bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3538: Analysis of error profiles in deep next-generation sequencing data
Authors: Ma Xiaotu , Zhang Jinghui
Published: August 2019

Background

Sequencing errors are a key confounding factor for detecting low frequency genetic variants for cancer screening, testing, treatment and surveillance through deep next-generation sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, enrichment PCR, and sequencing. In this study we systematically investigated the above question by using the current NGS technology.

Results

We discovered that the substitution error rate can be computationally suppressed to 10-5~10-4, which is 10~100-fold lower than current reports. We then quantified substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing using multiple deep sequencing datasets from multiple sequencing centers. We show that 1) error rate differs by nucleotide substitution types, ranging from 10-5 for A>C/T>G, C>A/G>T, and C>G/G>C changes to 10-4 for A>G/T>C changes; 2) C>T/G>A errors exhibit strong sequence context dependency; 3) sample-specific effects dominate elevated C>A/G>T errors; 4) target enrichment PCR lead to ~6-fold increase of overall error rate; 5) more than 70% of hotspot variants, such as BRAF V600E, can be detected at 0.1%~0.01% frequency with the current NGS technology by applying in-silico error suppression.

Conclusions

We present the first comprehensive analysis of error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS accuracy both experimentally and computationally which will lead to enhanced precision for deep sequencing.

Citation Format: Xiaotu Ma, Jinghui Zhang. Analysis of error profiles in deep next-generation sequencing data [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 3538.
Abstract 3542: Turbolase: A radically streamlined high throughput sample prep to sequencing workflow
Authors: Masser Dustin , Stedtfeld Robert , Lenhart Justin , Makarov Vladimir , Kurihara Laurie
Published: August 2019

Normalase is a novel enzymatic library normalization method that eliminates library quantification and manual concentration adjustment of each sample prior to library pooling, and the uniform sample processing eliminates numerous error-prone pipetting steps. When combined with Swift 2S Turbo rapid library kits, a highly streamlined ‘Turbolase’ workflow is created that is readily automated on the Hamilton Star and other platforms, where simple bulk processing improves throughput and reduces cost for NGS laboratories. Swift 2S Turbo kits comprise two enzymatic steps and a single purification that completes DNA fragmentation, end repair and adapter ligation. This is followed by standard library amplification using Normalase PCR primers to condition the libraries and produce a required minimum yield in excess of the 4 nM final concentration. This is followed by a single purification and two 15-minute Normalase incubation steps that; 1) enzymatically select 4 nM of each library, and 2) enzymatically normalize each library to 4 nM within a single pool. The pools can then be directly sequenced without further purification. Normalase allows for greater than 10-fold variation in input quantity, while generating less than 10% variation in sample representation within a pool resulting in optimal cluster density and sample balance for Illumina sequencing. Because of the unique workflow, Normalase also eliminates index hopping for libraries sequenced on patterned flow cells. The ‘Turbolase’ workflow is compatible with full-length indexed adapters that have been added by ligation as well as workflows that require indexing PCR primers to complete library construction during library amplification. Normalase is also compatible with library preparation kits available from other vendors. Normalase for pooling of libraries for pre-hybridization capture is currently under development, which will make multiplexed hybridization capture protocols streamlined and robust from library generation to sequencing.

Citation Format: Dustin Masser, Robert Stedtfeld, Justin Lenhart, Vladimir Makarov, Laurie Kurihara. Turbolase: A radically streamlined high throughput sample prep to sequencing workflow [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 3542.
Abstract 3543: Clinical whole genome sequencing at scale
Authors: MacBeth Alyssa , Bowers Tera , Woolf Betty , Harden Maegan , Lennon Niall , Gabriel Stacey
Published: August 2019

Whole genome sequencing (WGS) offers the greatest potential to comprehensively and accurately identify all forms of human genetic variation. The accessibility of WGS data for diagnostic use has increased due to cost reductions in recent years, driving the need for a high quality, clinically validated offering. Our platform has completed an extensive benchmarking study of the Illumina PCR-free whole genome pipeline to establish clinical validity for the end-to-end laboratory, analytical, and computational processes. Our benchmarking study is comprehensive in nature, including measuring the precision, robustness, limits of detection for variant classes, and contamination estimates. A cohort of well-characterized reference controls and clinical samples with previously identified pathogenic variants were used to establish the performance characteristics of our clinical WGS test, which at 30X coverage has >99% analytical sensitivity for SNVs and >98% analytical sensitivity for small insertions and deletions. Our platform is capable of operating at a scale that supports applications from individual clinics (cancer and medical genetics related applications) as well as large scale ambitious collaborative projects such as the All Of Us program which aims to generate clinical grade whole genome variant calls for 1 million healthy research participants.

Citation Format: Alyssa MacBeth, Tera Bowers, Betty Woolf, Maegan Harden, Niall Lennon, Stacey Gabriel. Clinical whole genome sequencing at scale [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 3543.
Abstract 3544: High performance multiplexed targeted enrichment sequencing from FFPE tissues
Authors: Consugar Mark , Arbiza Leonardo , Butcher Kristin , Chen Siyuan , Chilton Hutson , Gantt Richard , Hasin-Brumshtein Yehudit , Laugharn Jim , Simon Jayne , Thomann Ulrich , Thompson Christina , Zeitoun Ramsey
Published: August 2019

Library construction for Next-Generation Sequencing (NGS) using formalin-fixed paraffin-embedded (FFPE) samples offers unique challenges in acquiring high-quality sequencing data due to wide distribution of sample quality. Specifically, differences in formalin fixation methods lead to crosslinked and/or degraded nucleic acid and inconsistent extraction yields. Hence, FFPE extraction and library construction methods must be carefully considered for target enrichment applications. In collaboration, Covaris and Twist Bioscience demonstrate a complete library preparation and target enrichment solution that generates ready-to-sequence multiplexed libraries directly from FFPE tissue.This workflow leverages the Covaris truXTRAC FFPE total Nucleic Acid Plus Kit and oneTUBE-10 shearing with the world-class performance of Twist Bioscience’s Target Enrichment Solutions. Covaris, the Gold Standard for DNA shearing in NGS, also offers pre-analytical products that leverage Adaptive Focused Acoustics® (AFA)® technology. In this FFPE-specific application, the Covaris truXTRAC FFPE total Nucleic Acid Plus Kit and oneTUBE-10 shearing enable full emulsification of paraffin and disaggregation of tissue for highly efficient nucleic acid extraction and generation of size specific DNA libraries. With the Twist Bioscience Human Core Exome kit, the resulting libraries are indexed, pooled, and target enriched with uniquely optimized DNA probes to generate ready-to-sequence high quality multiplexed libraries.Using the aforementioned workflow, results from processing numerous FFPE tissue types are presented. Sequencing results demonstrate large improvements in general Picard metrics that include uniformity (Fold_80 < 1.8), sequencing depth (20X coverage >85% with 100X downsampling), and duplication rates (<6%) when compared to similar published studies. These results demonstrate a validated solution for library preparation and targeted exome sequencing of FFPE samples that can be integrated into automated workflows. The truXTRAC kit and AFA® technology from Covaris generate size specific DNA libraries from FFPE samples which, when paired with Twist Bioscience’s superior target enrichment workflow, deliver multiplexed libraries for high performance targeted sequencing.

Citation Format: Mark Consugar, Leonardo Arbiza, Kristin Butcher, Siyuan Chen, Hutson Chilton, Richard Gantt, Yehudit Hasin-Brumshtein, Jim Laugharn, Jayne Simon, Ulrich Thomann, Christina Thompson, Ramsey Zeitoun. High performance multiplexed targeted enrichment sequencing from 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 3544.
Abstract 3545: High expression of miR-30a is associated with favorable breast cancer survival
Authors: Obata Yuki , Katsaros Dionyssios , Biglia Nicoletta , Shen Yi , Fu Yuanyuan , Wang Zhanwei , Yu Herbert
Published: August 2019

Introduction: MicroRNAs (miRNAs) are single-stranded, small non-coding RNAs, consisting of about 20 nucleotides and studies have shown that miRNAs may play important roles in the development and progression of cancer. Members of the miR-30 family, including miR-30a, are reported to play different roles as oncogenes or tumor suppressor genes depending on the type of cancer in several studies. We studied miR-30a expression in breast cancer in relation to disease features and patient survival.

Methods: We measured miR-30a expression in tumor samples of 509 breast cancer patients by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Using the study-specific tertile distribution as cut-off, miR-30a expression data were grouped into low, medium, and high three categories. Hazards ratios (HRs) and 95% confidence intervals (CIs) were calculated to assess the association between miR-30a expression and breast cancer survival using the Cox proportional hazards regression model, and the analysis was adjusted for age at surgery, tumor grade, disease stage, and hormone receptor status. Overall survival (OS) was defined as the time interval from the date of surgery to the date of death or last follow-up. Disease-free survival (DFS) was the time interval from surgery to recurrence or last follow-up. We also retrieved breast cancer provisional data in The Cancer Genome Atlas (TCGA) using the web-based tool cBioPortal (http://www.cbioportal.org/index.do) and analyzed the association between miR-30a and breast cancer survival.

Results: Patients with estrogen receptor (ER)-positive tumors had higher expression of miR-30a compared to those with ER-negative tumors (p = 0.0080). Patients with progesterone receptor (PR)-positive tumors also had higher miR-30a expression than those with PR-negative tumors (p = 0.0038). Survival analysis showed that patients with high expression of miR-30a had 58% reduction in risk of relapse (HR = 0.42, 95% CI = 0.21-0.83, p = 0.013) compared to those with low expression. This association remained significant (HR = 0.43, 95% CI = 0.21-0.89, p = 0.023) after adjustment for age at surgery, disease stage, tumor grade, and hormone receptor status. A significant association between miR-30a and overall survival was also observed in the TCGA data (HR = 0.60, 95% CI = 0.41-0.88, p = 0.0088).

Conclusions: Our results suggest that high expression of miR-30a may reduce the risk of breast cancer relapse and miR-30a may act as a tumor suppressor in breast cancer.

Citation Format: Yuki Obata, Dionyssios Katsaros, Nicoletta Biglia, Yi Shen, Yuanyuan Fu, Zhanwei Wang, Herbert Yu. High expression of miR-30a is associated with favorable breast cancer survival [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 3545.
Abstract 3555: miR-374a-5p induces tumor progression in triple negative breast cancer
Authors: Kim Yesol , Son Dasom , Kim Do Yeon , Park Jong Hoon
Published: August 2019

Triple negative breast cancer (TNBC) has higher aggressiveness and poorer outcomes compared with other subtypes of breast cancer. However, the genomic and molecular aberrations of TNBC are largely unknown, with cytotoxic chemotherapy remaining the major treatment for TNBC patients. In this study, miR-374a-5p was discovered as a novel TNBC-specific miRNA and its functions and the molecular mechanisms involved were investigated. Combined gene expression profiling of miRNA-microarray and human transcriptome dataset analysis revealed that miR-374a-5p is specifically upregulated in TNBC patients. Functional studies using in vitro and in vivo TNBC models, as well as survival analysis of TNBC patients, indicated that upregulated miR-374a-5p promotes tumor progression in TNBC. miR-374a-5p was also found to directly target arrestin beta 1 (ARRB1) that is specifically downregulated in TNBC patients in several human genomic datasets. Overexpressed ARRB1 reduced TNBC cell growth and migration, and the ARRB1 expression level is inversely correlated with the histological grade of the breast cancer and positively associated with TNBC patient survival, suggestive of a tumor-suppressive function of ARRB1 in breast cancer. Interestingly, increased ARRB1 activates AMPK in TNBC cells, which is associated with the expression of miR-374a-5p. Taken together, the findings suggest that miR-374a-5p is a novel prognostic marker and therapeutic target in the treatment of TNBC.

Citation Format: Yesol Kim, Dasom Son, Do Yeon Kim, Jong Hoon Park. miR-374a-5p induces tumor progression in triple negative breast 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 3555.
Abstract 3574: Identification of long non-coding RNAs involved in chromosomal instability in a cellular model of prostate cancer
Authors: Manriquez Rogelio Montiel
Published: August 2019

Most of the human genome is transcribed into RNA, but only 2% encodes proteins, leading to a large group of non-coding RNA which, without constitutive RNA, is divided into 2 groups: small (sncRNA) and long (lncRNAs) non-coding RNAs. In this study we used RNA-seq data to identify new lncRNAs which could be involved in chromosomal instability (CIN) in prostate cancer (PCa). Our aim is to study their molecular mechanism and potential role as prognostic biomarkers in this disease.

Using RNA-seq data from two prostate cell lines, neoplastic (LNCaP) and non-neoplastic (PrEC), we have identified an unannotated long lncRNA adjacent to CEP55 (centrosomal protein 55), a gene associated with CIN. We have named this 1.6 kb transcript as lncRNA- CEP55. qPCR experiments showed it is downregulated in the neoplastic cell line, in contrast to the adjacent coding gene, CEP55, which is upregulated in the same cell line and downregulated in PrEC. Our data suggest this new lncRNA might be a possible repressor for CEP55. Changes in expression of CEP55 have been associated to CIN in other cancer cell lines.

LINC02043 is an annotated lncRNA. We identified a differential expression between PrEC and LNCaP using RNA-seq data. LINC02043 is adjacent to the protein-coding gene RFC4; this gene is associated with CIN in different human cancers. We performed qPCR experiments and found LINC02043 to be downregulated in LNCaP, whereas the adjacent coding gene RFC4 is upregulated; the opposite is found in PrEC cell line. Our findings suggest LINC02043 could have a possible repressor role in RFC4. This protein-coding gene is related to mismatch repair and DNA double-strand break repair, abnormalities in this coding gene have been associated with CIN in other cancer cell lines. LINC02043 could play an important role in the cis-regulation of RFC4.

lncRNA-CEP55 and LINC02043 are important targets of study, these lncRNAs could play an essential role in regulating the expression of adjacent genes related to CIN, an enabling characteristic that facilitates the development of the hallmarks of cancer. Further analysis is needed to validate their mechanisms of action and their potential use as prognostic biomarkers in PCa.

Particularly in PCa, CIN has lately been related to cancer progression. The role of lncRNAs as drivers of CIN in PCa is a promising field in order to understand PCa progression and may play a potential role as biomarkers in this disease.

Citation Format: Rogelio Montiel Manriquez. Identification of long non-coding RNAs involved in chromosomal instability in a cellular model of prostate 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 3574.
Abstract 3582: Circular RNA circPLEHM3 acts as the sponge of microRNA-9 to suppress ovarian carcinoma progression
Authors: Zhang Lei , Liu Pengyuan , Lu Bingjian , Lu Weiguo , Lu Yan
Published: August 2019

Circular RNAs(circRNAs) are a class of stable and widespread endogenous noncoding RNA. CircRNAs have been reported to play essential roles in cancer biology, and have potential as biomarkers and therapeutic targets for cancer intervention. Here, we screened the expression profiles of human circRNAs in 27 ovarian cancer and 26 normal ovary tissues, and found circPLEKHM3 (hsa_circ_0001095) was significant down-regulated in ovarian cancer tissues. Patients with lower circPLEKHM3 expression tended to have poorer prognosis. Functionally, circPLEKHM3 overexpression inhibited cell growth, migration and epithelial-mesenchymal transition (EMT) in vitro and in vivo, but its knockdown reversed these effects. Further analyses showed that circPLEKHM3 sponges miR-9 to regulate the expression of BRCA1, DNAJB6 and KLF4, and inactivates the AKT1 expression. In addition, AKT inhibitor MK-2206 blocked the tumor-promoting effect of circPLEKHM3 knockdown, and potentiated taxol induced growth inhibition of ovarian cancer cells. Taken together, our findings demonstrated that circPLEKHM3 functions as a tumor suppressor in ovarian cancer cells by targeting the miR- 9/BRCA1/DNAJB6 /KLF4/AKT1 axis and may provide a prognostic biomarker and therapeutic target in ovarian cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Lei Zhang, Pengyuan Liu, Bingjian Lu, Weiguo Lu, Yan Lu. Circular RNA circPLEHM3 acts as the sponge of microRNA-9 to suppress ovarian carcinoma progression [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 3582.
Abstract 3589: Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy
Authors: Cheng Gang , Zielonka Jacek , Hardy Micael , Dwinell Michael B. , Kalyanaraman Balaraman
Published: August 2019

Invasive melanoma is an aggressive form of skin cancer with poor clinical outcomes. Nearly 50% of melanoma patients exhibit oncogenic BRAFV600E mutations. Existing BRAF kinase inhibitor drugs provide a short-term benefit prior to the rapid onset of drug resistance. Emerging results suggest that BRAF and other kinase inhibitors induce metabolic reprogramming from glycolytic energy production to oxidative phosphorylation (OXPHOS) in a subset of malignant melanomas. Metabolic reprogramming to OXPHOS is emerging as an important mechanism of resistance to BRAF kinase inhibitors and is associated with worse prognosis. Consequently, there is a significant opportunity for multimodal therapeutic approaches that selectively target the metabolic reprogramming associated with resistance to BRAF kinase inhibitors and promote immune-targeted therapy. We have previously developed mitochondria-targeted drugs by exploiting the enhanced negative mitochondrial membrane potential of cancer cells to selectively target and disrupt tumor growth. We hypothesized that metabolic reprogramming from glycolysis to OXPHOS in BRAF inhibitor-resistant melanoma cells would make them susceptible to OXPHOS inhibitors. In this study, we have modified the structure of a naturally occurring polyphenol, magnolol into a mitochondria-targeted-magnolol (Mito-MGN). We tested the effects of Mito-MGN on mitochondrial respiration and bioenergetic function, cellular redox status, and mitophagy in wild type (WT) and BRAF-resistant (metabolically reprogrammed) melanoma cells. Our data show that Mito-MGN inhibits mitochondrial respiration, melanoma cell proliferation and invasion at concentrations more than 200-fold lower than that of magnolol. Furthermore, Mito-MGN increases ROS formation and oxidation of mitochondrial peroxiredoxin. Preliminary results also indicate that BRAF inhibitor resistant melanoma cells exhibit enhanced OXPHOS as a consequence of metabolic reprogramming and that Mito-MGN effectively inhibits proliferation and mitochondrial respiration in the resistant cells. Mito-MGN inhibited mitochondrial complex I activity in both WT and resistant cells. The IC50 values to inhibit complex I-mediated respiration was consistent with the values calculated for inhibition of cell respiration. We conclude that inhibition of complex I is a key initial event that is responsible for the anti-proliferative effects observed in the presence of Mito-MGN against wild-type as well as drug-resistant melanoma cells.

Citation Format: Gang Cheng, Jacek Zielonka, Micael Hardy, Michael B. Dwinell, Balaraman Kalyanaraman. Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy [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 3589.
Abstract 3597: BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of diffuse large B-cell lymphoma (DLBCL)
Authors: Eheim Ashley , Christian Sven , Meyer Hanna , Stoeckigt Detlef , Merz Claudia , Zimmermann Katja , Bauser Marcus , Haegebarth Andrea , Ferrara Steven , Sykes David B. , Scadden David T. , Gradl Stefan , Janzer Andreas
Published: August 2019

DLBCL is the most common type of non-Hodgkin lymphoma. It is an aggressive and fast growing tumor with two major molecular subtypes: germinal center (GCB) and activated B-cell like (ABC). While the majority of patients are 60 years or older, DLBCL can occur at any age.

Despite a cure rate of around 50% the need for novel therapies remains high, especially for relapsed/refractory DLBCL patients not eligible for stem cell transplant.

DHODH is a key enzyme in the de novo pyrimidine synthesis converting dihydroorotate to orotate. We recently discovered its role in AML differentiation (Sykes et al 2016, Cell) and we are investigating the novel DHODH inhibitor BAY 2402234 in an ongoing phase I study in myeloid malignancies (NCT03404726). Further screening of non-leukemia cancer types identified DLBCL as highly responsive to DHODH inhibition in preclinical studies.

Here, we disclose for the first time the functional preclinical characterization of BAY 2402234 in DLBCL. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro it potently inhibits proliferation of DLBCL cell lines in the sub-nanomolar to low-nanomolar range. The anti-proliferative effects can be rescued by uridine supplementation which bypasses DHODH via the salvage pathway and demonstrates the on-target specificity of the inhibitor. In vivo, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in subcutaneous models derived from patient-derived xenograft (PDX) and cell lines representing various DLBCL subtypes, including GCB and ABC. Dose dependent target engagement and drug exposure of BAY 2402234 could be observed by increases in plasma dihydroorotate levels and unbound plasma drug levels after treatment with the inhibitor.

Based on preclinical data presented herein we plan to start clinical investigations of BAY 2402234 in patients with DLBCL in early 2019.

Citation Format: Ashley Eheim, Sven Christian, Hanna Meyer, Detlef Stoeckigt, Claudia Merz, Katja Zimmermann, Marcus Bauser, Andrea Haegebarth, Steven Ferrara, David B. Sykes, David T. Scadden, Stefan Gradl, Andreas Janzer. BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of diffuse large B-cell lymphoma (DLBCL) [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 3597.
Abstract 3599: BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of colorectal carcinomas
Authors: Merz Claudia , Christian Sven , Eheim Ashley , Seidel Henrik , Lesche Ralf , Eversloh Merlin Luetke- , Meyer Hanna , Ferrara Steven , Bauser Marcus , Haegebarth Andrea , Gradl Stefan , Janzer Andreas
Published: August 2019

Colorectal carcinoma (CRC) is the third most common malignancy in the US, being responsible for approximately 50,000 deaths per year. The overall 5-year survival rates are high for localized disease (~90%), but low for metastatic disease (~14%), thereby underlining the high unmet medical need for novel therapeutics for CRC patients.

DHODH is a key enzyme in the de novo pyrimidine synthesis, converting dihydroorotate to orotate. We recently discovered its role in AML differentiation (Sykes et al 2016, Cell) and we are investigating BAY 2402234 in an ongoing phase I study in myeloid malignancies (NCT03404726). Since then, several publications have also described a role of DHODH in solid tumor indications.

Herein we disclose the functional preclinical characterization of the novel DHODH inhibitor BAY 2402234 in CRC. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro it potently inhibits proliferation of CRC cell lines in the sub-nanomolar to low-nanomolar range. The anti-proliferative effects can be rescued by uridine supplementation which is known to bypass DHODH via the salvage pathway and demonstrates the on-target specificity of the inhibitor. Interestingly, not all CRC cell lines are sensitive to the inhibitor. Similar to the in vitro observations, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in some, but not all, subcutaneous CRC xenograft models. A large CRC PDX in vivo screen revealed tumor growth inhibition in over 60% of the models in response to BAY 2402234 treatment. To elucidate the mode of action of BAY 2402234 in CRC we analyzed transcriptomic changes in CRC PDX models in vivo after single treatment with BAY 2402234, and identified several hundred differentially regulated genes at early time points. These in vivo PDX models were also used to explore potential effects of BAY 2402234 treatment on a large panel of metabolites.

These preclinical results support the clinical evaluation of BAY 2402234 in patients with CRC, and such a clinical study is planned for early in 2019.

Citation Format: Claudia Merz, Sven Christian, Ashley Eheim, Henrik Seidel, Ralf Lesche, Merlin Luetke- Eversloh, Hanna Meyer, Steven Ferrara, Marcus Bauser, Andrea Haegebarth, Stefan Gradl, Andreas Janzer. BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of colorectal carcinomas [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 3599.
Abstract 3615: Design for a flexible localized drug delivery system
Authors: Wiranowska Marzenna , Toomey Ryan , Falahat Rana , Alcantar Norma
Published: August 2019

This project focuses on studying the optimal design of a drug delivery system based on niosomes (non-ionic surfactants vesicles) embedded in a hydrogel network (chitosan) to ensure full control of the release time and dosage of cancer treatment drugs for localized delivery. Here we present data on the molecular interactions facilitating specific binding at tumor cell surfaces to aid in the treatment of ovarian, brain and lung cancers to name a few. We have done three types of characterizations to determine the basic design parameters for a personalized cancer treatment. First, we determined how fundamental surface interactions can be optimized to target and attack tumor integrity and boundaries. We used confocal microscopy and Xenogen imaging to determine how data from cells and in-vivo models compare. Second, we determined the parameters of chemotherapeutic agents that can act individually or in a cocktail configuration in both in-vitro and in-vivo studies. We used light scattering and transmission electron microscopy to determine the topography for guidance on the design of the drug delivery process. Third, we found that specific targeting can be accomplished by optimizing the binding between drug molecules and mucin 1 (MUC1) found on the surface of epithelial-derived tumors. We used attenuated total internal reflection Fourier transform infrared (ATR-FTIR) spectroscopy to elucidate the interactions that enhance the selectivity of our drug delivery system with respect to model cancer systems. In addition, we used chlorotoxin, a carrier-type polypeptide known to bind to a broad number of tumors. By identifying and optimizing these two methodologies, we have found that tumor and normal cells can be selectively targeted. The significant advantage of applying a localized treatment is the avoidance of systemic exposure to chemotherapy drugs thereby limiting their side effects.

Citation Format: Marzenna Wiranowska, Ryan Toomey, Rana Falahat, Norma Alcantar. Design for a flexible localized drug delivery system [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 3615.
Abstract 3619: Nanoparticle formulation of VERU 111 for pancreatic cancer treatment
Authors: Kashyap Vivek K. , Hafeez Bilal B. , Wang Qinghui , Chauhan Neeraj , Nagesh Prashanth K. , Yallapu Murali M. , Miller Duane D. , Li Wei , Jaggi Meena , Chauhan Subhash C.
Published: August 2019

Background: Pancreatic cancer (PanCa) is one of the leading causes of cancer-related mortality in the United States due to very limited therapeutic options. Thus, developing novel therapeutic strategies will help for the management of this disease. We recently identified VERU-111, a novel synthetic molecule which showed potent anti-cancer effect against PanCa via targeting clinically important βIII and βIV tubulin isoforms. In this study, we synthesized and characterized its novel nanoformulation (MNP-VERU) and evaluated its therapeutic effects in vitro and xenograft mouse model.

Methods: MNPs were prepared by chemical precipitation method and loaded with VERU-111 using diffusion method. This formulation was characterized for particle size, chemical composition, and drug loading efficiency, using various physico-chemical methods (TEM, FT-IR, DSC, TGA, and HPLC). The internalization of MNP-VERU was achieved after 6 hours incubation with MNP-VERU in PanCa cells. To determine therapeutic efficacy of MNP-VERU, we performed various in vitro (MTS, wound healing, boyden chamber real-time xCELLigence, and apoptosis assays) and in vivo (mouse tumor xenograft) studies using PanCa. Effect of MNP-VERU on various key oncogenic signaling pathways, and miRNAs was evaluated by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses respectively.

Result: Our novel MNP-VERU formulation provided average size of 110 nm in dynamic light scattering (DLS) and exhibited -8.23 to -11.65 mV zeta potential with an outstanding loading efficiency (94%). Cellular uptake and internalization studies demonstrate that MNP-VERU escape lysosomal degradation, providing efficient endosomal release to cytosol. MNP-VERU showed remarkable anti-cancer potential in various PanCa cells (Panc-1, AsPC-1, HPAF-II, BxPC-3, MiaPaca) and more effectively repressed βIII and βIV tubulin isoforms via restoring the expression of miR-200c. MNP-VERU more effectively suppressed AsPC-1 cells derived xenograft tumors in athymic nude mice.

Conclusions: Taken together, our results suggest that MNP-VERU has more anti-cancer potential than free VERU-111 against PanCa. MNP-VERU may reduce the toxicity and improve the bioavailability of free VERU-111 and could be used for the management of PanCa.

Citation Format: Vivek K. Kashyap, Bilal B. Hafeez, Qinghui Wang, Neeraj Chauhan, Prashanth K. Nagesh, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan. Nanoparticle formulation of VERU 111 for pancreatic cancer treatment [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 3619.
Abstract 362: Depolymerized fucosylated chondroitin sulfate in pancreatic cancer, metastasis and cancer-associated thrombosis
Authors: Yao Yiming , Godugu Kavitha , Jin Yongsheng , Jiang Yi , MOUSA SHAKER A.
Published: August 2019

The Depolymerized fucosylated chondroitin sulfate (dHG) prepared from natural fucosylated chondroitin sulfate, which was isolated from sea cucumber, showed remarkable anti-cancer efficacy in suppressing primary tumor growth, tumor angiogensis and metastasis. The anti-metastasis properties were demonstrated in multiple mouse cancer models, including B16F10 melanoma-induced lung metastasis model, 4T1 breast cancer induced lung metastasis model, Luciferase transfected SUIT-2 pancreatic cancer induced lung metastasis model as well as surgical removal SUIT-2 pancreatic tumor induced metastasis. Additionally, dHG demonstrated potent anti-angiogenesis efficacy against various growth factors-mediated angiogenesis. Cancer metastasis is a multiple process and regulated by a complex signaling network. Our In vitro studies demonstrated that dHG is a multiple targets agent. dHG inhibits angiogenesis and P-selectin, both are involved in cancer metastasis. dHG also induces TFPI release, especially TFPI-2 release. TFPI-2 has been recognized as a potent tumor suppressor. It inhibits the migration and invasion of human cancer cells. Low expression of TFPI-2 results in enhanced growth and metastasis of a variety of human tumors. Thrombelastography (TEG) showed that mixing of dHG and 1-10 picogram of TFPI-1 or TFPI-2 generated significant anti-thrombotic effect while dHG itself has very low anticoagulant activity. Thrombosis is a significant problem for cancer patient. dHG potentially inhibits thrombosis and normalizes hemostasis. Our studies demonstrated that dHG is a promising therapeutic agent for metastatic cancer and cancer-associated thrombosis management.

Citation Format: Yiming Yao, Kavitha Godugu, Yongsheng Jin, Yi Jiang, SHAKER A. MOUSA. Depolymerized fucosylated chondroitin sulfate in pancreatic cancer, metastasis and cancer-associated thrombosis [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 362.
Abstract 3627: Development of CLENs for future treatment & diagnostic detection using cell models of drug-resistant ovarian cancer
Authors: Amini Negar , Emerson Joshua , Clement Jenel
Published: August 2019

Current guidelines suggest the use of a platinum agent and a taxane as first line therapy for ovarian cancer where total surgical resection is limited. Liposomal preparations of these drugs have demonstrated therapeutic value by increasing drug uptake and reducing toxicity. The following research aims to improve target specificity by employing a multi-strategic approach to drug targeting and treatment in vitro. Cellular membrane lipid-extracted nanoliposomes (CLENs) were constructed in part from lipid extracts (LE) derived from the membrane of multidrug resistant ovarian cancer (SKOV-3-MDR) cell lines, and more conventional components of liposomes. Formulations of greatest interest were selected after comparing cellular uptake studies of conventional liposomes to various types of CLENs composed of mixed ratios of DOPC, cholesterol, and LE. Cellular uptake was measured by rhodamine fluorescence (RF) following 24 hours incubation in 48 well plates, where RF was used as an indicator of cellular uptake. To determine effectiveness of LE, formulations with and without synthetic cationic lipid DOTAP were compared. Flow cytometry was used to evaluate liposome binding. Additional studies include drug loading efficiency and SRB cytotoxicity studies involving cisplatin, and the effect of additional inclusion of MAG-C (magnetic material) in the SKOV-3 lipid extract-containing liposomes. CLENs containing 15% LE demonstrated greater uptake compared to CLENs prepared with 0% and 5% LE content. Minimal uptake was observed in non-target cancer cell lines (prostate cancer-CRL-1740 and gliobastoma-U87MG), as well as with normal pancreas, non-malignant (hTERT) cell line. The fluorescence intensity values reported for SKOV-3-MDR, CRL-1740, U87-MG and hTERT, were 437 ± 153, 295 ± 67, 57 ± 6, and 29 ± 6, respectively. The additional inclusion of DOTAP showed significant cellular uptake with and without LE. Flow cytometry showed greater initial binding of CLENs to target cells when LE was included in preparations. Incorporation efficiency of cisplatin into SKOV-3 CLENS was 67%. Chemotherapy drug-loaded CLENs is an appealing vehicle as it addresses many parameters. Early findings suggest that CLENS is a suitable vehicle for targeting and treatment using a drug-resistant ovarian cancer cell line. CLENS allows for faster initial nanoparticle binding, cell-type specific uptake, and enhances cytotoxicity. Our encouraging results corroborate this theorem, showing greater targeting with reduced uptake by non-target cells. Additional studies involving MAG-C are currently underway.

Citation Format: Negar Amini, Joshua Emerson, Jenel Clement. Development of CLENs for future treatment & diagnostic detection using cell models of drug-resistant ovarian 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 3627.
Abstract 363: CYT01B, a novel RAD51 inhibitor, acts synergistically with PARP inhibitors
Authors: Maclay Tyler , Day Melinda , Mills Kevin
Published: August 2019

Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. We have developed a novel RAD51 inhibitor, CYT01B, which we have previously shown to be preferentially active in Activation Induced Cytidine Deaminase (AID) expressing cells, and in other tumor derived cell lines with elevated levels of DNA damage and/or reduced DNA repair capabilities. CYT01B acts by reducing RAD51 focus formation, and depleting the nuclear pool of RAD51. We have observed anti-tumor effects with CYT01B in preclinical models, but have yet to determine if RAD51 could act as a sensitizer to current therapeutics. A main resistance mechanism to PARP inhibitors is the overexpression of RAD51, making it an attractive potential combination for our RAD51 inhibitor. To determine potential drug synergy, a matrix study was performed with CYT01B (concentration range of 20nM to 5µM) and 5 different PARP inhibitors including olaparib (78nM to 5µM), niraparib (78nM to 5µM), veliparib (780nM to 50µM), rucaparib (156nM to 10µM), and talazoparib (39nM to 2.5µM). The combination matrix was tested in cell lines of varying AID expression and PARP inhibitor sensitivity: ARPE19/HPV16 (HPV immortalized epithelial cell line), KYSE-70 (head and neck cancer cell line), Daudi (Burkitt’s Lymphoma cell line), HCC1143 (TNBC), and BT20 (TNBC). We used both the Loewe Additivity model and the Bliss Independence model to determine drug interaction (synergistic, independent, or antagonistic). In general, we observed synergy with all the combinations in the tumor derived cell lines. In the transformed epithelial cell line, synergy was observed only in the olaparib combination. The strength of the synergistic effect differed amongst the PARP inhibitors, with olaparib giving the strongest synergy scores. From this data we hypothesize that the synergistic activity with CYT01B is related to the PARP trapping efficiency of the PARP inhibitors; greater trapping efficiency the greater the synergy. These data suggest that CYT01B may be active as a combinatorial therapy with PARP inhibitors. Overall, we conclude that there is significant potential for combining RAD51 and PARP inhibition in future cancer treatment strategies, and warrants future exploration in vivo.

Citation Format: Tyler Maclay, Melinda Day, Kevin Mills. CYT01B, a novel RAD51 inhibitor, acts synergistically with PARP inhibitors [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 363.
Abstract 3632: Enhanced efficacy of optimized phenethyl isothiocyanate and cisplatin co-encapsulated liposome nanoparticle for treatment of non-small cell lung cancer
Authors: Sun Mengwei , Shi Yi , Pasqua Anthony Di
Published: August 2019

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, which is the leading cause of cancer-related death in the United States. Accounting for about 85% of all lung cancers, NSCLC is difficult to treat and its survival rates are low. After decades of basic and clinical research, the most effective treatments still involve the first-generation anticancer agent cisplatin (CDDP) in combination with other drugs. Naturally-occurring compound, phenethyl isothiocyanate (PEITC), can be used to sensitize NSCLC cells to CDDP. We have previously proved that co-encapsulation of PEITC and CDDP in liposomes enhances the cytoxicity toward NSCLC cells, compared to free PEITC or CDDP. In this study, liposomal-PEITC-CDDP is optimized and the release of PEITC and CDDP from the nanoparticle is characterized. In vitro studies show that liposomal-PEITC-CDDP has a higher cytotoxicity toward both A549 and H596 human NSCLC cell lines than toward WI-38 and BEAS-2B human normal lung cell lines. Therfore, we have prepared an efficacious therapy with a potential for fewer side-effects due to low off-target toxicity.

Citation Format: Mengwei Sun, Yi Shi, Anthony Di Pasqua. Enhanced efficacy of optimized phenethyl isothiocyanate and cisplatin co-encapsulated liposome nanoparticle for treatment of non-small cell lung 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 3632.
Abstract 3637: Development of a polyethylenimine conjugated liner multiblock polymer to deliver VEGF siRNA for triple negative breast cancer
Authors: Li Yuanke , Zhao Zhen , Cheng Kun
Published: August 2019

Vascular endothelial growth factor (VEGF) plays important roles in the angiogenesis process and is correlated with high proliferation and metastasis of breast cancer, particularly in triple negative breast cancer (TNBC). Downregulation of VEGF expression with siRNA in cancer cells is therefore a potential strategy for TNBC treatment. A polyethylene glycol based bio-degradable, linear multiblock polymer was synthesized and conjugated with branched low-molecular-weight poly(ethyleneimine) (PEI) to form nanocomplexes with a VEGF siRNA. The nanocomplex can protect siRNA from serum degradation and deliver the VEGF siRNA into TNBC cells with 72% silencing activity and low cytotoxicity. In vitro activity studies showed that the siRNA nanocomplexes significantly inhibit 64% migration and 67% invasion of TNBC cells. Moreover, the nanocomplex exhibited efficient tumor penetration in a 3D tumor spheroid model, suggesting a good penetration capability of the nanocomplex in tumor microenvironment in vivo. More importantly, the VEGF siRNA nanocomplex efficiently inhibit tumor growth in vivo and successfully downregulate VEGF expression in the tumor. These results suggested that VEGF siRNA is a promising anti-tumor agent for TNBC therapy, and the PEI 1800 conjugated bio-degradable multiblock polymer is a promising system to deliver siRNAs to TNBC cells.

Note: This abstract was not presented at the meeting.

Citation Format: Yuanke Li, Zhen Zhao, Kun Cheng. Development of a polyethylenimine conjugated liner multiblock polymer to deliver VEGF siRNA for triple negative breast 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 3637.