Salhia, Bodour

Introduction The Florida-California Cancer Research, Education, and Engagement (CaRE 2 ) Health Equity Center is a triad partnership committed to increasing institutional capacity for cancer disparity research, the diversity of the cancer workforce, and community empowerment. This article provides an overview of the structure, process innovations, and initial outcomes from the first 4 years of the CaRE 2 triad partnership. Methods CaRE 2 serves diverse populations in Florida and California using a “molecule to the community and back” model. We prioritize research on the complex intersection of biological, environmental, and social determinants health, working together with scientific and health disparities communities, sharing expertise across institutions, bidirectional training, and community outreach. Partnership progress and outcomes were assessed using mixed methods and four Program Steering Committee meetings. Results Research capacity was increased through development of a Living Repository of 81 cancer model systems from minority patients for novel cancer drug development. CaRE 2 funded 15 scientific projects resulting in 38 publications. Workforce diversity entailed supporting 94 cancer trainees (92 URM) and 34 ESIs (32 URM) who coauthored 313 CaRE 2 -related publications and received 48 grants. Community empowerment was promoted via outreaching to more than 3000 individuals, training 145 community cancer advocates (including 28 Community Scientist Advocates), and publishing 10 community reports. CaRE 2 members and trainees together have published 639 articles, received 61 grants, and 57 awards. Conclusion The CaRE 2 partnership has achieved its initial aims. Infrastructure for translational cancer research was expanded at one partner institution, and cancer disparities research was expanded at the two cancer centers.

Abstract Abstract 3527 Increasing evidence compiled from our lab and several others is illuminating the importance of epigenetic mechanisms in the pathogenesis of multiple myeloma (MM). The biochemical modifications that govern epigenetics are DNA methylation, and post-translational modifications of histone proteins on arginine and lysine residues. Modifications are generally associated with activation or repression of gene transcription depending upon the specific position of the modification. Histone methylation, catalyzed by histone methyltransferases (HMT) and histone demethylases (HDMT) are crucial for the proper programming of the genome during development. Deregulation of the methylation machinery can alter chromatin configuration and disrupt normal transcriptional programs, both features of cancer cells. Misregulated methylation may result from several genetic alterations in chromatin-modifying enzymes, which can include mutations, over-expression or chromosomal abnormalities. Indeed, genomic alterations of MLL, NSD1, CREBBP(CBP) MMSET and UTX, all encoding proteins involved in post-translational histone modifications, have been implicated in MM. The purpose of our study was to interrogate existing genomics datasets to identify additional HMT or HDMT genes that may be important in the pathogenesis of MM and, thus could become potential therapeutic targets. To identify novel HMT or HDMT in MM we mined existing expression, copy number and whole genome sequencing data generated as part of the Multiple Myeloma Research Consortium Genomics Initiative. Collectively, the datasets converged on numerous alterations involving histone methylation on lysine 9 (H3K9). One of these genes, a HDMT called JMJD1C, was over-expressed in approximately 15% of MM samples examined. We validated over-expression of JMJD1C expression by RT-PCR in over 50 clinical MM samples and 10 human myeloma cell lines (HMCLs). We also confirmed over-expression of JMJD1C by immunohistochemistry (IHC) of a tissue microarray (TMA) consisting of over 60 MM samples. Next we examined the levels of JMJD1C expression and H3K9 methylation by IHC, RT-PCR and western blot to determine the extent to which JMJD1C expression is associated with a decrease in H3K9 methylation (or vice versa). We demonstrated this negative correlation in 6/10 HMCL including OCIMY7 and KMS11 and in over 60% of clinical samples examined on the TMA. Furthermore, we demonstrated that H3K9 methylation, a repressive mark associated with heterochromatin, is low in a large percentage of samples. In addition, we generated double knockout isogenic cell line pairs using zinc finger nuclease technology to evaluate the direct role of JMJD1C on H3K9 methylation, cell function and chromatin and expression level changes using ChIP-seq and RNA-seq analyses. In summary our preliminary results demonstrate that JMJD1C is highly expressed in a significant percentage of MM patients. Our results demonstrate a noticeable inverse relationship of JMJD1C expression to H3K9. This reflects the importance of JMJD1C in modifying the H3K9 repressive mark and suggests that loss of repression due to JMJD1C-mediated histone demethylation disrupts the chromatin machinery and leads to neoplastic processes. Further studies are ongoing to realize JMJD1C as a potential and novel therapeutic target in MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 604 We have conducted a pilot study to interrogate both CpG hyper- and hypomethylation in Multiple Myeloma (MM) using the Illumina GoldenGate (GG) BeadArrayTM high-throughput assay. Specifically, we utilized the Illumina Methylation Cancer Panel I, which spans 1,505 CpG loci selected from 807 genes including known tumor suppressor genes and oncogenes. To date we have profiled 50 CD138+ MM specimens, 4 MM cell lines and 6 normal CD138+ plasma cell samples (each representing a pool of 3-5 individuals) obtained from the bone marrow of patients who have undergone hip or knee replacement surgeries. We have obtained highly reproducible and reliable DNA methylation profiles with an average r2 of greater than 0.98 when the β values were compared between technical replicates. Further, we generated an internal control panel for the array by performing methylation specific PCR (MSP) to determine the methylation status of several genes found on the array (CDKN2A, E-Cadherin, MGMT, hMLH1) in 4 MM cell lines. A differential methylation analysis was conducted between normal plasma cells and MM samples using the BeadStudio Methylation Module v3.2. Differential scores for each CpG locus on the array were generated that correspond to significant differences between groups. Scores greater than (hypermethylation) or less than (hypomethylation) 13 represent ‘Diff scores' that are statistically significant (p values < 0.05) and this cut-off threshold is used to determine differentially methylated loci. Globally, when comparing MM to normal plasma cells, hypomethylation occurred at 42% of probes on the array, and was more common than hypermethylation, which occurred at only 21% of probes. Interestingly, 77% of probes situated in non-CpG islands were hypomethylated compared to 26% or probes in CpG islands. On the contrary hypermethylation occurred mainly in CpG islands (26% of probes) whereas only 7.4% of probes in non-CpG islands were considered hypermethylated. A total of 946 CpG loci were differentially methylated between normal plasma cells and MM. Among the most significantly differentially methylated CpG loci between normal plasma cells and MM were FZD9, ALOX12, MMP-3, MMP-8, and p16. Array comparative genomic hybridization (aCGH) data availabel for this same sample set was used to generate a hyperdiploid index for each sample by identifying those samples with whole chromosome gains at minimally three odd numbered chromosomes. There were 751 CpG loci that were differentially methylated between hypderdiploid and non-hyperdiploid tumors with WEE1, FRZB, ELK1, CLDN4, and SHH being among the most significant. We also applied the pathway enrichment analysis tool GeneGo v5 (Agilent) to these data, which identified cell adhesion/ECM remodeling, developmental pathways and several immune response pathways as the most significantly enriched pathways between normal and plasma cells with ephrin signaling pathways in the context of cell adhesion as the most significantly enriched pathway between hyperdiploid and non-hyperdiploid tumors. We hope to integrate methylation data with orthogonal datasets including gene expression, aCGH, and DNA sequence data being developed as part of the MMRF Genomics Initiative. Further validation of hits will also be followed up by functional studies to assess the true biological relevance of differentially methylated genes. This study represents one of the first methylome interrogation studies in MM and points towards global hypomethylation as possibly an important mechanism driving myelomagenesis. Our data also suggest that differential methylation of genes may be a significant determinant of hyperdiploid biology. Determining the set of critical genes and pathways based on the myeloma methylome is likely to lead to an improved understanding of biological mechanisms involved in myelomagenesis, ultimately leading to new and improved methods of diagnosing and treating this disease. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1285 Introduction: Recent studies have now demonstrated that both genic and global hypomethylation characterizes the multiple myeloma (MM) epigenome. We previously conducted a study to measure changes in DNA methylation at approximately 1500 CpG (GoldenGate Methylation BeadArray, Illumina) loci in 193 MM samples used in the Multiple Myeloma Research Consortium Genomics Initiative. The purpose of this study was to determine the extent to which DNA methylation changes in MM correlate to changes at the gene expression level. Methods: To evaluate this correlation we performed three independent analyses. In the first approach we analyzed matching gene expression and DNA methylation data using a Pearson correlation to assess the linear relationship between the datasets. In the second approach methylation and gene expression data from 193 matching samples were quantile-normalized and standardized across genes and samples. Methylation data was then discretized into three groups (methylated, unchanged, unmethylated) according to the mean and standard deviation of the values for each probe on the array. We then computed a t-test to compare the gene expression differences for each gene represented on the methylation array between samples in the methylated and unmethylated groups. We also performed a class enrichment analysis to assess DNA methylation/gene expression correlations that might be associated with hyperdiploidy or molecular subtype. In the third approach we performed RT-PCR and methylation pyrosequencing to validate results in a subset of samples used for the analysis and in an independent cohort comprised of 50 MM samples. Results: By Pearson correlation, only 31 (2.1%) CpG loci (corresponding to 24 unique genes) had a gene expression probe with a statistically significant correlation. Using the discretization approach, we identified 382 loci (25.3%, 309 unique genes) with methylation-expression correlations. Although we identified more correlations by discretizing the data, methylated and unmethylated samples were represented by an average of only 30 and 27 samples per group. We identified correlations where the directionality of methylation and expression values were either in the opposite (negative) or same direction (positive). Among the genes identified, the tumor suppressor genes CDKN2A and DLC1 interestingly demonstrated a positive correlation (methylated and expressed) and the IGF1R and IL17RB genes were negatively correlated (unmethylated and expressed). A class enrichment analysis revealed that samples with DLC1, CDKN2A, IGF1R or IL17RB methylation were associated with hyperdiploid MM. Conversely, samples that were unmethylation for these genes were generally non-hyperdiploid. These findings were validated by RT-PCR and methylation pyrosequencing. Conclusion: Overall, our findings demonstrated, albeit in a limited number of genes, that DNA methylation changes are weakly associated to gene expression. These data suggest that CpG methylation may have other functional consequences such as predisposing the genome to global gene transcriptional changes or chromosomal instability. While future studies are needed to determine the exact role of DNA methylation, we identified a number of genes regulated by an epigenetic mechanism with important clinical and biological implications to MM and warrant further study. Disclosures: No relevant conflicts of interest to declare.

Abstract Background The dearth of relevant tumor models reflecting the heterogeneity of human central nervous system metastasis (CM) has hindered development of novel therapies. Methods We established 39 CM patient-derived xenograft (PDX) models representing the histological spectrum, and performed phenotypic and multi-omic characterization of PDXs and their original patient tumors. PDX clonal evolution was also reconstructed using allele-specific copy number and somatic variants. Results PDXs retained their metastatic potential, with flank-implanted PDXs forming spontaneous metastases in multiple organs, including brain, and CM subsequent to intracardiac injection. PDXs also retained the histological and molecular profiles of the original patient tumors, including retention of genomic aberrations and signaling pathways. Novel modes of clonal evolution involving rapid expansion by a minor clone were identified in 2 PDXs, including CM13, which was highly aggressive in vivo forming multiple spontaneous metastases, including to brain. These PDXs had little molecular resemblance to the patient donor tumor, including reversion to a copy number neutral genome, no shared nonsynonymous mutations, and no correlation by gene expression. Conclusions We generated a diverse and novel repertoire of PDXs that provides a new set of tools to enhance our knowledge of CM biology and improve preclinical testing. Furthermore, our study suggests that minor clone succession may confer tumor aggressiveness and potentiate brain metastasis.

Abstract Despite recent advances in cancer therapy, options for central nervous system metastasis (CM) remain limited and patient outcomes are dismal. The lack of tumor models that reflect the heterogeneity and complexity of human CM tumors is a barrier to understanding the underlying biology and to developing novel therapies. Here, we report the development and characterization of 39 patient derived xenograft (PDX) models of CM tumors with histological subtypes representing the disease spectrum. The PDX models successfully grew both in the flank and brains of mice, and retained their histology regardless of the site of implantation. Brain metastasis also formed in 5/12 models after intra-cardiac injection. Multi-omic analysis of the PDX tumor and the patient-matched CM tumor showed that PDXs largely retained the molecular profiles of the tumor from which they were derived, including the retention of aberrations in key driver genes and signaling pathways. PDXs differed primarily from the ungrafted patient tumors by a downregulation in immunity-related pathways, which was expected due to the use of an immuno-compromised host, arguing for the validity of using the flank as a site of implantation. Integrated analysis of mutations and gene expression changes in CM revealed an upregulation of neuronal signaling and DNA damage response, and downregulation of calcium signaling and inflammation. We also assessed the clonal evolution of the PDX from the original patient CM by tracing variant allele frequencies in copy number neutral regions. We found that the PDX tumors were heterogeneous and comprised of multiple tumor clones that were either unique or in common with the original tumor, and displayed evidence of clonal expansion. Two PDXs that underwent phylogeny assessment showed evidence of minor clone expansion. One such PDX was a tumor from a Her2+ breast cancer (CM13), which largely did not molecularly resemble its matched patient tumor but was confirmed to have some shared expressed variants. The PDX was nearly copy number neutral, which included loss of the Her2 amplicon, and had few mutations compared with its parent tumor. Most noteworthy, is that CM13 displayed unique molecular patterns, aggressive growth and was highly metastatic in vivo. From the flank, it led to multiple metastases, including micrometastasis in the brain. This suggests that minor clone takeover may confer tumor aggressiveness and potentiate brain metastasis. The large and diverse repertoire of PDXs developed and characterized in this study provides a new set of tools that will help deepen our understanding of CM and improve preclinical testing for CM therapies. Our data also show that expansion of minor clones could confer aggressive tumor behavior and may be important in tumor evolution in PDX models and human cancers. Citation Format: Ben Yi Tew, Christophe Legendre, Tim Triche, Gerald C. Gooden, Kyle N. Johnson, Rae Anne Martinez, Emanuel F. Petricoin, Mariaelena Pierobon, Joyce O'Shaughnessy, Cindy Osborne, Mark A. Schroeder, Daniel J. Ma, Mark Bernstein, Jann N. Sarkaria, Steven A. Toms, Bodour Salhia. Development and characterization of patient derived xenografts from central nervous system metastasis reveal minor clone expansion linked with aggressive tumor behavior [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 991.

Abstract Introduction: Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is arguably the most aggressive ovarian cancer. Most patients are diagnosed at an advanced stage, do not respond to chemotherapy, and die of disease within 1-2 years. It affects children and young women and is reported to occur in families. The cause of the disease is poorly understood. Therefore, we used next generation sequencing technology to identify the genetic basis of the disease. Method: We performed whole genome, whole exome sequencing or targeted sequencing on tumours and germline samples from 17 SCCOHT patients and on the SCCOHT cell line BIN-67 and SCCOHT-1. Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tumors from 23 patients and on a tissue microarray of 485 primary ovarian tumours of other subtypes. BIN-67 cells harbouring biallelic inactivation of SMARCA4 were transduced with a lentivirus expressing wild type SMARCA4. Result: We identified inactivating germline and somatic mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (11/14) of SCCOHT patients, 2 of whom bore germline mutations, and in both cell lines. SMARCA4 protein was lost in 87% (20/23) of SCCOHT tumours but in only 0.4% (2/485) of other primary ovarian tumours. Reintroduction of wild-type SMARCA4 into BIN-67 cells resulted in altered cell morphology and growth arrest. Conclusions: The mutation spectrum, IHC profile and cell culture phenotype implicate SMARCA4 as a critical tumour suppressor in SCCOHT pathogenesis. Citation Format: Pilar Ramos, Anthony N. Karnezis, David W. Craig, Aleksandar Sekulic, Megan L. Russell, William P.D. Hendricks, Jason J. Corneveaux, Michael T. Barrett, Karey Shumansky, Yidong Yang, Sohrab P. Shah, Leah M. Prentice, Marco A. Marra, Jeffrey Kiefer, Victoria L. Zismann, Troy A. McEachron, Bodour Salhia, Jaime Prat, Blaise A. Clarke, Joseph G. Pressey, John H. Farley, Stephen P. Anthony, Richard B.S. Roden, Heather E. Cunliffe, David G. Huntsman, Jeffrey M. Trent. Small cell carcinoma of the ovary, hypercalcemic type displays frequent inactivating germline and somatic mutations in SMARCA4 [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1327.

Abstract Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive malignancy that affects children and young women at a mean age of 24 (range 14 months - 58 years). SCCOHT is refractory to standard of care therapy for ovarian cancer, with ∼75% mortality within 18 months of diagnosis. The early age of onset of SCCOHT and reports of familial occurrence in some cases, strongly suggest an underlying hereditary etiology. To understand the molecular pathogenesis of SCCOHT, we performed next-generation genomic sequencing on a series of tumor and germline samples from SCCOHT patients. This analysis revealed germline and somatic inactivating mutations in SMARCA4, a subunit of the SWI/SNF chromatin-remodeling complex, in 75% (9/12) of SCCOHT patients. Moreover, immunohistochemical (IHC) analysis of 15 tumors revealed that 87% (13/15) of tumors lacked SMARCA4 protein. The high prevalence of SMARCA4 mutations in SCCOHT has not been previously reported in other, more common ovarian carcinomas. We therefore examined the expression of SMARCA4 protein in 300 ovarian carcinomas of different histologies by IHC and found SMARCA4 protein loss in only 6 tumors. In addition, the BIN-67 SCCOHT cell line, which harbors 2 splice site mutations in SMARCA4, showed complete absence of SMARCA4 protein by Western blot while representative cell lines from 4 other ovarian carcinoma subtypes as well as immortalized granulosa cells (SVOG) and adult granulosa tumor cells (KGN) all maintained SMARCA4 expression. The prevalence of germline and sporadic SMARCA4 mutations as well as frequent SMARCA4 protein loss in SCCOHTs implicates this gene as a tumor suppressor in this cancer and more broadly suggests a role for the SWI/SNF complex in its pathogenesis. In addition to providing evidence to the pathogenesis of SCCOHT, this finding provides the opportunity to develop treatment approaches for SCCOHT based on targeting vulnerabilities of SMARCA4-deficient cells. Citation Format: Pilar Ramos, Anthony Karnezis, David Craig, Aleksandar Sekulic, Megan Russell, William Hendricks, Michael Barrett, Karey Shumansky, Yidong Yang, Sohrab Shah, Leah Prentice, Marco Marra, Jeffrey Kiefer, Victoria Zismann, Troy McEachron, Bodour Salhia, Joseph Pressey, John Farley, Stephen Anthony, Richard Roden, Heather Cunliffe, David Huntsman, Jeffrey Trent. The rare, highly malignant small cell carcinoma of the ovary displays common inactivating germline and somatic mutations in the tumor suppressor SMARCA4. [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 LB-202. doi:10.1158/1538-7445.AM2014-LB-202

Abstract Increasing evidence points towards the importance of epigenetic changes in the pathogenesis of multiple myeloma (MM). The biochemical modifications that govern epigenetics are DNA methylation, and post-translational modifications of histone proteins. Histone methylation is catalyzed by histone methyltransferases (HMT) and histone demethylases (HDMT). The purpose of our study was to interrogate genomics data to identify HMT or HDMT genes that are altered in MM. We mined copy number, gene expression and whole genome sequencing data generated as part of the Multiple Myeloma Research Consortium Genomics Initiative. Collectively, the datasets converged on numerous alterations involving histone methylation of lysine 9 (H3K9). One of these genes, the HDMT JMJD1C, was over-expressed in approximately 15% of MM samples examined. We validated over-expression of JMJD1C expression by RT-PCR in over 50 clinical MM samples and 10 human myeloma cell lines (HMCLs). We also confirmed over-expression of JMJD1C by immunohistochemistry (IHC) in a tissue microarray (TMA) consisting of over 60 MM samples. Next we demonstrated a negative correlation between JMJD1C expression and H3K9 methylation by IHC and western blot in 6/10 HMCL and in over 60% of clinical samples examined on the TMA. This validates the H3K9 demethylase activity of JMJD1C. Furthermore, we demonstrated that, in general, H3K9 methylation, a repressive mark associated with heterochromatin, is low in a large percentage of samples. To study the functional significance, we generated a double knockout isogenic cell line pair in U266 cells using zinc finger nuclease technology. We performed ChIP-seq and RNA-seq analyses to identify genes affected by JMJD1C binding to H3K9. Our data indicate that loss of JMJD1C expression alleviates the repression conferred by H3K9 methylation in MM. Studies are ongoing to realize JMJD1C as a potential and novel therapeutic target in MM. Citation Format: Janine LoBello, Danielle DiPerna, Moly Gale, Aprill Watanabe, Galen Hostetter, Rafael Fonseca, Bodour Salhia. Regulation of H3K9 methylation by the demethyltransferase gene JMJD1C in multiple myeloma. [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 4243. doi:10.1158/1538-7445.AM2013-4243

Abstract Glioblastoma multiforme (GBM) is the most malignant primary adult brain tumor. Despite efforts at surgical resection of the glioma mass, invasive cells are always left behind and the tumor will inevitably recur and kill the patient. As such, novel therapeutics targeting pro-invasive factors could improve neurological outcomes and survival for these patients. This requires a detailed understanding of the mechanisms driving glioma migration and invasion. We have been investigating whether the multifunctional cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) regulate glioma cell invasive activity. We have previously demonstrated that Fn14 expression was elevated in the invasive rim of GBM specimens and migrating glioma cells in vitro. The Fn14 signaling axis is known to drive glioma invasion via Rac1. We have previously reported that Ect2, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, including Rac1 and Cdc42, is overexpressed in GBM, and that overexpression of Ect2 correlates directly with tumor grade and inversely with patient survival. In this study we show that Ect2 regulates Rac1 activation downstream of Fn14. Depletion of Ect2 by siRNA duplexes abrogates Fn14-induced Rac1 activation and subsequent glioma cell migration and invasion. We also found that Cdc42 activation by TWEAK is directly mediated by Ect2. Interestingly, depletion of Cdc42 expression impairs TWEAK-induced Rac1 activation and also results in a significant reduction of glioma cell migration and invasion. This suggests that Cdc42 is, in part, important for Rac1 activation downstream of the TWEAK-Fn14 signaling pathway and argues that another GEF(s) may be involved in the Fn14-Rac1 signaling axis. Recently, we have identified Trio as an additional GEF that activates Rac1 downstream of Cdc42. It is also known that Trio expression correlates directly with brain tumor grade and inversely with patient survival. siRNA-mediated depletion of Trio inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Therefore, delineating the mechanisms of Fn14-RhoGEF-RhoGTPase signaling pathway, may lead to identification of novel targets that can serve as possible points of therapeutic intervention. (Supported by NIH R01-CA130940) 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 5128.