Citations

Abstract Abstract 2462 Poster Board II-439 The eukaryotic translation initiation factor 5A (eIF5A) is highly conserved and the only known protein to contain the amino acid hypusine. Hypusinated eIF5A and deoxyhypusine synthase, one of the enzymes mediating eIF5A post-translational hypusination, have been identified as markers of neoplastic growth and metastasis, respectively. However, recent studies have indicated that, in its unhypusinated form, eIF5A is pro-apoptotic and thus functionally distinct from hypusine-modified eIF5A. SNS01 is a cancer gene therapy biologic targeted to the treatment of multiple myeloma. SNS01 is comprised of three components: a DNA vector containing a B-cell-specific (B29) promoter driving expression of a pro-apoptotic mutant of eIF5A (eIF5AK50R) that cannot be hypusinated; an siRNA that targets the native hypusinated eIF5A that promotes growth of cancer cells; and a synthetic polymer called polyethylenimine (PEI). RT-qPCR experiments demonstrated that treatment of the human myeloma cell line, KAS-6/1, with SNS01 results in a 90 % reduction in the expression of eIF5A mRNA (mediated by the siRNA) as well as the accumulation of ∼ 22,000 copies of the plasmid-derived eIF5AK50R mRNA per ng of total RNA. SNS01 was found to induce apoptosis in both IL-6-dependent (KAS-6/1) and IL-6-independent (U266) myeloma cell lines and was found to be more effective at inducing apoptosis than PEI nanoparticles carrying either the eIF5A siRNA or the eIF5AK50R plasmid alone. Apoptosis assays were used to optimize the ratio of DNA:siRNA in the nanoparticles and a ratio of 2:1 was found to be optimal for the induction of apoptosis in myeloma cells. The anti-tumoral activity of SNS01 was assessed in SCID mice bearing subcutaneous human multiple myeloma (KAS-6/1) tumors using twice-weekly intra-venous injections and a dose range between 0.15 mg/kg and 1.5 mg/kg. Control mice treated with PEI nanoparticles containing a non-expressing plasmid and a non-targeting siRNA had an average tumor volume of 284 mm3 at the time of sacrifice, whereas mice treated with 1.5 mg/kg or 0.75 mg/kg SNS01 exhibited significant tumor regression and had average tumor volumes of 13 mm3 (95 % inhibition; *p = 0.026) and 24.5 mm3 (91 % inhibition; *p = 0.03), respectively. In addition, for mice treated with 1.5 mg/kg SNS01, there was no evidence of tumors under the skin. Lower doses of SNS01 also demonstrated anti-tumoral activity with mice treated with 0.38 mg/kg and 0.15 mg/kg exhibiting a 73 % and 61 % inhibition of tumor growth, respectively. TUNEL-labeling revealed evidence of apoptotic cells in the treated tumors indicating that tumor regression occurs through the induction of apoptosis. Twice weekly dosing of SNS01 was found to be critical for effective tumor control. In a separate study, twice weekly dosing of SNS01 at 1.5 mg/kg was found to inhibit tumor growth by 88 % (p = 0.01) while once weekly dosing did not significantly reduce tumor burden. Since SNS01 is intended as a therapy for multiple myeloma, a bone marrow neoplasm, the biodistribution of PEI nanoparticles containing a GFP plasmid with the B-cell-specific B29 promoter and a fluorescently (DY547)-labeled siRNA was assessed following i.v. administration in Balb/c mice. The fluorescent siRNA was observed in the liver, lung, spleen, and bone marrow, while GFP expression was primarily observed in the bone marrow. In summary, our preclinical data indicate that systemic administration of SNS01 is an effective anti-cancer therapy in an animal model of multiple myeloma. Additionally, since PEI nanoparticles are taken up by cells of the bone marrow following systemic delivery, SNS01 may be an effective treatment option for multiple myeloma patients and is currently being evaluated for use in clinical trials. Disclosures: Taylor: Senesco Technologies Inc.: Patents & Royalties, stock options. Lust:Senesco Technologies Inc.: Research Funding. Dondero:Senesco Technologies Inc.: Employment, Equity Ownership, Patents & Royalties. Galton:Senesco Technologies Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Donovan:Senesco Technologies Inc.: Research Funding. Thompson:Senesco Technologies Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Abstract Abstract 591 Background: Survival of patients with MM has improved over the past two decades, in part due to the use of AHCT. Increasingly, second primary malignancies (SPMs) are observed in MM survivors. Determining the baseline incidence and risk factors associated with SPMs after AHCT is important to assess risk and to evaluate the risk-benefit ratio of newer therapies. Methods: We analyzed the incidence of SPMs in 3784 MM patients receiving (“upfront”) AHCT for MM within 18 months of diagnosis between 1990 and 2010 and reported to the CIBMTR. Cumulative incidence rates of SPMs were estimated taking into account the competing risk of death. For each transplant recipient, the number of person-years at risk was calculated from the date of transplantation until date of last contact, death, or diagnosis of SPM, whichever occurred first. Incidence rates for all invasive cancers in the general population were obtained from the SEER database. Age-, sex-, and race- specific incidence rates for overall SPMs and particular anatomical sites were applied to the appropriate person-years at risk to compute the expected numbers of cancers. Observed–to –expected (O/E) ratios were calculated, and Poisson distribution 99% confidence intervals (CIs) were generated. Poisson regression model was used to analyze risk factors for overall SPMs and AML/MDS. Results: Pre-transplant therapy included novel agents in 56% including thalidomide (35%), lenalidomide (9%), bortezomib (16%) or their combinations (11%). Majority (80%) received high dose melphalan conditioning. Post-transplant maintenance therapy included thalidomide (16%), lenalidomide (8%), bortezomib (9%) and interferon (6%). Median follow-up of survivors was 52 months (range 3 to 192 months).With 12707 person years of follow up, 153 new malignancies were reported with a crude rate of 1.2 SPM per 100 person years of follow up. Observed/Expected [O/E] ratio for all SPMs was 0.99 (99% CI, 0.80–1.22). Cumulative incidence of SPM overall was 2.48% (95% CI, 1.96–3.05) at 3 years and 6.0% (95% CI, 4.96–7.10) at 7 years [Figure 1]. Individual SPMs observed significantly more frequently than expected are summarized in Table 1. The cumulative incidence of MDS/AML was 0.5% (95% CI, 0.28–0.78) at 3 years and 1.3 (95% CI, 0.85– 1.9%) at 7 years. Majority had MM progression prior to diagnosis of SPM (65 of 102 patients overall and 15 of 23 patients for MDS/AML). In multivariate analysis, significant risk factors for development of SPMs included: obesity [Hazard ratio = HR 1.89(95%CI, 1.21–2.93), p=0.0047 for BMI>30 vs. BMI<25], older age: [HR10.53 (95%CI, 1.46–75.82), p=0.0195] for 60–69 year olds and HR14.4 (95%CI, 1.89–109.75), p=0.01 for 70+ year olds compared to the 18–39 year old group. Specific conditioning regimens did not correlate with the risk of SPM. The low number of MDS/AML (33 events out of 3784 cases) limited the power of multivariate analysis. Increasing age was significantly associated with development of MDS (HR10.77, (95%CI,92.09–55.51), p=0.004 for 70+ year old vs. 40–49 year olds). Conclusion: In this large cohort of AHCT recipients for MM, the incidence of MDS/AML, melanoma and other skin cancers was significantly higher compared to age and sex matched general population. However the overall risk of SPM was similar to that expected for age and sex matched population. It was also similar to the placebo arms of recent reports by McCarthy Pl et al and Attal M et al (N Engl J Med. 10; 366(19):1770–91). Lenalidomide (8%) or thalidomide maintenance (16%) used in a small subset of patients with comparatively short follow up, was not associated with risk of SPM in the analysis of the overall cohort. Disclosures: Gale: Celgene: Employment. Brandenburg:Celgene: Employment, Equity Ownership. Lonial:Millennium, Celgene, Novartis, BMS, Onyx, Merck all Consultancy. Krishnan:Celgene and Millennium: Consultancy, Speakers Bureau. Dispenzieri:Celgene and Millennium: Research Funding. Hari:Celgene: Consultancy, Honoraria.

Abstract Abstract 596 Background Large population-based studies have demonstrated a significant improvement in survival (OS) for patients (pts) with multiple myeloma (MM) over time since the mid-1990s. The improvement has mostly affected younger patients suggesting a beneficial impact of AHCT. The availability of novel drugs within the last decade and their impact on the utilization and outcomes of AHCT recipients is unknown. Methods We retrospectively reviewed the characteristics and outcomes of 20,278 patients undergoing AHCT within 12 months of diagnosis in USA and Canada and reported to CIBMTR in the periods of 1995–1999 (n=2226), 2000–2004 (n=6408) and 2005–2010 (n=11644) including a subgroup of 4373 individuals with detailed patient, disease and treatment information. We subsequently compared survival and analyzed patient, disease and treatment characteristics across the three periods with the time period serving as a surrogate for pre- and post-transplant exposure to novel agents. Results Across the 3 time periods, there was a marked increase in the number of upfront transplants across all age groups (Figure 1a). Patients receiving upfront HSCT in the more recent period were older, less likely to have stage 3 MM at diagnosis and more likely to have received thalidomide (<1 % vs. 22% vs. 52%), lenalidomide (0% vs. <1% vs. 21%) or bortezomib (0% vs. 2% vs. 35%) and have chemosensitive disease at the time of AHCT. The proportion of African-American patients remained low and stable across the three periods (14 vs. 13 vs. 15%). Over the 3 periods, there was a decrease in chemotherapy based stem cell mobilization and increase of single agent G-CSF based mobilization (24% vs. 24% vs. 41%). The use of single agent Melphalan conditioning increased from 54% in 1995–1999 to nearly 100% in the latest period concomitant with near disappearance of total body irradiation-based and multi-drug conditioning regimens. Compared with 1995–99 cohort, survival was superior in the 2000–04 and 2005–09 cohorts at 24 (72% vs. 79% vs. 84%, P<0.001), 48 (54% vs. 60% vs. 64%, P<0.001) and 60 months (47% vs. 52% vs. 55%, P<0.001) (Figure 1b). In multivariate analysis, we found that transplant in the 2000–04 period (HR=0.77, 95% C.I. 0.67–0.89) or in the 2005–10 period (HR=0.68, 95% C.I. 0.59–0.79) along with Karnofsky performance score of 80–100 (HR=0.59, 95% CI 0.51–0.70) and chemosensitivity (HR= 0.76, 95% C.I. 0.66–0.87) were associated with lower risk of death. Factors associated with higher risk of death were age 50–65 (HR=1.29 95% 1.13–1.47), age 65–80 (HR=1.45, 1.23–1.72), Durie-Salmon or ISS stage III at diagnosis (HR 1.56, 95% C.I. 1.39–1.76) and more than one prior line of therapy (HR=1.15, 95% C.I. 1.03–1.28). In comparison to patients transplanted in 1995–99, post relapse survival was superior for patients transplanted in 2000–04 and 2005–10 at 24 months (58% vs. 65% vs. 72%, P=0.005 and <0.001 respectively) and 48 months (33% vs. 39% vs. 40%, P=0.023 and 0.033 respectively). The use of maintenance therapy has changed with 27% in 1995–99 receiving planned interferon vs. 23% in 2005–09 receiving planned thalidomide, lenalidomide or bortezomib maintenance. Conclusions Utilization of upfront AHCT is increasing in the USA, particularly in older patients. Improvement in survival for MM patients seen in population studies results not only from greater utilization of AHCT but also from the improvement in outcomes after AHCT and a significant recent improvement in post-relapse survival. Pre- and post-transplant exposure to novel agents likely contributes to survival. AHCT and novel agents are complementary and should be combined for the optimal management of newly diagnosed MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples and are hampered by low chromatin yield in FFPE samples due to the lack of reliable and efficient chromatin preparation methods. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays. Results: During rehydration of FFPE tissues, applying tissue-level cross-link reversal to the deparaffinized tissue at 65°C dramatically increased chromatin yield in the soluble fraction. We found the resulting chromatin to be compatible with ChIP-qPCR and ChIP-seq analysis of histone marks (H3K4me3, H3K27me3, and H3K27Ac) and RNA polymerase II. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37°C, the majority of soluble chromatin was over 1 kb in size. The soluble chromatin prepared at 45-60°C showed a typical nucleosomal pattern shown in cell lines and frozen tissues. The majority of the chromatin prepared at 65°C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays. Conclusions: Our method achieves efficient extraction of high-quality chromatin from FFPE samples suitable for targeted and genome-wide epigenetic assays while minimizing degradation and damages. This approach provides a new platform for developing chromatin-based assays applicable to archived FFPE samples to circumvent the over-fixed nature of FFPE tissues. This work was supported, in part, by National Institutes of Health grant P01DK068055 and the Mayo Clinic Center for Individualized Medicine Epigenomics Program. Citation Format: Jian Zhong, Zhenqing Ye, Chad Clark, Samuel Lenz, Justin Nguyen, Huihuang Yan, Keith Robertson, Gianrico Farrugia, Zhiguo Zhang, Tamas Ordog, Jeong-Heon Lee. Enhanced and controlled chromatin extraction for chromatin-based epigenetic assays in FFPE tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5180.

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in United States with a 5-year survival rate of only 8%. Inherited predisposition plays an important role in PDAC risk. Rare, moderately to highly penetrant mutations in hereditary cancer and pancreatitis genes, identified in families with a high incidence of disease, account for a small fraction of cases. At the other end of the spectrum, common risk variants of low penetrance have been discovered through genome-wide association studies (GWAS). Currently, over 20 common PDAC risk signals have been identified through GWAS in populations of European ancestry. However, these loci explain only a small fraction of genetic heritability for PDAC, and the causal genes for the large majority of these are unknown. Most susceptibility alleles discovered through GWAS are located in noncoding regions of the genome and are likely to function through allele specific regulation of gene expression. Transcriptome-wide association studies integrate expression quantitative trait loci (eQTL) with GWAS data to discover genes whose cis-regulated expression is associated with complex traits. To identify novel susceptibility loci and likely causal genes, we applied this approach to PDAC. Firstly, we used two reference datasets generated from histologically normal pancreatic tissue samples from individuals of European ancestry, the DCEG/LTG (n=95) and Genotype-Tissue Expression (GTEx v7, n=174), to build robust predictors for gene expression (n=2,872-5,906; r2&gt;0.01), using 5 different models (Top1, Lasso, Enet, Blup and Bslmm) in FUSION and PrediXcan. Gene expression predictors (n=2,043-21,422) were also generated by Enet in 48 different tissues (n=74-421 samples) from 608 individuals of European ancestry (GTEx). We then applied these predictor models to PDAC GWAS summary statistics from 9,040 PC cases and 12,496 controls. We discovered 24 genes whose genetically predicted expression is significantly associated with PDAC risk (FDR &lt; 0.05), including 13 candidate genes at 10 novel loci (CELA3B, SMC2, SMC2-AS1, RP11-80H5.9, SMUG1, BTBD6, RCCD1, PNMT, CDK12, PGAP3, SUPT4H1, RP11-888D10.3 and PGPEP1) and 11 at 5 known risk loci (TERT, CLPTM1L, ZDHHC11B, INHBA, ABO, PDX1, KLF5, WDR59, CFDP1, BCAR1 and TMEM170A). Pathway enrichment analysis for genes differentially expressed in the top vs. bottom quartile of CELA3B expression highlighted higher expression of inflammatory and immune response genes in both the DCEG/LTG and GTEx pancreatic transcriptome datasets, indicating that low expression of this gene may be associated with an inflammatory state in the pancreas. Our findings showcase the utility of integrating multiple datasets to generate hypotheses and provide novel clues for the genetic mechanisms underlying pancreatic cancer risk. Citation Format: Jun Zhong, Ashley Jermusyk, Lang Wu, Jason W. Hoskins, Irene Collins, Mingfeng Zhang, Song Lei, Charles C. Chung, Tongwu Zhang, Wenming Xiao, Rachael Stolzenberg-Solomon, Alison P. Klein, Brian M. Wolpin, Xiao-Ou Shu, Stephen J. Chanock, Sara Olson, Nilanjan Chatterjee, Jill Smith, Jianxin Shi, Peter Kraft, Gloria M. Petersen, Wei Zheng, Laufey T. Amundadottir. Large-scale transcriptome-wide association study (TWAS) identifies novel candidate susceptibility genes for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1591.

AbstractAlterations in global DNA methylation patterns are a major hallmark of cancer and represent attractive biomarkers for personalized risk stratification. Chronic lymphocytic leukemia (CLL) risk stratification studies typically focus on time to first treatment (TTFT), time to progression (TTP) after treatment, and overall survival (OS). Whereas TTFT risk stratification remains similar over time, TTP and OS have changed dramatically with the introduction of targeted therapies, such as the Bruton tyrosine kinase inhibitor ibrutinib. We have shown that genome-wide DNA methylation patterns in CLL are strongly associated with phenotypic differentiation and patient outcomes. Here, we developed a novel assay, termed methylation-iPLEX (Me-iPLEX), for high-throughput quantification of targeted panels of single cytosine guanine dinucleotides from multiple independent loci. Me-iPLEX was used to classify CLL samples into 1 of 3 known epigenetic subtypes (epitypes). We examined the impact of epitype in 1286 CLL patients from 4 independent cohorts representing a comprehensive view of CLL disease course and therapies. We found that epitype significantly predicted TTFT and OS among newly diagnosed CLL patients. Additionally, epitype predicted TTP and OS with 2 common CLL therapies: chemoimmunotherapy and ibrutinib. Epitype retained significance after stratifying by biologically related biomarkers, immunoglobulin heavy chain mutational status, and ZAP70 expression, as well as other common prognostic markers. Furthermore, among several biological traits enriched between epitypes, we found highly biased immunogenetic features, including IGLV3-21 usage in the poorly characterized intermediate-programmed CLL epitype. In summary, Me-iPLEX is an elegant method to assess epigenetic signatures, including robust classification of CLL epitypes that independently stratify patient risk at diagnosis and time of treatment.

Marrow adipocytes reprogrammed by myeloma cells induce bone lesions.

Abstract Depression is a seriously disabling psychiatric disorder with a significant burden of disease. Metabolic abnormalities have been widely reported in depressed patients and animal models. However, there are few systematic efforts that integrate meaningful biological insights from these studies. Herein, available metabolic knowledge in the context of depression was integrated to provide a systematic and panoramic view of metabolic characterization. After screening more than 10 000 citations from five electronic literature databases and five metabolomics databases, we manually curated 5675 metabolite entries from 464 studies, including human, rat, mouse and non-human primate, to develop a new metabolite-disease association database, called MENDA (http://menda.cqmu.edu.cn:8080/index.php). The standardized data extraction process was used for data collection, a multi-faceted annotation scheme was developed, and a user-friendly search engine and web interface were integrated for database access. To facilitate data analysis and interpretation based on MENDA, we also proposed a systematic analytical framework, including data integration and biological function analysis. Case studies were provided that identified the consistently altered metabolites using the vote-counting method, and that captured the underlying molecular mechanism using pathway and network analyses. Collectively, we provided a comprehensive curation of metabolic characterization in depression. Our model of a specific psychiatry disorder may be replicated to study other complex diseases.

Abstract The oncolytic reovirus (RV) has demonstrated clinical efficacy and minimal toxicity in a variety of cancers, including multiple myeloma (MM). MM is a malignancy of plasma cells that is considered treatable but incurable because of the 90% relapse rate that is primarily from drug resistance. The systemic nature of MM and the antitumor immunosuppression by its tumor microenvironment presents an ongoing therapeutic challenge. In the present study, we demonstrate that RV synergizes with the standard-of-care MM drug bortezomib (BTZ) and, importantly, enhances its therapeutic potential in therapy-resistant human MM cell lines in vitro. Using the syngeneic Vk*MYC BTZ-resistant immunocompetent transplantable MM murine model, we also demonstrate that mice harboring BTZ-insensitive MM tumors respond to the RV/BTZ combination treatment in terms of decreased tumor burden and improved overall survival (P &lt; .00001). We demonstrate that BTZ augments RV replication in tumor-associated endothelial cells and myeloma cells, leading to enhanced viral delivery and thereby stimulating cytokine release, immune activity, apoptosis, and reduction of the MM-associated immune suppression. We conclude that combined RV/BTZ is an attractive therapeutic strategy with no safety signals for the treatment of MM.