LoBello, Janine

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 Background: Whole genome/exome/RNA sequencing has revolutionized the ability to assess the genomic landscape of cancer and is increasingly being utilized for clinical decision-making. Initial clinical applications have been constrained by specimen quantity, analyte quality and the time from sample acquisition to results report. Methods: Patients with advanced cancers underwent surgical resection, excisional/core biopsies, or bone marrow biopsy. Samples were analyzed by whole genome or exome sequencing in addition to RNA sequencing, bioinformatics analysis, and therapeutic target prioritization by a multi-disciplinary Clinical Genomics Board. All prioritized targets were CLIA validated using Sanger sequencing, RT-qPCR, FISH, or IHC as appropriate. Treatment was delivered using off-label FDA approved drugs, clinical trials, or single patient INDs. Results: We have enrolled 40 patients for whom sequencing data is available on 33. The initial 6 patients were evaluated in a non-CLIA pilot phase and 27 in a CLIA-enabled phase. Tumor types in the CLIA-enabled phase with the highest enrollment were pancreatic cancer (n=8) and cholangiocarcinoma (n=8). We sought to quantify the targets identified along with clinical benefit, defining these as the “Actionable Index” (AI) (proportion of patients with ≥ 1 putative drug target) and “Utility Index” (UI) (proportion of patients who derive clinical benefit). Putative therapeutic targets were identified in 7/8 (AI=0.88) cholangiocarcinoma (CC) patients and in 5/8 (AI=0.63) pancreatic cancer (PC) patients. All 3 CC patients who received target directed treatment achieved a partial response (UI=0.38). In contrast, none of the 4 PC patients who received target directed therapy had treatment response (UI=0.0). Interestingly no actionable targets were identified in 1 CC and in 2 PCs. One CC with an identified target was unable to access the drug and subsequently died. A CC patient and a PC patient, each with identified targets, expired prior to the initiation of therapy. Conclusions: While whole genome/exome/RNA sequencing is providing unparalleled detail of tumor genomes, the application to the clinic must be carefully considered. Actionability of targets will eventually need to be defined in close relation to eventual clinical utility and appropriate refinements to disease-gene-drug databases implemented. Preliminary observations in pancreatic cancer and cholangiocarcinoma demonstrate disparity in correlation between utility indices and actionable indices. Application of these tools in larger cohorts and types of tumors will need to be conducted to ascertain more precise estimates. Additional measures that are organ-site agnostic but pertain to specific targets (e.g. BRAF) will also need to be developed in order to facilitate more judicious application of sequencing in the clinical setting. Citation Format: Jan B. Egan, Alan H. Bryce, Mia D. Champion, Winnie S. Liang, Rafael Fonseca, Ann E. McCullough, Michael T. Barrett, Katherine Hunt, Rachel M. Condjella, Robert R. McWilliams, Stephen D. Mastrian, Janine LoBello, Daniel Von Hoff, David W. Craig, A. Keith Stewart, John D. Carpten, Mitesh J. Borad. Indices of actionability and clinical utility in a CLIA-enabled study of whole genome/exome/RNA sequencing in 33 cancer patients: Actionable vs. utility. [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 4694. doi:10.1158/1538-7445.AM2014-4694

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