bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3462: The F-box protein FBXL16 regulates the stability of c-myc oncoprotein
Authors: Morel Marion , Long Weiwen
Published: August 2019

F-box proteins are major components of the SCF (SKP1-CUL1-F-box) E3 ubiquitin ligases as they are responsible for substrate recognition. In this complex, F-box proteins bind to SKP1 through the F-box motif to bring the ubiquitination machinery and mediate protein ubiquitination of the substrates. So far, 69 F-box proteins are identified in humans, and they fall into 3 families depending on their substrate recognition domains: FBXLs (Leucine-Rich Repeats or LRR), FBXWs (WD repeats) and FBXOs (Other domains). FBXL16 is a poorly studied F-box protein which consists of an N-terminal Proline-rich domain, an F-box motif and a C-terminal domain of Leucine-rich repeats. FBXL16 was first identified as a transcriptional target of E2F1 (Sato et al, 2010) and was then showed as a binding partner of PP2A (Protein Phosphatase 2A) and as a regulator of its phosphatase activity (Honarpour et al, 2014). Interestingly, FBXL16 was shown to be overexpressed in a number of cancers, particularly invasive breast carcinoma (Oncomine) indicating that FBXL16 may play important roles in cancers. In this study, by both knockdown and overexpression experiments, we found that FBXL16 promotes cancer cell migration. To our surprise, we observed that knockdown of FBXL16 in cancer cells resulted in a strong decrease of c-myc protein level. Importantly, we also found that FBXL16 binds to and stabilizes c-myc protein. Mechanistically, FBXL16 overexpression decreased c-myc protein ubiquitination. Taken together, our study demonstrates a positive role of FBXL16 in regulating c-myc protein stability by inhibiting its ubiquitination.

Citation Format: Marion Morel, Weiwen Long. The F-box protein FBXL16 regulates the stability of c-myc oncoprotein [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 3462.
Abstract 3464: CBAP: A novel rheostat molecule for regulation of TSC GAP activity and mTORC1 signaling in cancer cells
Authors: Yen Jong-Young J. , Chiang Yun-Jung , Liao Wei-Ting , Wang Shih-Hao , Yang-Yen Hsin-Fang
Published: August 2019

The PI3K-mTOR signaling pathway is one of the most frequently dysregulated signaling cascades in cancer. Understanding the molecular wiring of the PI3K-mTOR signaling network and its activation in cancer will improve our understanding of their contribution to cancer pathology and provide novel therapeutic strategies by targeting this network. Our previous works demonstrated that Common receptor Beta chain Associated Protein (CBAP) is required for Jurkat cell leukemogenesis and regulates Akt-dependent TSC2 phosphorylation and lysosomal dissociation of TSC complexes, and in turn enhances mTORC1 signalling and expression of c-MYC and HIF-1a in Jurkat T-ALL leukemic cells. In this report, we tackle the question how TSC/Rheb/mTORC1 pathway is regulated by CBAP via cell-based functional assay, immunoprecipitation and in vitro GTPase assay. Our data revealed that CBAP interacted with TSC2 via tuberin-binding domain and could compete for TSC2 binding with TSC1 and suppressed the GTPase activation activity of TSC1/2 complexes. Akt could also associate with TSC2 complexes and CBAP in both Jurkat and CBAP-knockout Jurkat cells, except that TSC1 could be detected within the complexes only in the absence of CBAP. Moreover, the Rheb-GAP activity of this Akt-associated TSC2 complexes was sensitive to the presence of CBAP and TSC1/2 GAP activity was strongly suppressed by recombinant CBAP proteins in the in vitro reconstituted assay. Finally, a peptide domain of CBAP interacting with TSC2 was mapped and disruption of CBAP-TSC2 interaction by overexpressing this peptide inhibits the Akt/Rheb signal axis and suppresses leukemia cell growth. In summary, our data revealed an important role of the interaction between CBAP and TSC2 proteins in promoting Rheb/mTORC1 signaling activity, and suggested a crucial role of overexpression of CBAP protein in tumor cell proliferation.

Citation Format: Jong-Young J. Yen, Yun-Jung Chiang, Wei-Ting Liao, Shih-Hao Wang, Hsin-Fang Yang-Yen. CBAP: A novel rheostat molecule for regulation of TSC GAP activity and mTORC1 signaling in cancer cells [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 3464.
Abstract 3472: The role of mutant p53 R248W in oral squamous cell carcinoma
Authors: Enaka Mayu , Nakanishi Masako , Muragaki Yasuteru
Published: August 2019

TP53 is the most commonly mutated gene in oral squamous cell carcinoma (OSCC). It is known that mutant p53 has not only loss of function but gain of function. Cytokeratin 17 (K17) has been shown to promote tumorigenesis and aggressiveness in OSCCs and other various carcinomas. Although p53 reportedly induces K17 transcription by irradiation, the molecular interaction between p53 and K17 in OSCCs remains unknown. To address this issue, we investigated the relationship between p53 and K17 using SAS cells with nonsense mutation and Ca9-22 cells with R248W mutation in the TP53 gene. Western blot and immunohistochemistry showed higher K17 expression in SAS cells and virtually no K17 expression in Ca9-22 cells, suggesting negative correlation between p53 and K17 expression. ChIP analysis revealed that p53 bound specifically to the tentative p53 binding site in the promoter region of the K17 gene. Unexpectedly, however, overexpression of wild-type (wt) p53 did not suppress K17 expression in SAS cells, whereas knock down of mutant (mt) p53 significantly upregulated K17 expression in Ca9-22 cells, raising a possibility that only R248W mtp53 suppresses K17 expression. Furthermore, overexpression of wtp53 and R248Q mtp53 did not suppressed K17 expression in Ca9-22 cells with knock down of p53, whereas R248W mtp53 overexpression clearly showed suppression of K17. Taken together, R248W mtp53 acts as a direct transcriptional repressor of K17 in OSCCs, reducing aggressiveness of the carcinoma cells.

Citation Format: Mayu Enaka, Masako Nakanishi, Yasuteru Muragaki. The role of mutant p53 R248W in oral squamous cell carcinoma [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 3472.
Abstract 3473: Comprehensive genetic screening in hereditary colorectal cancer
Authors: Rohlin Anna , Eiengård Frida , Mårtensson Emma , Zagoras Theofanis , Gebre-Medhin Samuel , Nordling Margareta
Published: August 2019

High-penetrant pathogenic variants in established genes associated with hereditary colorectal cancer (CRC) explain the disease in approximately 5-6% of cases. In as much as 20%-30% of all, including sporadic CRC cases, genetic factors are thought to play a significant role. Dependent on technical as well as limitations due to interpretation challenges, the high-penetrant pathogenic variants are gathered in the exonic regions of the genes. Contributions of pathogenic variants in genes, not traditionally associated with hereditary CRC have also recently been recognized. It is crucial for the patients to get a correct molecular diagnosis that allows for adequate follow-up since the majority of the syndromes include predisposition also for tumors in other organs. A comprehensive panel including high-penetrant susceptibility genes as well as genes not generally associated with hereditary CRC was used for the analyses. Sequencing of 50 kb upstream and downstream of all genes including intronic regions were performed. All SNVs and CNVs in exons were thoroughly evaluated concerning pathogenicity and also intronic and intragenic sequences were screened for the presence of possibly pathogenic variants. In addition, cDNA were analysed to detect variants in splice-sites as well as aberrations causing other effects on mRNA level. The study included 206 patients referred to the Cancer Genetics Counselling Clinic in Gothenburg, Sweden. The spectra of pathogenic variants in well-known high-penetrance predisposition genes as well as the contribution of pathogenic variants in low-penetrant genes were determined. The contribution of pathogenic variants also in “non-CRC” genes was examined. The findings include identification of previously unreported variants and putative causative variants in unexplained patients with hereditary CRC. An attempt to identify putative genetic variants that could explain the variability in cancer risk among carriers of specific variants in high-penetrance genes was also made.

Citation Format: Anna Rohlin, Frida Eiengård, Emma Mårtensson, Theofanis Zagoras, Samuel Gebre-Medhin, Margareta Nordling. Comprehensive genetic screening in hereditary colorectal 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 3473.
Abstract 3483: The African-specific S47 variant of the p53 tumor suppressor gene alters cell metabolism
Authors: Gnanapradeepan Keerthana , Basu Subhasree , Kung Che-Pei , Barnoud Thibaut , Good Madeline , Quinn William , Lee Joyce , Wellen Kathryn , Schug Zachary , Baur Joseph , George Donna , Murphy Maureen
Published: August 2019

The TP53 gene, often referred to as the guardian of the genome, is the most frequently mutated gene in human cancer. This gene encodes the tumor suppressor p53, a master regulator of various processes such as programmed cell death, growth arrest and senescence. However, there is increasing evidence that highlights the role of p53 in tumor suppression with regards to maintaining metabolic homeostasis. Our lab has identified a polymorphic variant of p53 that encodes a serine residue instead of a proline at amino acid 47 (hereafter S47). This variant is most prevalent in African and African-American individuals, and mice and humans carrying this variant have increased cancer risk and impaired response to therapy. Using both mouse models and human cells, we have found that S47 cells have significantly altered metabolism compared to the WT counterpart. Specifically, S47 mice have increased weight and increased lean content compared to WT mice. These mice also show increased fitness in treadmill studies. Analyses of cell metabolism in S47 murine and human cells indicate that S47 cells show increased glycolysis and glycolytic flux, along with increased oxygen consumption rate on a Seahorse analyzer. The combined data support the premise that the S47 variant confers increased cancer risk but also increased metabolic fitness; the latter may explain the high rate of this variant in certain regions of Africa. By clearly elucidating the roles of WT p53 and the S47 variant in the context of metabolism, we hope to uncover new therapeutic avenues and ultimately enable more personalized medicine approaches for individuals who carry this variant.

Citation Format: Keerthana Gnanapradeepan, Subhasree Basu, Che-Pei Kung, Thibaut Barnoud, Madeline Good, William Quinn, Joyce Lee, Kathryn Wellen, Zachary Schug, Joseph Baur, Donna George, Maureen Murphy. The African-specific S47 variant of the p53 tumor suppressor gene alters cell metabolism [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 3483.
Abstract 3484: Ras recruits oncogenic serine protease hepsin to disrupt mammary epithelial integrity
Authors: Tervonen Topi A. , Pant Shishir M. , Belitskin Denis , Englund Johanna , Närhi Katja , Verschuren Emmy , Kovanen Panu , Klefström Juha
Published: August 2019

Type II transmembrane serine protease hepsin is overexpressed and redistributed in clinical breast cancer samples, although the HPN gene is not a frequent target of cancer-specific genetic alterations. Here, we sought to identify cancer relevant upstream factors responsible for oncogenic deregulation of hepsin. We explored the effects of ectopic expression of major oncogenes and tumor suppressors on hepsin protein expression levels in non-malignant mammary epithelial cells, finding that HrasV12, KrasV12, ectopic wild-type Kras, endogenous mutant KrasD12 and E2F-1 induce the expression of active form of hepsin. Oncogenic Ras proteins also reduced expression levels of cognate hepsin inhibitor, HAI-1. Concomitantly, oncogenic Ras expression led to disappearance of desmoplakin and desmoglein 2 from desmosomal junctions, which correlated with mislocalization of hepsin from its predominant localization in desmosomes to cytosol. To explore the effects of mutated Ras expressed at endogenous levels we used lung tumors derived from lox-stop-lox (LSL)-KrasD12; p53-/- and LSL-KrasV12 mammary epithelial structures in ex vivo 3D cultures. In both cases, hepsin was highly expressed and predominantly localized to cytosol. Knockdown of hepsin by shRNA rescued HrasV12-induced epithelial integrity defects in mammary epithelial 3D culture and furthermore, MEK and ERK inhibitors prevented HrasV12-induced hepsin deregulation as well as desmosomal and basement membrane defects. Critical pathways downstream of MAPK pathway are being studied and will be discussed. These findings suggest a critical role for hepsin in mediating Ras-MAPK-mediated disruption of epithelial integrity during tumorigenesis.

Citation Format: Topi A. Tervonen, Shishir M. Pant, Denis Belitskin, Johanna Englund, Katja Närhi, Emmy Verschuren, Panu Kovanen, Juha Klefström. Ras recruits oncogenic serine protease hepsin to disrupt mammary epithelial integrity [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 3484.
Abstract 3492: A targeted semi-conductor based next-generation sequencing (NGS) test to characterize microsatellite instability in FFPE tumor samples
Authors: Kraltcheva Anelia , El-Difrawy Sameh , Kamat Asha , Au-Young Janice , Cawley Simon , Sadis Seth
Published: August 2019

Microsatellite instability (MSI) arises from defects in the mismatch repair (MMR) system and is associated with hypermutability of short DNA sequence repeats. Defects in MMR are observed in diverse cancer types but are common in colorectal, gastric and endometrial cancers. MSI testing is increasingly important for patient management. For example, in diverse cancer types, MSI is associated with favorable response to immune checkpoint inhibitors. The mainstay of MSI testing has been PCR/fragment analysis or IHC to monitor loss of expression of MMR proteins. Although generally robust, these methods are single biomarker tests that may consume limited biopsy samples. For this reason, MSI testing has been recently incorporated into NGS tests. However, targeted amplicon based NGS tests for MSI that consume limited sample input and provide fast turn-around time remain an unmet need. Herein, we describe a targeted NGS-based method to assess MSI that leverages Ion AmpliSeq™ or Ion AmpliSeq™ HD multiplex PCR and Ion GeneStudio™ S5 next-generation sequencing. The test is comprised of diverse microsatellite markers including mono- and di-nucleotide repeats that range from 10 to 40 bp. A novel algorithm was developed that leverages the unique signal processing properties inherent in semi-conductor sequencing and workflows were developed for tumor only samples as well as paired tumor-normal samples. The test provides results for individual microsatellites and generates an MSI score for the sample. The performance of the assay was verified over a large cohort of colorectal, gastric and endometrial cancer samples with MSI status independently assigned by orthogonal on-market tests. The MSI panel can be used by itself or integrated into larger targeted sequencing panels. MSI tests that leverage the inherent advantages of targeted semiconductor sequencing, low sample input and fast turn-around time, will support expanded research opportunities into the association of MSI with other targeted alterations and help elucidate the interaction of MSI, DNA repair defects, and the response to immune checkpoint inhibition.

Citation Format: Anelia Kraltcheva, Sameh El-Difrawy, Asha Kamat, Janice Au-Young, Simon Cawley, Seth Sadis. A targeted semi-conductor based next-generation sequencing (NGS) test to characterize microsatellite instability in FFPE tumor samples [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 3492.
Abstract 3493: Tumor treating fields (TTFields) significantly alters how tumor cells repair double stranded breaks using homeologous Alu sequences
Authors: Morales Maria E. , Kaul Tiffany K. , Deininger Prescott L.
Published: August 2019

Tumor Treating fields (TTfields) are an FDA-approved therapeutic modality that utilizes low intensity alternating electric fields. Although the therapy’s primary mechanism seems to cause aberrant mitosis in dividing cells and subsequently cellular death, TTfields have also been reported to alter DNA repair pathways. Recent reports showed that chromosomal double strand breaks (DSBs) repair is delayed after TTfields treatment of glioma cells. DSBs are often repaired by error-prone pathways that can be influenced by proximity of repetitive sequences, such as Alu elements. Alu elements are known to contribute to genetic instability though homeologous recombination due to their sequence similarity and high density in the genome. We find that different tumor cells resolve the Alu heteroduplexes formed during DNA repair in different ways. To understand the molecular mechanisms underlying the biological effects of TTfields exposure, we examined mechanistic aspects of DNA repair using our previously published Alu/Alu recombination system in HEK293 and HeLa cells. Of note, our assay probes the recombination properties that occur in the cells that survive TTfields treatment, thus providing potential insights into therapeutic resistance. Our Alu/Alu recombination system is able to efficiently measure both the rate of Alu/Alu recombination (Single Strand Annealing-SSA), as well as Alt-Non Homologous End Joining (Alt-NHEJ) deletions that cause a deletion in the vicinity of the two Alu elements in the system. Sequence verification confirmed that most of the genetic deletions that occurred post TTfields treatment were the result of Alu/Alu recombination events that predominantly arose from repair by SSA in HEK293 and HeLa. This increase in SSA repair could be the result of SSA-intermediates that escaped heteroduplex rejection. These observations suggest the possibility that treatment of TTfields significantly alters how tumor cells repair DSBs using homeologous Alu sequences.

Citation Format: Maria E. Morales, Tiffany K. Kaul, Prescott L. Deininger. Tumor treating fields (TTFields) significantly alters how tumor cells repair double stranded breaks using homeologous Alu sequences [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 3493.
Abstract 3498: Highly specific macrocyclic ATR inhibitors for the targeted treatment of a broad spectrum of cancers showing lack of anemia or neutropenia in pre-clinical animal models
Authors: Pamarthy Sahithi , Li Dansu , Goliadze Ekaterine , Gill Tina , Jia Lanqi , George Erin , Butler Laura R. , Ragland Ryan L. , Afargan Michel , Simpkins Fiona A. , Brown Eric J. , Gilad Oren
Published: August 2019

Ataxia Telangiectasia and Rad3-related (ATR) and its downstream effector Checkpoint Kinase 1 (CHK1) are central to the protection of stalled replication forks.Specific targeting of the ATR is synthetically lethal with multiple cancer-associated changes including oncogenic stress and defects in the DDR pathway and represents an emerging strategy to treat a broad spectrum of cancers. Atrin Pharmaceuticals has rationally designed a novel series of conformationally constrained macrocyclic ATR inhibitors with higher potency and selectivity than other ATR inhibitors currently in clinical development. An in-depth characterization of the ATRN series identified ATRN-119 as our lead compound with an in vitro enzyme IC50 of 20 nM and inhibition of ATR substrate CHK1 Ser345 phosphorylation in cells at an IC50 of 5 nM. ATRN-119 inhibits ATR in cells at much lower concentrations and demonstrates higher selectivity (>2000) for ATR over other closely-related PIK-kinases like ATM, DNA-PK and mTOR. Additionally, ATRN series have favorable ADME properties with increased water solubility and metabolic stability in human serum of up to 4 hours. Oral dosing of ATRN-119 showed significant antitumor effects in human pancreatic and colon cancer xenografts and in orthotopic ovarian Patient Derived Xenograft (PDX) tumors. Notably, hematological analysis of mice treated with daily oral dosing of ATRN-119 indicated no thrombocytopenia, anemia or neutropenia up to 4 weeks of treatment. Exploratory multiple high dosing toxicity studies in rats and dogs indicate significant exposure and good tolerability with lack of anemia or neutropenia. In a series of in-vitro cell viability assays, three dimensional organoid cultures and in-vivo combinationstudies, ATRN-119 showed significant synergism with various PARP inhibitors as well as restoration of PARPi sensitivity in PARPi resistant tumors. Our data suggests a new generation of highly potent and selective ATR inhibitors with favorable safety profile and a broad clinical therapeutic potential either as monotherapy, in combination with PARP inhibitors or as a synthetic lethal approach with key DDR mutations.

Citation Format: Sahithi Pamarthy, Dansu Li, Ekaterine Goliadze, Tina Gill, Lanqi Jia, Erin George, Laura R. Butler, Ryan L. Ragland, Michel Afargan, Fiona A. Simpkins, Eric J. Brown, Oren Gilad. Highly specific macrocyclic ATR inhibitors for the targeted treatment of a broad spectrum of cancers showing lack of anemia or neutropenia in pre-clinical animal models [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 3498.
Abstract 352: The effect of anti-TNF-alpha Ab in AOM/DSS-induced colitic cancer mouse model
Authors: Ohnuma Shinobu , Yang Wang Dang , Suzuki Hideyuki , Kanehara Keigo , Karasawa Hideaki , Kohyama Atsushi , Watanabe Kazuhiro , Miura Koh , Kamei Takashi , Naitoh Takeshi , Unno Michiaki
Published: August 2019

Aim: To reveal whether anti TNF-α antibody (Ab) induces colon carcinogenesis or not in colitic cancer models.

Methods: The effects of TNF-α and anti TNF-α Ab were analyzed with cell-proliferation, migration, and invasion assays in human colon cancer cell lines, in vitro. Then, the effects of anti TNF-α Ab, infliximab (10 mg/kg, i.p.) were investigated with drug-induced colitis-associated colon cancer model by azoxymethane (AOM: 10 mg/kg, i.p.)/dextran sodium sulfate (DSS: 2.5%, oral) in vivo.

Results: 50 ng/ml of TNF-α inhibited cell proliferation, migration, and invasion of HCT8 and COLO205, however, anti TNF-α Ab offset the TNF-α mediated-inhibition in vitro. Infliximab significantly attenuated the development of colon cancers in AOM/DSS treated mice. The microarray analyses revealed that mast cell related genes (mast cell protease1, mast cell protease 2, chymase 1, etc) were down-regulated in cancer tissues of the infliximab-treated AOM/DSS mice. The results of microarray were validated by real-time RT-PCR. Finally, the number of mast cells were also reduced in those mice.

Conclusions: Mast cell may have a pivotal role in the development of colitic cancer. Anti TNF-α Ab may prevent colitic cancer in the patients with inflammatory bowel diseases.

Citation Format: Shinobu Ohnuma, Wang Dang Yang, Hideyuki Suzuki, Keigo Kanehara, Hideaki Karasawa, Atsushi Kohyama, Kazuhiro Watanabe, Koh Miura, Takashi Kamei, Takeshi Naitoh, Michiaki Unno. The effect of anti-TNF-alpha Ab in AOM/DSS-induced colitic cancer mouse model [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 352.
Abstract 3522: Enzymatic DNA repair enables high quality library preparation and accurate sequencing from highly damaged DNA inputs
Authors: Heider Margaret R. , Chen Lixin , Song Chen , Sun Luo , Liu Pingfang , Ettwiller Laurence , Higgins Lauren , Dimalanta Eileen , Davis Theodore , Evans Thomas
Published: August 2019

High-throughput sequencing is key for clinical diagnostic applications such as disease mutation profiling and identifying pathogens with high taxonomic specificity. Preparation of high quality next-generation sequencing (NGS) libraries is critical for obtaining accurate patient data, but clinical scientists are often limited by input DNA quality. In cancer genomics, formalin-fixed, paraffin-embedded (FFPE) tissue from patient biopsy is a common source of DNA, but the extracted DNA is often poor in both yield and quality due to fixation- and storage-induced damage. Additionally, these damage-induced sequencing artifacts raise the background level of mutations, making it difficult to discern true, low frequency, disease-related variants from noise. Even relatively high-quality DNA inputs contain nicks and gaps limiting library yields and read lengths for long read sequencing platforms including Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio).

We developed a second-generation DNA repair enzyme mix (V2) that efficiently repairs the most prevalent damage types found in FFPE DNA and further improves the quality and yield of NGS libraries through efficient nick and gap repair. Illumina library yield and quality were improved when DNA samples were treated with FFPE DNA Repair V2 before library preparation. Target enrichment and whole exome enrichment experiments showed that the V2 repair mix did not alter the overall frequency of variants identified, thus it did not introduce bias, but significantly improved the sequencing accuracy by reducing the number of false variant calls due to damage-induced sequence artifacts. True mutation frequencies were validated using droplet digital PCR (ddPCR). Repaired ONT libraries using DNA inputs of varying quality also showed improved library quality as well as greater read lengths. Therefore, enzymatic DNA repair prior to NGS library preparation is a critical first step for improving the quality and accuracy of short- and long-read sequencing libraries.

Citation Format: Margaret R. Heider, Lixin Chen, Chen Song, Luo Sun, Pingfang Liu, Laurence Ettwiller, Lauren Higgins, Eileen Dimalanta, Theodore Davis, Thomas Evans. Enzymatic DNA repair enables high quality library preparation and accurate sequencing from highly damaged DNA inputs [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 3522.
Abstract 3527: Combined high-throughput DNA genotyping and protein quantification in single cancer cells
Authors: Demaree Benjamin , Delley Cyrille , Ruff David , Treusch Sebastian , Eastburn Dennis , Jones Keith , Abate Adam
Published: August 2019

Clonal genetic diversity is a hallmark of tumor biology. In many cancers, clonal evolution has been implicated as a driver of tumor initiation, progression, and relapse, and can be a prognostic indicator of disease outcome. Cancer cells are also phenotypically heterogeneous, constituting features which can enable intravasation and metastasis, or which can impart immunoevasive capabilities. Clinically, a combination of single-cell genotype and phenotype data from cancer biopsies could provide valuable insights into oncological disease states, such as metastatic risk or drug response. Here we present a multiomic technology for the simultaneous, high-throughput capture of DNA genotype and protein information from single cells, demonstrated through the analysis of acute myeloid leukemia (AML) derived cell lines. The experimental workflow leverages the high-throughput single-cell microfluidic droplet technology on the Mission Bio Tapestri platform, a system optimized to conduct multiplex targeted genomic DNA amplification from >50 AML-associated loci. In our modified workflow, cells are stained with a pool of monoclonal antibodies conjugated with barcoded oligonucleotides, enabling a sequencing-based readout of single-cell protein signatures. The sequencing data is analyzed with a bioinformatic pipeline that separates antibody signal from targeted genotyping data on a cell-by-cell basis. We find that on a mixed population of myelogenous leukemia cells and B-cells, single-cell genotype information effectively distinguishes between cell lines, while the protein data independently recapitulates the biological signal from conventional flow cytometric methods. In conclusion, this technology opens up a new era for discovery using multiomic analysis of the complex relationships between genotype and phenotype in cancer, paving the way for future studies analyzing primary tissue samples.

Citation Format: Benjamin Demaree, Cyrille Delley, David Ruff, Sebastian Treusch, Dennis Eastburn, Keith Jones, Adam Abate. Combined high-throughput DNA genotyping and protein quantification in single cancer cells [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 3527.
Abstract 3528: Novel enzymatic DNA fragmentation integrated into library construction
Authors: Panchapakesa Vaishnavi , Apone Lynne , Duggan Karen , Langhorst Bradley , Song Chen , Liu Pingfang , Shtatland Timur , Rozzi Christine , Sumner Christine , Stewart Fiona , Dimalanta Eileen , Davis Theodore
Published: August 2019

Next Generation Sequencing (NGS) is rapidly becoming a powerful tool, by providing a high resolution and global view of the cancer genome mainly through whole genome, whole exome and transcriptome sequencing. This knowledge is important to support drug development targeted towards precision medicine. Obtaining high quality sequencing data begins with optimal sample preparation. DNA fragmentation is one of the critical first steps in the construction of high quality libraries for NGS, but current fragmentation methods create a bottleneck on library preparation throughput. We have developed an efficient, one tube fragmentation system that integrates enzyme-based DNA fragmentation with end-repair and dA-tailing in a single step, followed by adaptor ligation in the same tube. This streamlined method is compatible with a wide range of DNA samples and input amounts, resulting in reproducible and reliable fragment sizes.

Genomic DNA (100pg to 500ng) with various GC content, was used in the construction of libraries, that were sequenced on an Illumina platform. For low input samples, PCR amplification was performed prior to sequencing. Libraries constructed using intact genomic DNA and this novel library preparation method produced substantially higher yields than standard methods and mechanically-fragmented DNA. Enzyme-based shearing increases library yield by reducing DNA damage and sample loss. High library yields also enable PCR-free workflows from as little as 50ng starting material. This method can be used for DNA extraction and library preparation from whole blood and dried blood samples.Sequencing quality of libraries generated with inputs ranging from 100pg to 500ng DNA show similar coverage uniformity and fragment size distribution, with minimal GC bias.

We have developed an enzymatic DNA fragmentation kit that enables the construction of high quality libraries from a range of sample types and inputs with minimal sequence bias, high yields and low duplication rates. This method eliminates the need for expensive equipment to fragment DNA as well as numerous cleanup and liquid transfer steps thereby reducing the time, cost and errors associated with library construction. These advances in NGS technologies, supported by powerful bioinformatics tools, promises to revolutionize cancer research, diagnosis and therapy.

Citation Format: Vaishnavi Panchapakesa, Lynne Apone, Karen Duggan, Bradley Langhorst, Chen Song, Pingfang Liu, Timur Shtatland, Christine Rozzi, Christine Sumner, Fiona Stewart, Eileen Dimalanta, Theodore Davis. Novel enzymatic DNA fragmentation integrated into library construction [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 3528.
Abstract 3535: Identification of multiple hepatocellular carcinoma using genomic approach
Authors: Midorikawa Yutaka , Yamamoto Shogo , Tatsuno Kenji , Ueda Hiroki , Takayama Tadatoshi , Aburatani Hiroyuki
Published: August 2019

Multiple hepatocellular carcinoma (HCC) is categorized into two types; multicentric hepatocarcinogenesis (MC) and intrahepatic metastasis (IM). Although clinical discrimination of the types of multiple HCC has been proposed, it is quite difficult to determine it correctly even after histological diagnosis. In order to diagnose multiple HCC as MC or IM, we compared pairs of multiple HCC samples from 60 patients with multiple HCC and 2 patients with pairs of primary HCC and extrahepatic metastasis such as adrenal grand and lung metastasis with regard to molecular aberrations. Exome or capture sequences showed that more than 70% of somatic mutations were common in the pairs of IM, which were consistent with the result of a pair of primary HCC and extrahepatic metastasis, while no common somatic mutations was detected in genomic MC pairs. Notably, more than 60% of patients who were clinically diagnosed metachronous MC were genetically IM due to the concordance of mutation and shows significantly frequent tumor thrombus, suggesting that genomic IM pairs developed from the common ancestor. Epigenetically, methylation profiles were similar in a pair of IM, while those of MC were different each other. Consequently the accuracy of clinical diagnosis of multiple HCC was only 53.3%. In addition, cell-free DNA was analyzed using exome sequence in five patients with recurrent HCC, and found that somatic mutations of cell free DNA were similar to the primary HCC only in genomic IM but not in genomic MC cases. Intriguingly, overall survivals of patients with clinical IM was significantly shorter than those with clinical MC, while overall survivals of patients with genomic IM and genomic MC were almost similar. Taken together, comparison of mutations in a pair of HCCs makes it possible to classify multiple HCC into MC and IM, which is difficult to be correctly determined in clinical practice. In addition our data showed that it is not clinically important to determine the types of multiple HCC. Furthermore liquid biopsy for mutation analysis is available to make treatment plans for multiple HCC patients.

Citation Format: Yutaka Midorikawa, Shogo Yamamoto, Kenji Tatsuno, Hiroki Ueda, Tadatoshi Takayama, Hiroyuki Aburatani. Identification of multiple hepatocellular carcinoma using genomic approach [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 3535.
Abstract 3537: Targeted deep sequencing of driver mutations in airway epithelial cells from smokers
Authors: Craig Daniel J. , Blomquist Thomas M. , Crawford Erin L. , Willey James C.
Published: August 2019

Background: Inter-individual variation in risk for lung cancer is based on variation in both exposure to environmental factors and genetic predisposition. As such, it is reasonable to hypothesize that prevalence of mutations in the airway epithelium will vary based on the combined effects of these lung cancer risk factors. Genes most commonly mutated in non-small cell lung cancer were recently reported from The Cancer Genome Atlas (TCGA) project. The purpose of this study was to use targeted deep sequencing to assess the prevalence of these mutations in bronchial epithelial cells (BEC) of individuals at high demographic risk for lung cancer.

Methods: We enrolled 19 individuals with varying demographic risk for lung cancer, who were undergoing standard-of-care bronchoscopy into an IRB-approved research study in which we collected 1-5 million BEC via bronchoscopic brush biopsy and extracted genomic DNA. 10 subjects were smokers with lung cancer, 6 subjects were smokers without lung cancer, and 3 subjects were non-smokers without lung cancer. For targeted deep sequencing, we prepared synthetic spike-in competitive internal standards for each of 12 gene loci commonly mutated in lung cancer based on TCGA results and combined them into an IS mixture (ISM). DNA aliquots from each patient were quantified and mixed with equivalent genome equivalents of ISM prior to library preparation to control for technical artifacts associated with library preparation and NGS platform-specific error. The DNA input varied depending on sample quantity; there were 5 samples with 50,000 genome copies loaded, 12 samples with 100,000 genome copies loaded, and 2 samples with 1 million genome copies loaded. This approach enabled reliable measurement of 95% confidence limits of variant allele frequency (VAF), controlled for technical sequencing error, and allowed confident call of variants at a VAF of 0.1% and lower. Libraries for specimens from each of the 19 subjects were first sequenced using the Illumina MiSeq platform to ensure equal coverage across targets and samples. Deeper sequencing was then obtained using the Illumina NextSeq platform.

Results: Preliminary results showed that smokers harbored mutations up to 1.6% in gene loci associated with non-small cell lung cancer according to TCGA, while none were identified in non-smoking controls.

Conclusion: Initial results suggest that airway epithelial cells in smokers acquire somatic mutations that gradually lead to malignant conversion. Deep sequencing replicate experiments are underway to expand these results.

Citation Format: Daniel J. Craig, Thomas M. Blomquist, Erin L. Crawford, James C. Willey. Targeted deep sequencing of driver mutations in airway epithelial cells from smokers [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 3537.
Abstract 3539: Impact of formalin time on targeted NGS performance in FFPE tissue
Authors: Blomquist Thomas M. , Crawford Erin , Willey James , Xu Joshua ,   Onco-panel Working Group of the Sequencing Quality Control Phase 2 (SEQC2) Consortium
Published: August 2019

Targeted next generation sequencing (NGS) panel testing is now a standard of care for management of advanced stage cancer. At present, most targeted NGS oncology testing occurs with formalin fixed paraffin embedded (FFPE) tissue. This is due to the established utility and prevalence of FFPE in routine histologic classification, grading, staging, and ancillary testing. In addition, FFPE materials are an optimally suited medium for determining adequacy of the specimen prior to downstream NGS targeted panel testing. For these reasons, as well as a growing trend of minimally invasive procedures, clinical specimens are increasingly prioritized for FFPE to meet the bulk of standard oncology care. Currently, formalin fixation time guidelines do exist for specific immunohistochemical and in-situ hybridization oncology tests. However, specific FFPE processing guidelines have yet to be established that would optimize targeted NGS panel testing performance.

In this study, our aim was to develop contrived FFPE materials using the FDA-led Sequencing Quality Control Phase II (SEQC2) consortium working group 2 (WG2) cell line materials. The overarching goal was to use these FFPE materials to identify an upper limit of formalin time associated with minimal impact on somatic variant calling performance in targeted NGS oncology panels. The SEQC2 - WG2 normal lymphoblast cell line was selected as a well-characterized test material to undergo systematic alteration in formalin fixation time (1, 2, 6 and 24 hours) prior to “routine” tissue processing and embedding (FFPE). These FFPE cell-block materials, derived from a single sample source (with both extracted FFPE DNA as well as shaved sections), were distributed to multiple labs running various targeted NGS amplicon and hybridization oncology panels.

The expected outcome of these studies is to provide general guidance to the community on how formalin time in FFPE specimens impacts targeted NGS oncology panel testing performance. In addition, we hope to identify genomic regions (internal control regions) that serve as markers for the full range of formalin time FFPE-effect. This information will assist in improving the lower limit of detection for variant calling (<5% variant allele frequency).

Citation Format: Thomas M. Blomquist, Erin Crawford, James Willey, Joshua Xu, Onco-panel Working Group of the Sequencing Quality Control Phase 2 (SEQC2) Consortium. Impact of formalin time on targeted NGS performance in FFPE tissue [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 3539.
Abstract 3540: A complete solution for high throughput single cell targeted multiomic DNA and RNA sequencing for cancer research
Authors: Dhingra Dalia , Gokhale Kaustubh , Li Nianzhen , Mendez Pedro , Wang Shu , Manivannan Manimozhi , Sciambi Adam , Jones Keith , Silver Charlie , Eastburn Dennis , Ruff David
Published: August 2019

Recent advancements in single cell analysis technologies are now able to provide insights into genomic DNA content, RNA expression and protein surface markers. In bulk assays, the effect of genetic variation on gene expression would be masked by the heterogeneity inherent in tumor cells. Additionally, for cancer immunotherapy studies that rely on gene editing, single cell resolution is necessary to minimize possible off target effects. However, the ability to simultaneously interrogate multiple intracellular analytes, such as genomic DNA and RNA, have proved difficult to implement in a high throughput single cell workflow. We report the development of a complete solution that enables both targeted genomic DNA and RNA sequencing from individual cells.

This workflow relies on the Tapestri microfluidic droplet platform, where up to 20,000 cells can be sequenced in each run. Leveraging proprietary cell barcoding, novel primer design strategies and enzymatic manipulation of cellular contents, DNA and RNA multiplex targeted sequencing panels provide for independent barcoded sequence information from both overlapping mRNA and corresponding genomic DNA regions. In addition, amplification primers can be designed to target separate gDNA and non-overlapping RNA transcript. Sequencing is followed by an integrated analysis solution that assigns the reads from both the gDNA and RNA to each cell.

Feasibility of the targeted nucleic acid workflow has been shown with inputs from mixed cancer cell lines. A targeted sequencing panel with overlapping mRNA and gDNA regions was designed covering oncogenes, tumor suppressor genes, and known fusions. Expected SNVs and indels were detected and gene expression measured for thousands of cells per run with high cell recovery. This complete solution for single cell multiomics on the Tapestri platform has the power to quantitatively and unambiguously link genotypic and phenotypic data, giving insight into cancer progression.

Citation Format: Dalia Dhingra, Kaustubh Gokhale, Nianzhen Li, Pedro Mendez, Shu Wang, Manimozhi Manivannan, Adam Sciambi, Keith Jones, Charlie Silver, Dennis Eastburn, David Ruff. A complete solution for high throughput single cell targeted multiomic DNA and RNA sequencing for cancer research [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 3540.
Abstract 3556: MiR-506 suppresses tumor progression by reprogramming macrophage polarization in pancreatic ductal adenocarcinoma
Authors: Sun Longhao , Song Qianqian , Tian Weijun , Zhang Zhixiang
Published: August 2019

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancy owing to its aggressive nature and tumor-associated macrophages (TAMs) significantly promote disease progression by representing an immunosuppressive phenotype in PDAC. Macrophages could be functionally reprogrammed to opposite phenotype owning to different microenvironment and STAT3 (signal transducer and activator of transcription 3) activation may play an essential role in TAM polarization, directly contributing to local immunosuppression. Our recent study demonstrated that MiR-506 functioned as a tumor suppressor in many cancer types through the regulation of multiple pathways and directly targeted STAT3 in PDAC cells. In this study, we hypothesized that MiR-506 could affect tumor microenvironment and exert a tumor suppressive function in PDAC by reprograming TAM polarization through targeting STAT3 in TAM. Our results provided evidence that MiR-506 downregulation was associated with less immunosuppressive TAMs accumulation and better prognosis in human PDAC. MiR-506 could reduce the infiltration of immunosuppressive TAMs and inhibit the PDAC progression in mouse model. MiR-506 reprogramed macrophage polarization from an immunosuppressive phenotype to immune-activated phenotype through direct targeting the STAT3 axis. Silencing and inhibiting STAT3 recapitulated the effects of MiR-506. These findings expand the known mechanisms of MiR-506-mediated tumor suppression to reprogramming TAM polarization and suggest a strategy for using MiR-506 as an anti-STAT3 approach to PDAC immunotherapy.

Citation Format: Longhao Sun, Qianqian Song, Weijun Tian, Zhixiang Zhang. MiR-506 suppresses tumor progression by reprogramming macrophage polarization in pancreatic ductal adenocarcinoma [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 3556.