukaryotic cells that modifies proteins with ubiquitin leading to protein degradation via the ubiquitin-proteosome pathway. Cellular levels of ubiquitin are driven by the expression of RPS27A, UBA52, UBB and UBC. In cancer, the dysregulation of ubiquitination can affect various aspects of tumor progression including cell cycle regulation and gene expression. Ubiquitin B (UBB) gene has been shown to be over-expressed in some tumor types, including NSCLC and cervical cancer, and under-expressed in other tumors, in particularly a subset of ovarian cancers. A role for ubiquitin C (UBC) in cancer has not been well established but UBC has been described as a biomarker in renal cancer and CLL. In recent studies, repression of UBB in ovarian cancer cells was associated with sensitivity to UBC knockdown and demonstrated a synthetic lethal relationship. Since both UBB and UBC show substantial sequence homology, we explored the possibility of developing dual targeting siRNA that would silence both UBB and UBC. We identified unique individual siRNA sequences that efficiently silenced both UBB and UBC and led to rapid cytotoxicity in several cancer cell lines including the colon cancer cell line HCT-116, the breast cancer cell line SK-BR3, and the prostate cancer cell line 22Rv1. Treatment with either UBB-specific or UBC-specific siRNA did not show the same lethal phenotype. Dose response assays using the dual targeting siRNA designated U21 indicated EC50 for cytotoxicity between 50–500 pM in several cancer lines. Treatment of cancer cell lines with U21 leads to rapid apoptosis and cell death. Chemical modifications to pharmacologically stabilize the U21 siRNA sequence did not change its activity. The efficient cytotoxic activity of the U21 siRNA sequence has been integrated into our SeekR™ platform to create aptamer-siRNA chimeras for targeted delivery of the lethal U21 payload to specific cancer cell types. Citation Format: David O. Azorsa, David W. Lee, Marvin O'Ketch, Nathalie A. Azorsa, Ioanna Papacharalampous, Lizette A. Castaño, Jeffrey A. Kiefer, Spyro Mousses. Development of novel anticancer RNA therapeutics with dual-targeting siRNA that silences ubiquitin family members [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 4096.

T-cell exhaustion describes the dysfunctional state of effector T cells in the tumor microenvironment that results from chronic and prolonged exposure to antigen. It also represents an important mechanism of clinical resistance to immune checkpoint blockade. Recently, the transcription factor NR4A1 has been shown to be upregulated in tumor-specific T cells and plays a role in driving exhaustion during chronic antigen stimulation of T cells. Pro-oncogenic activities of NR4A1 have long been observed in various solid tumors including colorectal and breast cancers, and co-expression of NR4A1 with known immune checkpoint markers such as PD-L1 have also been observed in various cancer cell lines. These findings have identified NR4A1 as an important target for overcoming resistance to cancer immunotherapy. We hypothesize that multi-targeting to simultaneously block PD-1 signaling and silence NR4A1 gene expression can reverse T-cell exhaustion and expand the clinical benefit of PD-1 blockade in immune therapy resistant solid tumors. Using our SeekR™ RNA therapeutics platform, we combined aptamer binders to PD-1 with siRNA against NR4A1, andNR4A1 and evaluated their ability to reverse T-cell exhaustion and re-activate T-cell based anticancer immunity in solid tumors. Specifically, we created SeekR™ therapeutic RNA molecules with dual flanking RNA aptamers binders to PD-1 and other checkpoint inhibitors, connected by a double stranded bridge that encodes for siRNA silencers that target multiple immunomodulatory genes, such as NR4A1. The novel anti-PD-1 RNA aptamer component serve to: A) direct and deliver the SeekR™ RNA oligo therapeutics to exhausted T-cells that express PD-1 receptors; and B) internalize the siRNA component into the T-cells. The siRNA components of the SeekR™ serve to silence NR4A1 which lead to reversal of the cytotoxic T-cell exhausted phenotype following chronic in-vitro stimulation. These results demonstrate that dual targeting of PD-1 and NR4A1 has the potential to reverse T-cell exhaustion and the potential to overcome cancer immunotherapy resistance in the clinic. Citation Format: Marvin O'ketch, Victoria A. Love, Jeffrey A. Kiefer, David W. Lee, Lizette A. Castaño, Spyro Mousses, David O. Azorsa. Development of multi-targeting immunomodulatory SeekR RNA therapeutics to overcome resistance to PD-1 blockade [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 4239.

Pancreatic cancer is a highly aggressive disease characterized by poor prognosis and vast genetic instability. Recent microarray-based, genome-wide surveys have identified multiple recurrent copy number aberrations in pancreatic cancer; however, the target genes are, for the most part, unknown. Here, we characterized the 19q13 amplicon in pancreatic cancer to identify putative new drug targets. Copy number increases at 19q13 were quantitated in 16 pancreatic cancer cell lines and 31 primary tumors by fluorescence in situ hybridization. Cell line copy number data delineated a 1.1 Mb amplicon, the presence of which was also validated in 10% of primary pancreatic tumors. Comprehensive expression analysis by quantitative real-time reverse transcription-PCR indicated that seven transcripts within this region had consistently elevated expression levels in the amplified versus nonamplified cell lines. High-throughput loss-of-function screen by RNA interference was applied across the amplicon to identify genes whose down-regulation affected cell viability. This screen revealed five genes whose down-regulation led to significantly decreased cell viability in the amplified PANC-1 cells but not in the nonamplified MiaPaca-2 cells, suggesting the presence of multiple biologically interesting genes in this region. Of these, the transcriptional regulator intersex-like (IXL) was consistently overexpressed in amplified cells and had the most dramatic effect on cell viability. IXL silencing also resulted in G0-G1 cell cycle arrest and increased apoptosis in PANC-1 cells. These findings implicate IXL as a novel amplification target gene in pancreatic cancer and suggest that IXL is required for cancer cell survival in 19q13-amplified tumors. [Cancer Res 2007;67(5):1943–9]

Background: The current study explores T-cell (TC) clonality and molecular factors associated with this metric. Tumors employ multiple mechanisms to evade antitumor immune responses. One process involves TC inhibition via upregulation of immune checkpoint (IC) ligands in the tumor microenvironment (TME). Gradual upregulation of inhibitory receptor at their cellular surface results in a decreased capacity to proliferate and activate cytotoxic pathways against tumor cells presenting antigenic peptides.1 In melanoma, this subset of exhausted TCs has been described as highly clonal, where the majority of PD-1-expressing TC population shares the same TCR sequence specific against the same antigenic fragment.2 Previous studies have demonstrated that a clonal TCR repertoire appears to be associated in part with therapeutic responses during IC blockade.3 Methods: We performed TCR sequencing on 82 blood and 73 archival tumor tissue samples collected from ED SCLC patients (pts) in an ongoing longitudinal cohort study in US community oncology practices. Of these, 82 blood and 48 tissue samples had sufficient material available to quantify a clonality metric. To quantify TC abundance as a fraction of total nucleated cells, 82 blood and 58 tissue samples had sufficient material available. Results: Within the subset of 48 tumor samples, a more clonal (ie, less diverse) TCR repertoire was associated with less necrosis (P≤0.012) and lower levels of inflammatory cell infiltration in the local TME (P≤0.021). When pts were divided into 2 equal groups according to the median clonality level, pts with a less clonal TCR repertoire (n = 24/48) who were treated with non-immune-targeted therapy trended toward a longer overall survival (OS; 446 vs 301 days; P≤0.039). In contrast, the percentage of TCs in the TME did not correlate with improved survival (P≤0.412), necrosis (P≤0.131), and inflammation (P≤0.615). This observation differs from results in melanoma describing the impact of IC blockade where pts responding to therapy were associated with a more clonal TME TC population and increased CD8 TCs in the tumor compartment and at the invasive margin.3 In blood, while a less clonal TCR repertoire was associated with a similar but non-significant trend toward longer survival (P≤0.148), pts with increased TC abundance had longer OS (P≤0.025). No association was observed between clonality in TME and clonality (P≤0.571) or TC abundance (P≤0.965) in blood. Conclusion: We hypothesize that a diverse TCR repertoire in the TME and increased peripheral TC abundance are 2 predictors of longer OS in ED SCLC. To further explore factors that may influence TC responses in ED SCLC, the current TCR sequencing results will be integrated with transcriptome and whole genome sequencing analyses. References 1. Wherry EJ. Nat Immunol. 2011;12:492-99. 2. Gros A, et al. J Clin Invest. 2014;124:2246-59. 3. Tumeh PC, et al. Nature. 2014;515:568-71. Citation Format: Maen Hussein, Sharon Wilks, Marc Monte, Donald A. Richards, Jerome H. Goldschmidt, David Waterhouse, Lisu Wang, Saumya Pant, Erik Yusko, Ryan O. Emerson, Darin M. Taverna, Kaushal Desai, Spyro Mousses, Zhenhao Qi, Jason D. Hipp, Harlan Robins, Kimary Kulig, Cory Batenchuk. Characterization of the T-cell receptor (TCR) repertoire in extensive disease small cell lung cancer (ED SCLC). [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 4159.

Despite recent advances in targeted treatments for multiple myeloma, optimal molecular therapeutic targets have yet to be identified. To functionally identify critical molecular targets, we conducted a genome-scale lethality study in multiple myeloma cells using siRNAs. We validated the top 160 lethal hits with four siRNAs per gene in three multiple myeloma cell lines and two non-myeloma cell lines, cataloging a total of 57 potent multiple myeloma survival genes. We identified the Bcl2 family member MCL1 and several 26S proteasome subunits among the most important and selective multiple myeloma survival genes. These results provided biologic validation of our screening strategy. Other essential targets included genes involved in RNA splicing, ubiquitination, transcription, translation, and mitosis. Several of the multiple myeloma survival genes, especially MCL1, TNK2, CDK11, and WBSCR22, exhibited differential expression in primary plasma cells compared with other human primary somatic tissues. Overall, the most striking differential functional vulnerabilities between multiple myeloma and non–multiple myeloma cells were found to occur within the 20S proteasome subunits, MCL1, RRM1, USP8, and CKAP5. We propose that these genes should be investigated further as potential therapeutic targets in multiple myeloma. Cancer Res; 72(3); 757–68. ©2011 AACR.

Genetic changes underlie tumor progression and may lead to cancer-specific expression of critical genes. Over 1100 publications have described the use of comparative genomic hybridization (CGH) to analyze the pattern of copy number alterations in cancer, but very few of the genes affected are known. Here, we performed high-resolution CGH analysis on cDNA microarrays in breast cancer and directly compared copy number and mRNA expression levels of 13,824 genes to quantitate the impact of genomic changes on gene expression. We identified and mapped the boundaries of 24 independent amplicons, ranging in size from 0.2 to 12 Mb. Throughout the genome, both high- and low-level copy number changes had a substantial impact on gene expression, with 44% of the highly amplified genes showing overexpression and 10.5% of the highly overexpressed genes being amplified. Statistical analysis with random permutation tests identified 270 genes whose expression levels across 14 samples were systematically attributable to gene amplification. These included most previously described amplified genes in breast cancer and many novel targets for genomic alterations, including the HOXB7 gene, the presence of which in a novel amplicon at 17q21.3 was validated in 10.2% of primary breast cancers and associated with poor patient prognosis. In conclusion, CGH on cDNA microarrays revealed hundreds of novel genes whose overexpression is attributable to gene amplification. These genes may provide insights to the clonal evolution and progression of breast cancer and highlight promising therapeutic targets.

We have shown recently that about half of the human TGCTs(3) reveal DNA copy number increases affecting two distinct regions on chromosome arm 17q. To identify potential target genes with elevated expressions attributable to the extra copies, we constructed a cDNA microarray containing 636 genes and expressed sequence tags from chromosome 17. The expression patterns of 14 TGCTs, 1 carcinoma in situ, and 3 normal testis samples were examined, all with known chromosome 17 copy numbers. The growth factor receptor-bound protein 7 (GRB7) and junction plakoglobin (JUP) were the two most highly overexpressed genes in the TGCTs. GRB7 is tightly linked to ERBB2 and is coamplified and coexpressed with this gene in several cancer types. Interestingly, the expression levels of ERBB2 were not elevated in the TGCTs, suggesting that GRB7 might be the target for the increased DNA copy number in TGCTs. Because of the limited knowledge of altered gene expression in the development of TGCTs, we also examined the expression levels of 512 additional genes located throughout the genome. Several genes novel to testicular tumorigenesis were consistently up- or down-regulated, including POV1, MYCL1, MYBL2, MXI1, and DNMT2. Additionally, overexpression of the proto-oncogenes CCND2 and MYCN were confirmed from the literature. The overexpressions were for some of the target genes closely associated to either seminoma or nonseminoma TGCTs, and hierarchical cluster analysis of the gene expression data effectively distinguished among the known histological subtypes. In summary, this focused functional genomic characterization of TGCTs has lead to the identification of new gene targets associated with a common genomic rearrangement as well as other genes with potential importance to testicular tumorigenesis.

To explore molecular mechanisms of prostate cancer progression, we applied tissue microarrays (TMAs) to analyze expression of candidate gene targets discovered by cDNA microarray analysis of the CWR22 xenograft model system. A TMA with 544 clinical specimens from different stages of disease progression was probed by mRNA in situ hybridization and protein immunohistochemistry. There was an excellent correlation (r = 0.96; n = 16) between the expression levels of the genes in the xenografts by cDNA microarray and mRNA in situ hybridization on a TMA. One of the most highly overexpressed genes in hormone-refractory CWR22R xenografts was the S100P gene. This gene, coding for a calcium signaling molecule implicated in the loss of senescence, was also significantly associated with progression in clinical tumors by TMA analysis (P0.001), suggesting dysregulation of this pathway in hormone-refractory and metastatic prostate cancers. Conversely, two genes that were down-regulated during tumor progression in the CWR22 model system were validated in vivo: crystallin mu (CRYM) and a LIM-domain protein LMO4 both showed significantly lower mRNA levels in hormone-refractory tumors as compared with primary tumors (P0.001). These results illustrate a strategy for rapid clinical validation at the mRNA and protein level of gene targets found to be differentially expressed in cDNA microarray experiments of model systems of cancer.

Ewing sarcoma is the second most common cancer of bone and soft tissue arising in children and young adults. Standard treatment of Ewing sarcoma includes surgery, radiation, and chemotherapy largely consisting of combinations of non-targeted cytotoxic agents. Although the survival rate has improved for patients treated for localized disease, the survival rate of patients with metastatic tumor remains lower than 30%. In order to improve therapeutic options for Ewing Sarcoma, we employed a functional genomics approach based on RNA interference (RNAi) screening to identify genes whose silencing affected the proliferation and survival of Ewing sarcoma cells. Four Ewing sarcoma cell lines, TC-32, TC-71, SK-ES-1 and RD-ES, were transfected with a library of siRNA targeting 287 cancer-associated genes. The siRNA screening data were normalized and statistical cut-offs were determined for each cell line. From the list of siRNA ‘hits,’ we identified siRNAs targeting Fibroblast growth factor 4, FGFR4, to be among the most effective in inducing cytotoxicity in Ewing sarcoma cells. Further validation was conducted showing that siRNAs to FGFR4, but not FGFR1, FGFR2, or FGFR3, were able to significantly reduce Ewing Sarcoma cell viability. Furthermore, siRNA targeting FGFR4 were able to induce Caspase 3 activity. FGFR4 protein was shown to be expressed in Ewing sarcoma by western blot analysis, with expression levels lower than FGFR4 expression on rhabdomyosarcoma cells. Targeting FGFR4 activity in Ewing sarcoma cells using a pan-FGFR inhibitor, PD-173074, demonstrated that Ewing sarcoma cells were sensitive to FGFR inhibition. Since FGFR4 in a potential therapeutic target in rhadomyosarcoma, we compared the response of the Ewing sarcoma cells to PD-173074 to that of the rhabdomyosarcoma cell lines, RH-30 and RD. The IC50s of the four Ewing sarcoma cells ranged from 3.8-4.7 µM and were slightly lower than the IC50 for the rhabdomyosarcoma cell lines RH-30 (5.6 µM) and RD (7.9 µM). These results indicate that FGFR4 is a potential therapeutic target in Ewing sarcoma and that FGFR4 targeted therapy should be tested in preclinical xenograft models of Ewing sarcoma. Citation Format: David O. Azorsa, Irma M. Gonzales, Shilpi Arora, R. Tanner Hagelstrom, Tanya H. Little, Robert J. Arceci, Spyro Mousses. RNAi screening identifies FGFR4 as a modulator of growth and survival in Ewing sarcoma. [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 3422. doi:10.1158/1538-7445.AM2014-3422

Background Acute myeloid leukemia (AML) is a complex and heterogeneous disease leading to a wide range of response variability to conventional therapy, excessive treatment related toxicity, and an overall poor outcome. The NCI supported Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative provided initial support for large-scale genomics to identify novel disease-associated genomic and epigenomic alterations that could be used to develop novel, targeted therapies for pediatric AML. Patients Diagnostic (Dx), remission (Rm), and relapse (Rel) samples were obtained from over 200 children with AML treated on the most recent Children's Oncology Group clinical trials. Data analyses were performed using Partek Genomics Suite 6.6 and Ingenuity Pathway Analysis. Results Methylation profiling (Illumina HumanMethylation27) identified 603 CpG sites significantly differentially methylated between Dx and Rm. In addition, 514 CpG sites were significantly differentially methylated between matched Rel and Rm (p Gene expression profiling (Affymetrix Human Gene 1.1 ST) revealed 1,142 significantly differentially expressed between Dx and normal bone marrow obatined from the blood bank. Similarly, there were 1,242 significantly differentially expressed genes between Rel and normal bone marrow (p Copy-number data analysis (Affymetrix Genome-Wide Human SNP 6.0) identified 180,379 significantly different regions in 254 Dx vs matched Rm. Similarly, 4,707 regions a significantly different in 49 Rel vs matched Rm (p Whole-genome sequencing (Complete Genomics, Inc) was performed on 100 cases with matched Dx and Rm pairs and on 86 cases with matched Dx, Rel, and Rm samples. The frequencies of either somatic or germline variants were not significantly different when comparing Dx vs. Rel samples. Pathway analysis compared 1,528 mutated genes in the pediatric (TARGET) cohort with 1,963 mutated genes in adult de novo AML (TCGA cohort). Of the 85 and 80 significantly (p Conclusion A combination of genomic approaches have identified genes and pathways that appear deregulated in pediatric AML. Many changes are specific to Dx or Rel sample groups, or to pediatric vs. adult AML. Further study into these genes and pathways may reveal candidate targets for therapeutic intervention. Citation Format: Daniel H. Wai, Rhonda E. Ries, Michael Considine, David W. Lee, Sun-Mei Liu, Nicole M. O'Connor, Oliver Pepper, Ranjan Bista, Eiman Aleem, David O. Azorsa, Tanja M. Davidsen, Toni Farley, Jaime Guidry-Auvil, Jason E. Farrar, Malcolm A. Smith, Daniela S. Gerhard, MIchael F. Ochs, Spyro Mousses, Soheil Meshinchi, Robert J. Arceci. Integrative genomic analyses on pediatric acute myeloid leukemia. [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 5583. doi:10.1158/1538-7445.AM2014-5583

Triple negative breast cancer (TNBC), is characterized by a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2). TNBC comprises about 15 of all breast cancer diagnoses, but ∼25% of deaths. TNBC has a more aggressive clinical course and long-term disease-free survival is poor. As is well known, tumors that develop in women who carry deleterious germline BRCA mutations typically have tumors characteristic of the basal-like TNBC phenotype associated with defects in double stand break repair and homologous recombination. These observations have led to the development of therapeutic strategies aimed at these mechanisms, such as PARP inhibitors, which are in mature stages of drug development for the treatment of a subset of patients diagnosed with mTNBC. Iniparib, although not believed to be a true PARP inhibitor, is believed to act through a similar mechanism of action. We have completed a study of whole genome and transcriptome sequencing of 14 patients with metastatic triple negative breast cancer (mTNBC). One patient who was sequenced had a complete response to a combination including gemcitabine, carboplatin, and iniparib prior to sequencing. Sequencing analysis revealed a collection of somatic mutations and alterations in genes associated with DNA repair and homologous recombination, including DCLRE1C, MEI1, TP53, DDB1, RIF1, ZBTB40, BRIP1, and BRCA2. Furthermore, RNA-seq expression analysis demonstrated significant underexpression of BRCA1. Thus, these alterations may serve as markers that can be predictive of responders from non-responders of treatment regimens including iniparib or possibly PARP inhibitors. To begin to identify a genomic profile indicative of PARP response, tumors and blood of four additional mTNBC patient samples were collected and subjected to whole exome and RNA sequencing. We have also obtained the mutational data of TNBC tumors sequenced by the Cancer Genome Atlas. The genetic profiles of these tumors will be compared to the previously sequence mTNBC sample that responded to iniparib to determine the frequency of these specific events in mTNBC. Through this work we hope to identify a genetic profile that can serve as a possible predictor of response to iniparib or true PARP inhibitors. Citation Format: Shukmei Wong, Joyce A. O'Shaughnessy, Patricia LoRusso, Jessica Aldrich, Ahmet Kurdoglu, Alexis Christoforides, Jennifer Dinh, Tyler Izatt, Shripad Sinari, Angela Baker, Tracy M. Moses, Bodour Salhia, Spyro Mousses, Jeffrey Kiefer, Jeffrey M. Trent, Daniel Von Hoff, Geoffrey Shapiro, Winnie Liang, David W. Craig, John D. Carpten. Whole exome and RNA sequencing reveals genetic alterations in DNA damage and homologous recombination pathways in triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2023. doi:10.1158/1538-7445.AM2013-2023

Tumor suppressor genes (TSGs) have been classically defined as genes whose loss of function in tumor cells contributes to the formation and/or maintenance of the tumor phenotype. Typically but not always, tumors neutralize both alleles of TSGs by deleting one copy and mutating the other. One molecular strategy to discover potential tumor suppressor gene candidates involves the “re-expression” of RNA transcripts containing nonsense mutations by inhibition of the nonsense-mediated RNA decay (NMD) process. Combining NMD inhibition with array CGH has lead to the genome-wide identification of genes with biallelic inactivation (involving nonsense mutations on one allele and loss of the second allele) in prostate cancer cell lines and identified EphB2 as a novel candidate TSG in prostate cancer. Using such a strategy in 5 breast cancer cell lines, we identified ARID1A as a NMD target in the T47D breast cancer cell line. ARID1A encodes a human homologue of yeast SWI1, which contains a DNA-binding motif (AT-rich interactive domain, ARID) and is an integral member of the hSWI/SNF complex, an ATP-dependent chromatin-remodeling multiple-subunit enzyme. Mutations in the ARID1A gene were recently reported to be present in 50% of clear cell tumors of the ovary. We found the ARID1A gene contains a nonsense mutation in exon 9, which introduces a premature stop codon, changing Q944 to a stop codon in the T47D breast cancer cell line. Although we did not find any somatic mutations in breast tumors, we show that low ARID1A RNA expression or low ARID1A nuclear protein expression is associated with more aggressive breast cancer phenotypes in 2 independent cohorts of over 200 breast cancers each. We also found that low or absent ARID1A nuclear expression becomes more prevalent during the later stages of breast tumor progression. Finally, we found that ARID1A re-expression in the breast cancer cell line in which it is mutated results in significant inhibition of colony formation in soft agar. These results suggest that the ARID1A gene may be a candidate tumor suppressor gene in breast cancer, and that it warrants further investigation as potential diagnostic and therapeutic marker in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4023. doi:10.1158/1538-7445.AM2011-4023

Introduction: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and Her2-neu, and accounts for ∼15% of all breast cancer diagnoses. Adjuvant chremotherapy and surgery can be effective in many women, although relapse with chemoresistant tumors that are difficult to treat is common. Importantly, these tumors may disproportionately affect African American women. Here, we present initial integrated analysis of matched normal and tumor whole genome and tumor transcriptome sequencing data from three African American patients with metastatic chemo-resistant TNBC, to better understand the compendium of somatic events occurring in these tumors within this high-risk population. Methods: Next Generation Sequencing was performed using the SOLiD version 4.0 system. Patient matched tumor and germline genomes are sequenced to 300 million mappable mate-pair reads (30X) and analyzed using custom paired analysis tools to uncover somatic alterations. Tumor transcriptomes are sequenced (RNA-seq) to 30 million uniquely aligned reads using Life Technologies Bioscope. For expression analysis, patient RNA-seq data are compared to data generated from ethnicity-matched population-based control hyperplastic breast tissue using EdgeR and DEGseq. Results: On average, we detected >3 million germline variants in these three African American patients. Review of germline results also provides information on BRCA1/2 status. Somatic paired analysis has revealed point mutations, small indels, copy number alterations, and translocations, in known cancer genes, and has revealed potential novel genes as well. Among three patients sequenced thus far, we have uncovered both unique and common genomic and transcriptomic perturbations. In both patients, unique somatic genomic alterations are associated with upregulation of specific signaling pathways. RNA-seq-based expression analysis has revealed a profile dominated by perturbations in cell cycle mitotic checkpoint defects. Furthermore, integrated analysis has provided key insights into the transcriptional consequences of a number of genomic alterations including exon skipping events and the discovery of potential fusion transcripts. Importantly, DNA and RNA changes have been validated by independent methods and technologies. Conclusions: Deep genome and transcriptome profiling of chemo-resistant TNBC has provided insights into events occurring in this difficult to treat cancer. These data are being leveraged to provide an opportunity for informed therapeutic options for intervention of this difficult to treat form of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-267. doi:10.1158/1538-7445.AM2011-LB-267

Patients with advanced prostate cancer are usually treated with androgen withdrawal. Although this therapy is effective at the beginning, nearly all prostate cancers become refractory to it. Approximately 15% of these hormone-refractory prostate cancers contain a genomic amplification at 10q22. Aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving gene. We applied high-resolution array-CGH using the 244k Agilent microarrays to cell lines harboring 10q22 amplification. We identified a common amplified region (CAR) and silenced each of the 26 genes in this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22rv1. Genes with a significant growth reduction in the 10q22 amplified cell line PC-3 but not in the non-amplified 22rv1 cells were selected as putative candidate genes of this amplicon. They were further investigated in vivo by functional assays and in vitro by immunohistochemical analysis of the protein expression in more than 500 human prostate cancers on a tissue microarray (TMA). We could narrow down the CAR to a region of 5.8 Mb. The siRNA screening experiments revealed Vinculin (VCL) as the most promising candidate gene of this amplicon. Immunohistochemical analysis of the Vinculin protein expression on a TMA enriched for 10q22 amplified prostate cancers showed a strong association between VCL gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that Vinculin expression was highest in hormone-refractory prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Notably, high tumor cell proliferation measured by Ki67 expression was significantly associated with high Vinculin expression in prostate cancer (p < 0.0001). Although there are countless reports on Vinculin as a cytoskeletal protein, its role in prostate cancer has previously not been investigated. Our data strongly suggest Vinculin as a major driving gene of the 10q22 amplification in prostate cancer and that Vinculin overexpression might contribute to prostate cancer progression by enhancing tumor cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 250.

Pancreatic cancer is a deadly disease that kills more than 30,000 people in the United States each year. Pancreatic cancer often presents as an advanced, therapy resistant cancer that is refractory to many forms of chemotherapy. Current therapeutics to treat pancreatic cancer include gemcitabine, yet the one-year survival of pancreatic cancer patients treated with gemcitabine is about 18%, representing a significant but modest advancement in the quality of life. In order to improve gemcitabine response in pancreatic cancer cells, we utilized high-throughput RNAi (HT-RNAi) to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine. This screen also identified genes that represent “Achilles Heel” targets, which when silenced, results in a decrease in pancreatic cancer cell viability. A HT-RNAi assay was developed to monitor the growth inhibition of BxPC3 pancreatic cancer cells transfected with siRNA and treated with gemcitabine. For screening, BxPC3 cells were transfected with siRNA from several two siRNA libraries including kinases (574 kinases) and druggable-genome targets (∼7000 genes). Twenty-four hours after siRNA transfection, an EC30 dose of gemcitabine was added and growth was assessed after 72 hours of drug exposure. Results from the screen showed that silencing the TNK1 resulted in a significant decrease in cell viability and also modestly increased sensitivity to gemcitabine. These results were consistent with previous HT-RNAi screens performed on MIA PaCa-2 cells. To confirm TNK1 as a potential target, a panel of pancreatic cancer cells were transfected with one of four different TNK1 siRNA and then treated with a range of gemcitabine doses to evaluate gemcitabine sensitivity following knockdown of TNK1. The TNK1 protein has previously been identified as a putative inhibitor of Ras and an inhibitor of TNFα-induced translocation of NFkB. We were able to confirm this in pancreatic cancer cells by demonstrating an increase in p-MEK 1/2 as well as an increase in NFkB gene targets in TNK1 siRNA-treated cells. Furthermore, we have characterized TNK1 expression in a panel of pancreatic cell lines. Although previous studies suggest that TNK1 may serve as a tumor suppressor, here, we suggest that TNK1 also demonstrates oncogenic properties in pancreatic cancer cells. The role that TNK1 plays in pathways such as K-ras and NFkB indicate it is likely to be an important factor in the development and/or progression of pancreatic cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2204.

Ewing sarcoma family tumors (ESFT) represent ∼ 3% of pediatric cancers and are the second most common bone malignancy in children and adolescents. Rearrangement of the EWS gene on chromosome 22q12 with an ETS gene family member is the underlying molecular genetic abnormality for Ewing's sarcoma. These translocations provide a valuable tool for accurate diagnosis of ESFTs and also represent ideal targets for the development of targeted therapeutics. The most common translocation is t(11;22)(q24;q12), which involves the proteins EWS and Friend Leukemia Integration Site 1 (FLI1). The newly formed EWS-FLI1 fusion protein is a transcription factor that could then lead to aberrant transcription and gives the ES cells a specific tumorigenic context. We employed a functional genomics approach based on high throughput RNA interference (HT-RNAi) analysis to identify “Achilles heel” targets or genes who's silencing affect the proliferation and survival of Ewing's sarcoma cells. We used a human “kinome” library targeting 572 kinases and four ES cell lines, TC-32, TC-71, SK-ES-1 and RD-ES for the HT-RNAi screening. All the screens were done in duplicate and the data was normalized and analyzed using the Z-score method. One gene that was a significant “hit” across all the four cell lines was serine threonine kinase 10 or STK10. Our validation confirmed that reduction in ES cell growth by STK10 siRNA was due to specific STK10 silencing and was not an off target effect. Moreover, silencing of STK10 in ES cells resulted in increased apoptosis. We also showed that normal human fibroblasts did not depend on STK10 for cell growth. Our results have confirmed that STK10, which belongs to the polo like kinase family, is required for the growth of ES cells. After further validation we will use these results for the development of specific drugs targeting STK10 and thereby accelerate the development of improved therapeutics for Ewing's sarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3408.