bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1242: RAB20 is a stress-induced regulator of mitochondrial homeostasis
Authors: Nayak Sunayana Govind , Shaw Samantha , Stets Colin , Dent Anthony , Strohecker Anne Marie
Published: September 2020

Mitochondrial homeostasis is tightly regulated via a combination of biogenesis and elimination via a form of selective autophagy called mitophagy. Given the importance of mitochondria for energy production, cellular redox status, and apoptotic signaling, the mitophagy pathway is of particular interest in cancer. While mitophagy has been reported to both assist and hinder tumorigenesis in different studies, the emerging consensus is that mitophagy supports tumor growth. Intriguingly, only a subset of the canonical autophagy machinery is required for mitophagy. Upstream regulators including ULK1, the phosphatidyl-inositol-3-phosphate (PI3P) effectors and components of the omegaosome DFCP1 and WIPI, and ATG9 are recruited to mitochondria independent of LC3. How these components are recruited to mitochondria and the mechanisms involved in engulfment/creation/scaling of the autophagosome around the mitochondrion are unclear. Here we report a novel role for the small GTPase RAB20 in mitochondrial homeostasis. Rab GTPases are master regulators of vesicle trafficking, templating membrane identity, coordinating vesicle docking, fusion, and cargo selection in concert with SNAREs and membrane tethers. Rab20 is normally expressed at low levels but can be induced by in vitro simulated metabolic stress (1% oxygen, glucose deprivation) or secondary to the stress of an actively growing tumor in vivo. Surprisingly, we discovered that silencing RAB20 in multiple Non-Small Cell Lung Cancer (NSCLC) cell lines impairs ischemia-induced mitophagy without impacting autophagic flux measured by LC3 lipidation. This defect is accompanied by an increase in mitochondrial mass. RAB20 silencing is associated with increased Reactive Oxygen Species both under nutrient replete and ischemic conditions. Further, Rab20 appears to influence mitochondrial network organization, manifest as elevated numbers of fragmented mitochondria in the Rab20 silenced cells compared to non-targeting vector isogenic vector control cells. Finally, mitochondrial respiration is impaired in RAB20 silenced cells. Taken together, this work indicates an important role for RAB20 in maintaining mitochondrial fitness. We are actively assessing the functional consequences of Rab20 ablation on lung tumorigenesis in an established mouse model of lung cancer driven by BrafV600E; Trp53−/−. These studies will be important in determining the role of RAB20 and its involvement in mitophagy on tumor progression in vivo.

Citation Format: Sunayana Govind Nayak, Samantha Shaw, Colin Stets, Anthony Dent, Anne Marie Strohecker. RAB20 is a stress-induced regulator of mitochondrial homeostasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1242.
Abstract 1277: SPARCL1 exhibits different expression pattern between left- and right- sided colon cancer and it was down-regulated via DNA methylation
Authors: Wu Dehao , Cai Wen , Xu Junxi , Liu Xue , Bai Rui , Zheng Shu , Ge Weiting , Hu Hanguang
Published: September 2020

Background: We previously identified SPARC-like 1 (SPARCL1) as a suppressor for colon cancer metastasis. Recent studies confirmed its epigenetic modification in prostate cancer and osteosarcoma. Here we conducted in vivo and in vitro studies to investigate the mechanism of SPARCL1 epigenetic regulation and the relationship with clinicopathological features.

Method: Tissue array (271 left- [LCC] and 257 right-sided colon cancer [RCC]) and immunohistochemistry were performed to measure SPARCL1 expression. Methylation status was compared by bisulfite sequencing PCR between 17 LCC and 16 RCC. Epigenetic alteration and rescue experiment were performed in colon cancer cell lines (HCT-116, RKO and HT-29) using decitabine, panobinostat, or siRNA. Transwell assay was employed to compare cell mobility. Chi-square test, Mann-Whitney U test were used for comparing SPARCL1 expression or other clinical characteristics, and t-test for cell motility.

Result: RCC significantly lower expressed SPARCL1 protein than LCC (P =0.007). High methylated CpG site of SPARCL1 enriched in RCC not in LCC (56.25% vs. 29.41%). In vitro studies confirmed that decitabine could up regulate SPARCL1 transcription by 72-fold. Transwell assays further revealed that significant inhibition of decitabine in decreasing cells invasion and migration properties, which could be rescued by siRNA against SPARCL1. In addition, we didn't observe transcriptional alteration with panobinostat treatment.

Conclusion: DNA methylation not histone acetylation modulated SPARCL1 expression and suppress tumor metastasis, which may contribute to aberrant expression pattern and different clinical outcomes varying in left and right-colon cancer.

Citation Format: Dehao Wu, Wen Cai, Junxi Xu, Xue Liu, Rui Bai, Shu Zheng, Weiting Ge, Hanguang Hu. SPARCL1 exhibits different expression pattern between left- and right- sided colon cancer and it was down-regulated via DNA methylation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1277.
Abstract 1285: Functional analysis of ZFHX4 as a novel therapeutic target in ovarian cancer
Authors: Oyama Yoshiko , Shigeta Shogo , Tsuji Keita , Tokunaga Hideki , Shimada Muneaki , Yaegashi Nobuo
Published: September 2020

[Introduction] Ovarian cancer is one of the most lethal gynecologic malignancies. More than half of the patients are diagnosed in advanced stages of disease where the prognosis is poor due to the inevitable acquired resistance to platinum agents. Although maintenance therapy with PARP inhibitors or bevacizumab has been proven to improve patient survival, platinum sensitivity is still the most important prognostic factor. Thus, novel therapeutic strategies effective for platinum-resistant cases or to overcome platinum-resistance are required.

Zinc Finger Homeobox 4 (ZFHX4) has been known as one of the major transcription regulators in nerve and muscle differentiation. Although not fully elucidated, recent researches including our genome-wide exploration indicate ZFHX4 is also involved in carcinogenesis and cancer progression. The objective of this study was to investigate the potential of ZFHX4 as a novel therapeutic target in ovarian cancer by analyzing clinical database and cell model experiments.

[Methods] Correlations between ZFHX4 mRNA expression and overall survival in 61 ovarian cancer cases treated at our institute and in the cases registered in TCGA database respectively were assessed by log-lank test. RNAi knockdown was introduced to several ovarian cancer cells to investigate the influence of ZFHX4 on cancer cell proliferation. Scratch assay was performed to assess the effect of RNAi knockdown on cell migration ability. Cells were also treated with cisplatin after 72 hours of siRNA transfection to assess the influence of ZHFX4 on platinum sensitivity.

[Results] Both in our clinical dataset and TCGA data, the overall survival was significantly poor in the group in which ZFHX4 is highly expressed. In cell model assay, ZFHX4 knockdown significantly suppressed ovarian cancer cell proliferation. Decreased motility was observed in the cells treated with siRNA targeting ZFHX4 by scratch assay. Furthermore, ZFHX4 knockdown increased cisplatin sensitivity in ovarian cancer cells.

[Conclusions] Altogether, our results suggest ZFHX4 functions to promote cancer cell proliferation, migration and platinum-resistance. Interruption of ZFHX4 function independently or in combination with platinum agents has a potential to bring benefit for the treatment of ovarian cancer.

Citation Format: Yoshiko Oyama, Shogo Shigeta, Keita Tsuji, Hideki Tokunaga, Muneaki Shimada, Nobuo Yaegashi. Functional analysis of ZFHX4 as a novel therapeutic target in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1285.
Abstract 1308: CDK12 alterations define a distinct molecular subtype of prostate cancers with unique genomic and clinical characteristics
Authors: Nguyen Bastien , Mota Jose Mauricio , Nandakumar Subhiksha , Stopsack Konrad H. , Weg Emily , Rathkopf Dana , Morris Michael J. , Scher Howard , Kantoff Philip W. , Gopalan Anuradha , Zamarin Dmitriy , Solit David B. , Schultz Nikolaus , Abida Wassim
Published: September 2020

Background: CDK12 alterations are present in several tumor types but little is known regarding their oncogenic role and their clinical significance. Here we sought to describe the landscape of CDK12 alterations across solid cancers and the clinical features of CDK12-altered prostate cancer.

Methods: A total of 26,743 patients across 25 solid tumor types with available targeted sequencing data from MSK-IMPACT as part of their clinical care were included. Clinicopathological features and outcomes were assessed in prostate cancer. CDK12 alterations and association with genomic characteristics are described. For prostate cancer patients, overall survival from the date of metastasis and time to castration-resistance from the start of androgen deprivation therapy were assessed using univariable and multivariable Cox regression analysis.

Results: CDK12 alterations were identified in 404/26,743 (1.5%) patients overall, but were most frequent in prostate (100/1875, 5.3%) and ovarian cancer (43/1034, 4.2%) where they were associated with a high prevalence of truncating variants and biallelic inactivation. CDK12 alterations defined a distinct subtype of prostate cancer with more aggressive clinical features, a unique copy number alteration profile and involvement of distinct oncogenic pathway alterations, including cell cycle pathway genes. Independent of standard clinical factors and genomic instability, CDK12-altered prostate cancer patients were associated with shorter overall survival (median 64.4 vs 74.9 months, adjusted hazard ratio [aHR] 1.65, 95% CI 1.07-2.53, p = 0.03) and shorter time to the development of castration-resistant disease (median 10.8 months vs 13.1, aHR 1.43, 95%CI 1.07-1.8, p = 0.02).

Conclusions: CDK12 loss of function is a rare event across solid cancers but defines a distinct molecular subtype of prostate cancer that is associated with more aggressive clinical features and unique genomic alterations.

Citation Format: Bastien Nguyen, Jose Mauricio Mota, Subhiksha Nandakumar, Konrad H. Stopsack, Emily Weg, Dana Rathkopf, Michael J. Morris, Howard Scher, Philip W. Kantoff, Anuradha Gopalan, Dmitriy Zamarin, David B. Solit, Nikolaus Schultz, Wassim Abida. CDK12 alterations define a distinct molecular subtype of prostate cancers with unique genomic and clinical characteristics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1308.
Abstract 1313: Genomic and transcriptomic profiles of primary prostate cancer identify unique targets for therapeutic intervention
Authors: Chakravarty Dimple , Wang Zichen , Rossi Michael , Ratnani Parita , Reva Boris , Rykunov Dmitry , Bhatt Kamala , Wagaskar Vinayak , Weil Rachel , Beaumont Kristin , Beaumont Michael , Maayan Avi , Sebra Robert , Bhardwaj Nina , Nair Sujit S. , Tewari Ashutosh K.
Published: September 2020

The tumor microenvironment and genomic landscape of intermediate and high-risk primary localized prostate cancers are clinically heterogeneous and result in variable treatment response in individuals. Cancer-specific alterations at DNA and RNA level is a critical driver of intra-tumoral heterogeneity that significantly impacts the molecular processes which influence the path of disease progression and outcome. In this study, we have performed genomic and transcriptomic analysis of 267 primary prostate cancer. Our analysis revealed actionable alterations and gene signatures for risk stratification and decision support. Clinically significant variants were detected in DNA repair (HR and Mismatch pathway), PI3/AKT, and TP53 signaling pathways with a strong association with disease progression. Further, single-cell analysis (scRNA-seq) of prostate cancer that progressed to metastasis post-therapy identified the unique tumor and immune cell-clusters. Functional annotation of these clusters and drug repurposing studies revealed novel targets for therapeutic intervention that was validated using organoid cultures. Our studies show that an Integrated analysis using genomic, clinical, and pathological features has the potential to define prostate cancer subtypes or phenotypes associated with poor prognosis. Our approach can advance prostate cancer treatment in patients with adverse clinical features by providing treatment options beyond the standard of care (surgery or radiation) and with better therapeutic benefit.

Citation Format: Dimple Chakravarty, Zichen Wang, Michael Rossi, Parita Ratnani, Boris Reva, Dmitry Rykunov, Kamala Bhatt, Vinayak Wagaskar, Rachel Weil, Kristin Beaumont, Michael Beaumont, Avi Maayan, Robert Sebra, Nina Bhardwaj, Sujit S. Nair, Ashutosh K. Tewari. Genomic and transcriptomic profiles of primary prostate cancer identify unique targets for therapeutic intervention [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1313.
Abstract 1320: The landscape of somatic substitutions in the repetitive genome across cancer types
Authors: Tarabichi Maxime , Demeulemeester Jonas , Verfaillie Annelien , Van Loo Peter , Konopka Tomasz
Published: September 2020

Somatic mutation calling from bulk DNA sequencing is a complex problem susceptible to elevated false positive rates. High mapping quality is considered an important feature of reliable variant calls. At the resolution of short reads, ~10% of the genome displays high sequence similarity with at least one other genomic region and is assigned low mapping quality by alignment algorithms. These low-mapping-quality regions represent recurrent blind spots for mutation callers, which discard many of the variants they harbor, overlooking true biological variation.

Here, we developed a pipeline to call substitutions in the low-mapping-quality genome. We used a published thesaurus approach to annotate the variant positions with their high-similarity links. We trained a classifier to emulate high-quality consensus calls made in unique regions using 20 features unrelated to mapping quality, reaching ~95% accuracy in those regions. In an independent sample more than 90% of the thesaurus calls were validated through linked-read sequencing. We then applied the classifier to all candidate substitutions of 2,658 cancer whole genomes from the PCAWG/ICGC consortium including variants falling in low-mapability regions.

We retrieve hidden thesaurus variants genome-wide in ~6% of the genome, including genic, coding, and promoter regions. Thesaurus calls are directly proportional in numbers to somatic calls falling in the low-mapping-quality genome and share a similar trinucleotide context spectrum. Rescuing these mutations reveal hidden signal in known cancer genes, including PIK3CA, and excess of mutations genome-wide in promoter, untranslated, and coding regions of many other genes. We also find potential excess of non-synonymous mutations, including in genes from the TRIM and POTE families, having been previously implicated in multiple cancer types.

Altogether, we developed a pipeline to call somatic substitutions in the low-mapping-quality genome and uncovered hidden somatic changes along the genomes of human cancers. In the future, this pipeline could be extended to indels and structural variants, and applied to the study of de novo germline variants.

Citation Format: Maxime Tarabichi, Jonas Demeulemeester, Annelien Verfaillie, Peter Van Loo, Tomasz Konopka. The landscape of somatic substitutions in the repetitive genome across cancer types [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1320.
Abstract 1330: Scalable single-day workflows for variant calling with the Genexus Integrated Sequencer
Authors: Gioia Jason , Casuga Iris R. , Chan Frances , Commins James , Conners David , Kratzer Marla , Seeger Collyn , Yaccarino David
Published: September 2020

The purpose of this study is to test the speed, scalability, and flexibility of variant detection with the Genexus Integrated Sequencer. This research use only instrument uses nucleic acid input and executes the steps of library preparation, sequencing, analysis, and variant calling. Taking advantage of the instrument's flexibility in sample plexy, the authors used the Oncomine™ Comprehensive Assay v3 and commercially available control samples to detect SNPs, InDels, and fusions reliably at up to 6-plex paired DNA and RNA samples. The instrument can accommodate 1, 2, 4, or 6 paired DNA and RNA samples when using 1, 2, 3, or 4 lanes of the GX5 chip. Regardless of the number of samples analyzed, the system achieved at least 98% sensitivity, 98% positive predictive value, 95% agreement to known truth for de novo variants, and 98% agreement to known truth for hotspot variants in the Acrometrix™ Oncomine Hotspot Control. Also regardless of sample plexy, the system achieved at least 97% agreement to known truth for Fusions within the SeraSeq® Fusion RNA Mix v3. The flexible workflows allow for fast turn-around times of as little as 18 hours for a single sample pair and less than 30 hours for 6 paired DNA and RNA samples. Stability of system initialization is an important feature for flexible use of an NGS system, and read length is a limiting performance metric for evaluating stability. In order to test the Genexus system's read length and stability features the Oncomine™ TCR Beta-LR Assay GX was used. Instruments were initialized with sequencing consumables and tested for performance on day 1 and day 15. There was no statistically significant difference in total reads (3.5 ± 1.0 M at day 1 vs. 3.1 ± 1.0 M at day 15), the fraction of reads that are low quality reads (13% ± 2% vs. 14% ± 2%), or mean read length (301 ± 6 bases vs. 296 ± 6 bases) between the two time points. In conclusion, the Genexus integrated sequencer is a robust NGS system that facilitates rapid turn-around time, flexibility in sample plexy, and stability in system consumables use.

Citation Format: Jason Gioia, Iris R. Casuga, Frances Chan, James Commins, David Conners, Marla Kratzer, Collyn Seeger, David Yaccarino. Scalable single-day workflows for variant calling with the Genexus Integrated Sequencer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1330.
Abstract 1335: Analytical evaluation of a targeted solid tumor next-generation sequencing assay
Authors: Tsai Jeff H. , Montel Valerie , Mouttham Nathalie
Published: September 2020

With the growing interest in personalized medicine for cancer patients, next generation sequencing (NGS) variant screening assays are becoming a powerful clinical tool for tumor classification. The AmpliSeqTM for Illumina Focus Panel is a targeted sequencing approach that interrogate 52 of the most common solid tumor genes for single nucleotide variants (SNVs), insertion/deletions (indels), and copy number variants (CNVs) in DNA and gene fusions in RNA concurrently. To evaluate the performance of the Focus assay, an analytical performance study was conducted. Using an automated workflow with high quality DNA reference samples, both intra-assay and inter-assay experiments resulted in high reproducibility (R2 >0.95) for detecting expected variants at various frequencies. Similarly, using high quality RNA reference samples, 100% of all expected fusions were detected in all experiments, demonstrating accuracy and precision of the Focus panel assay with high quality samples. To evaluate assay sensitivity, limit of detection (LOD) studies were carried out by titrating the nucleic acid input into the assay. Using a 5% variant allele frequency threshold (empirically established in the analysis pipeline), the assay detected all expected SNVs and indels across sample across sample input amounts. Using extracted DNA from a human cell line model (HL-60) with a confirmed MYC gene gain, results showed reliable MYC gain detection down to 4 copies in a diploid background. Similarly, extracted RNA from a confirmed EML4-ALK fusion positive cell line (H2228) diluted into background RNA showed 100% positive fusion calling down to 250pg or 133 copies. Finally, to understand the assay performance on clinically-relevant FFPE samples, somatic variant detection in 8 human cell lines and 68 total FFPE samples was evaluated. For FFPE DNA samples, the libraries averaged >90% on-target alignment and coverage uniformity with >3000x mean coverage across all samples. While FFPE RNA showed lower alignment for poor quality samples, the assay showed overall high positive and negative predictive value for variant and fusion calling. In summary, the AmpliSeqTM for Illumina Focus panel is an automation-compatible small panel NGS assay with robust performance to assess somatic variants in solid tumors.

Citation Format: Jeff H. Tsai, Valerie Montel, Nathalie Mouttham. Analytical evaluation of a targeted solid tumor next-generation sequencing assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1335.
Abstract 1344: Simultaneous mRNA, protein, and immune repertoire profiling of antigen-specific T cells by single cell sequencing
Authors: Siddique Imteaz , Shi Xiaoshan , Nakamoto Margaret , Mortimer Stefanie
Published: September 2020

High-throughput single cell RNA-seq (scRNA-seq) has transformed our understanding of complex and heterogenous immune populations. New advances in scRNA-seq are expanding the molecules that can be profiled at the single cell level, such as oligo-conjugated antibody technologies that enable protein expression profiling alongside mRNA. Although the ability to sequence the immune repertoire can provide crucial insights into understanding the complexities of the adaptive immune system and advancing discoveries in immuno-oncology, the ability to extract this information from single cells requires new technology to profile regions of the mRNA that are missed by conventional 3' scRNA-seq. In this study we utilized an ex vivo antigen stimulation system to measure antigen-specific T-cell activation and clonal amplification. Stimulated T cells from two donors were loaded onto the BD Rhapsody™ Single-Cell Analysis System to extract immune repertoire information in addition to gene and protein expression information from the same cells. In a single workflow, we profiled a panel of 400 mRNA targets, 20 BD® AbSeq protein markers associated with different status of T-cell activation, and the hypervariable region of T-cell receptors at the single cell resolution. The study is a proof of concept of combining mRNA, protein and immune repertoire analysis at the single cell level to deconvolute the heterogeneity and different activation of stimulated T cells.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

Citation Format: Imteaz Siddique, Xiaoshan Shi, Margaret Nakamoto, Stefanie Mortimer. Simultaneous mRNA, protein, and immune repertoire profiling of antigen-specific T cells by single cell sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1344.
Abstract 1368: 4NQO shows formation of yH2AX foci, oxidative base damage and activates DNA repair pathway preferentially in HPV positive HNSCC cells
Authors: Shishodia Gauri , Toledo Rhodee Ric G. , Rong Xiahuao , Xiao Adam Y. , Harrison Lynn , Nathan Cherie Ann
Published: September 2020

Background: There is an epidemic of HPV associated head and neck squamous cell carcinoma (HNSCC) There is controversy as to whether HPV positive SCC patients who have a >10 pack year smoking history have a worse prognosis than HPV associated non-smokers. Tobacco exposure is associated with alteration of DNA damage repair (DDR) pathway which is often activated in several cancers including HNSCC. A mutational profile of HPV associated smokers determined there was no significant difference in the mutational profile between HPV associated non-smokers and smokers. Hence we wanted to test this observation by analyzing the effects of 4NQO, a mutagen and carcinogen which serves as a surrogate for tobacco exposure, in HPV positive and HPV negative HNSCC cells.

Methods: Protein and mRNA expression were analyzed by western blotting and real time PCR respectively. yH2AX staining and oxidative DNA damage analysis were performed using immunofluorescence and FPG-modified alkaline comet assay respectively.

Results: Our preliminary data shows that 4NQO activates DDR pathway preferably in NOK E6/E7 and HPV positive UMSCC47 and SCC104 HNSCC cells as seen by increased phosphorylation of BRCA1, Rad50 and RAD51, chk1 and increased p21 levels in a time- and concentration-dependent fashion. Real time PCR data showed higher mRNA transcript levels of RAD51 and BRCA1 in NOK E6/E7 and HPV positive UMSCC47 and SCC104 HNSCC cells as compared to NOK and HPV negative SCC40 HNSCC cells upon 4NQO treatment. 4NQO causes yH2AX foci formation suggesting double strand breaks (DSBs) formation and an increase in DNA damage in HNSCC cells. 4NQO increased oxidative base damage in NOK E6/E7, UMSCC47 and SCC104 HPV positive HNSCC cells, and to a lesser degree in NOK and SCC40 HPV negative HNSCC cells.

Conclusion: Our preliminary study shows for the first time that HPV E6/E7 oncogenes promote DNA repair in NOK and HPV positive HNSCC cells in response to 4NQO treatment. This could explain why HPV associated HNSCC have a better prognosis irrespective of smoking history.

Citation Format: Gauri Shishodia, Rhodee Ric G. Toledo, Xiahuao Rong, Adam Y. Xiao, Lynn Harrison, Cherie Ann Nathan. 4NQO shows formation of yH2AX foci, oxidative base damage and activates DNA repair pathway preferentially in HPV positive HNSCC cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1368.
Abstract 1377: Identification and validation of WRN as a novel synthetic lethality target in context of microsatellite instability
Authors: Ding Zhihu (Jeff) , Zhang Jing , Pedamallu Chandra Sekhar , Rowley Steve , Cheng Jane , Dai Shujia , Zhou Bridget , Koundinya Malvika , Guo Zhuyan , Poirier Stephane , Hopke Joern , Lennon Amanda , Buell Jennifer , Cindhuchao May , Wong Karen , Wang Emma , Protopopov Alexei , Zhang Bailin , Hoffmann Dietmar , Sun Fangxian , Pollard Jack , Debussche Laurent , Bouaboula Monsif
Published: September 2020

Despite the significant clinical success of the Immuno-Oncology treatment of microsatellite instability (MSI) patients, there remains huge unmet medical need because of mechanisms of resistance. Through an effort to analyze publicly available large-scale shRNA screening and CRISPR screening, we surprisingly found that WRN is a potent synthetic lethality target in the context of MSI. WRN is an enzyme known as the “Werner syndrome ATP-dependent helicase”. WRN is involved in multiple cellular functions, including DNA repair and telomere maintenance. Silencing of WRN by RNAi and CRISPRi in a panel of MSI-H cells lead to tumor cell growth inhibition in vitro and in vivo, thus demonstrated WRN is a novel synthetic lethality target in context of MSI. Our discovery has recently been cross-validated by multiple independent studies1-4. Furthermore, we also report here the first time that silencing of WRN by RNAi and CRISPRi in DLD1, a MSI-H cell, does not lead to tumor cell inhibition. Taken together, our data using independent genetic approach CRISPRi in vitro and in vivo, further highlight fundamental importance of WRN as a synthetic lethality target in some, but not all, MSI context.

References: 1 Chan E.M. et al. (2019). Nature. 568(7753):551-556. 2 Behan F.M. et al (2019). Nature. 2019 568(7753):511-516. 3 Kategaya L. et al. (2019). iScience. 13:488-497. 4 Lieb S. et al. (2019). Elife. 8. pii: e43333.

Citation Format: Zhihu (Jeff) Ding, Jing Zhang, Chandra Sekhar Pedamallu, Steve Rowley, Jane Cheng, Shujia Dai, Bridget Zhou, Malvika Koundinya, Zhuyan Guo, Stephane Poirier, Joern Hopke, Amanda Lennon, Jennifer Buell, May Cindhuchao, Karen Wong, Emma Wang, Alexei Protopopov, Bailin Zhang, Dietmar Hoffmann, Fangxian Sun, Jack Pollard, Laurent Debussche, Monsif Bouaboula. Identification and validation of WRN as a novel synthetic lethality target in context of microsatellite instability [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1377.
Abstract 1402: Preclinical evaluation of a novel senescence-associated miRNA miR-3140-3p for malignant pleural mesothelioma
Authors: Yamamoto Yuki , Nishiura Ayaka , Yano Kimiyoshi , Matsuda Noriaki , Takahashi Ryou-u , Tsutani Yasuhiro , Okada Morihito , Tahara Hidetoshi
Published: September 2020

Cellular senescence acts as a natural barrier against tumor progression Our previous study showed that selective induction of senescence using miR-22, one of senescence-associated microRNAs (SA-miRNAs), suppressed tumor progression (JCB 2011) In this study, we developed functional screening system using miRNA library to identify a potent SA-miRNA that suppress malignant tumor such as anti-cancer drug resistant and cancer stem cells.

We focused on microRNA inducing the senescent phenotype in normal human fibroblast cell After transfection of 2028 miRNA mimics library in normal human fibroblast TIG-3, we evaluate the senescence phenotypes such as cell growth arrest and size enlargement using high content analyzer, and found that 349 miRNAs induced cellular senescence More importantly, we identified miR-3140-3p as potent SA-miRNA that significantly suppressed tumor cell proliferation in several types of cancer cells including cancer stem cells and anti-cancer drug resistant cells.

We focused on malignant pleural mesothelioma (MPM) which is an aggressive tumor developed from the mesothelial lining cells of the pleura MPM is caused by direct asbestos exposure and is an incurable tumor type for the lack of effective treatment In addition, as Japan is one of the world's highest importers and users of asbestos, the cases of mesothelioma are expected to increase in the near future Therefore, we investigated the possibility of whether SA-miRNAs showed tumor suppressive effects against MPM cell lines Moreover, we tried to develop a new treatment or medicine for the MPM patients using SA-miRNA In vitro and in vivo analysis revealed that several SA-miRNAs drastically suppressed the tumor cell proliferation

Based on these findings, the modulation of SA-miRNA is a promising approach to improve the treatment of MPM patients In addition, we performed and completed pre-clinical trials and design first-in-human study in Japan.

Citation Format: Yuki Yamamoto, Ayaka Nishiura, Kimiyoshi Yano, Noriaki Matsuda, Ryou-u Takahashi, Yasuhiro Tsutani, Morihito Okada, Hidetoshi Tahara. Preclinical evaluation of a novel senescence-associated miRNA miR-3140-3p for malignant pleural mesothelioma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1402.
Abstract 1403: HOTAIR functions through NF-κB pathway in regulating ovarian cancer stem cells
Authors: Wang Weini , Fang Fang , Özeş Ali R. , Nephew Kenneth P.
Published: September 2020

Ovarian cancer (OC) is the fifth leading cause of cancer-related death among American women. Persistence of OC stem cells (OCSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse. Long non-coding RNA HOXC transcript antisense RNA (HOTAIR) has been shown to be associated with chemoresistance and overexpressed in many types of cancers, including high-grade serous OC (HGSOC). Previously published work has demonstrated that NF-κB was activated by HOTAIR through I-κB inhibition via trimethylation of histone H3 lysine K27, which contributes to chemoresistance in HGSOC. NF-κB-medicated signaling pathways involved in maintaining characteristics of CSCs, such as targeting stem cell markers, CD44, CD133, and ALDH1 has been demonstrated but not well defined in OC. The goals of this study are to understand the mechanism of HOTAIR-mediated NF-κB signaling pathway in regulating OCSCs and develop novel strategies to target OCSCs and overcome OC recurrence and drug resistance. Quantitative RT-PCR analysis revealed that HOTAIR was overexpressed in OCSCs compared to non-OCSCs. In order to produce loss-of-function phenotypes of HOTAIR and investigate the function of this gene, we utilized the paired CRISPR guide RNA design to delete the functional sites of HOTAIR without affecting nearby protein-coding gene. Knockout of HOTAIR re-sensitized OC cells to platinum treatment and significantly decreased (P<0.001) OCSC population and stemness-related phenotypes, including spheroid formation and colony formation ability. ALDH1A1 and expression of other stemness-related genes, including Notch3, PROM1 were significantly decreased by HOTAIR knockout. Overexpression of HOTAIR in OC cells significantly increased (P<0.05) these stem-like characteristics. Furthermore, we showed NF-κB nuclear accumulation and activation in OCSCs compared to non-OCSCs. Knockout of HOTAIR decreased (P<0.01) nuclear localization of NF-κB in OCSCs. Inhibiting NF-κB in using a commercially available pharmacological approach as well as NF-κB knockout both significantly decreased OCSC population and stemness-related phenotypes, as well as ALDH1 protein expression (P< 0.01). However, inhibition of NF-κB has no effect on OCSC characteristics in HOTAIR knockout cells, indicating that HOTAIR may function through constitutively activated NF-κB in regulating OCSCs. We suggest a better understanding of HOTAIR- NF-κB axis in regulating OCSCs will facilitate identifying the key therapeutic target to eliminate residual tumor cells after conventional chemotherapy and prevent OC recurrence and drug resistance.

Citation Format: Weini Wang, Fang Fang, Ali R. Özeş, Kenneth P. Nephew. HOTAIR functions through NF-κB pathway in regulating ovarian cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1403.
Abstract 141: G9a regulates tumorigenicity and stemness through genome-wide DNA methylation reprogramming in non-small cell lung cancer
Authors: Pangeni Rajendra P. , Yang Lu , Zhang Keqiang , Wang Jinhui , Li Wendong , Guo Chao , Yun Xinwei , Sun Ting , Wang Jami , Raz Dan J.
Published: September 2020

Eukaryotic Histone Methyltransferase 2 (EHMT2 or G9A) has been regarded as a potential target for Non-Small Cell Lung Cancer (NSCLC) therapy. To investigate the regulatory roles of G9A in tumorigenesis, and stemness, we isolated and enriched tumor-initiating cells (TIC) from surgically resected NSCLC tissues by FACS and sphere formation assays. We then knocked down G9A using shRNA and carried out genome-wide 850K methylation array and RNA sequence analyses. We identified 67 genes hypomethylated and 143 genes upregulated following G9A knockdown of which 43 genes were both hypomethylated and upregulated. We selected 6 genes (CDYL2, DPP4, SP5, FOXP1, STAMBPL1 and ROBO1) for validation. These genes are involved in epigenetic regulation in human cancers, signaling pathways, other biological processes and have roles in patients' prognosis. In addition, G9A expression was higher in TICs and targeting G9a by shRNA knockdown or by selective inhibitor UNC0638 significantly inhibited the expression of cancer stem cell markers and sphere forming capacity, in vitro proliferation and in vivo growth. Further, transient overexpression of FOXP1 in TICs resulted in downregulation of stem cell markers and sphere forming capacity and cell proliferation in vitro indicating that the genes we identified are directly regulated by G9A through aberrant DNA methylation and subsequent expression. These data suggest that G9A contributes to epigenetic regulation by maintaining DNA methylation of multiple genes genome-wide and their expression.

Citation Format: Rajendra P. Pangeni, Lu Yang, Keqiang Zhang, Jinhui Wang, Wendong Li, Chao Guo, Xinwei Yun, Ting Sun, Jami Wang, Dan J. Raz. G9a regulates tumorigenicity and stemness through genome-wide DNA methylation reprogramming in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 141.
Abstract 1411: Population level genetic differentiation at the PVT1 gene locus: Implications for prostate cancer
Authors: Pal Gargi , Di Lia , Orunmuyi Akintunde , Olapade-Olaopa E. Oluwabunmi , Qiu Weigang , Ogunwobi Olorunseun O.
Published: September 2020

The incidence and mortality rate of prostate cancer (PCa) is highest in males of African ancestry. PCa is a multi-factorial, complex disease, but the exact mechanisms for its development and progression are unclear. Analysis of data from the one thousand genomes project revealed that a 26-kb region spanning PVT1 exons 4A and 4B shows a run of 75 SNPs with distinct allelic frequencies between African and non-African populations. The gene desert located on chromosome 8q24 is associated with aggressiveness of PCa in males of African ancestry. Interestingly, the non-protein coding gene locus Plasmacytoma Variant Translocation (PVT1) present at 8q24 is overexpressed in PCa. PVT1 gives rise to multiple transcripts with potentially different functions. To investigate if our observed population-level differences at the PVT1 gene locus have implications for PCa, we determined the expression of PVT1 exons 4A and 4B in histologically confirmed normal prostate (n=22), benign tumor prostate (n=35), and malignant tumor prostate tissue (n=28) samples obtained from males who had undergone prostatectomy or transrectal ultrasound-guided biopsy in Nigeria, a sub-Saharan Black African population. We have observed that PVT1 exons 4A and 4B are significantly overexpressed in PCa tissues in comparison to others. Transient and stable overexpression of PVT1 exons 4A and 4B significantly induce greater prostate epithelial cell migration and proliferation. We anticipate that further exploration of the role of PVT1 exons 4A and 4B may lead to the possibility of exploiting them for diagnosis, therapy, and other clinical applications in PCa.

Citation Format: Gargi Pal, Lia Di, Akintunde Orunmuyi, E. Oluwabunmi Olapade-Olaopa, Weigang Qiu, Olorunseun O. Ogunwobi. Population level genetic differentiation at the PVT1 gene locus: Implications for prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1411.
Abstract 1426: A PI3K/AKT scaffolding protein, IQ motif-containing GTPase associating protein 1 (IQGAP1), promotes head and neck carcinogenesis
Authors: Wei Tao , Choi Suyong , Buehler Darya , Anderson Richard A. , Lambert Paul F.
Published: September 2020

Purpose: Head and neck cancer (HNC) is the sixth most common cancer worldwide with a 5-year survival rate of less than 50%. The PI3K/AKT/mTOR signaling pathway is frequently implicated in HNC. Recently, IQ motif-containing GTPase activating protein 1 (IQGAP1) was discovered to scaffold the PI3K/AKT signaling pathway. IQGAP1 gene expression is increased in HNC, raising the hypothesis that IQGAP1 contributes to HNC.

Experimental Design: We performed a combination of in vitro studies using human cancer cell lines treated with a cell permeable peptide that interferes with IQGAP1's ability to bind to PI3K, and in vivo studies utilizing mice genetically knocked out for the Iqgap1 (Iqgap1-/-). In vivo EGF-stimulation assays were used to evaluate PI3K signaling. To study the role of IQGAP1 in HNC, we used a well-validated mouse model that drives HNC via a synthetic oral carcinogen, 4-nitroquinoline 1-oxide (4NQO).

Results: IQGAP1 is necessary for efficient PI3K signaling in vitro and in vivo. Disruption of IQGAP1-scaffolded PI3K/AKT signaling reduced HNC cell survival. Iqgap1-/- mice had significantly lower cancer incidences, lesser disease severity and fewer cancer foci. IQGAP1 protein levels were increased in HNC arising in Iqgap1+/+ mice. The level of PI3K signaling in 4NQO-induced HNC arising in Iqgap1-/- mice was significantly reduced, consistent with the hypothesis that IQGAP1 contributes to HNC at least partly through PI3K signaling. High IQGAP1 expression correlated with reduced survival, and high pS6 levels correlated with high IQGAP1 levels in HNC patients.

Conclusions: These data demonstrate that IQGAP1 contributes to head and neck carcinogenesis.

Citation Format: Tao Wei, Suyong Choi, Darya Buehler, Richard A. Anderson, Paul F. Lambert. A PI3K/AKT scaffolding protein, IQ motif-containing GTPase associating protein 1 (IQGAP1), promotes head and neck carcinogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1426.
Abstract 1432: TORC2 dependent control of cell growth and size in cancer cells
Authors: DeWitt Jerry T. , Kellogg Douglas
Published: September 2020

Severe defects in cell growth and size are a nearly universal feature of cancer cells and have provided a basis for cancer pathology for over 100 years; however, the underlying mechanisms remain largely unknown. Recent work in budding yeast has shown that a highly conserved signaling network surrounding Tor complex 2 (TORC2) controls cell growth and size. A key component of the network is the yeast homolog of serum and glucocorticoid-regulated kinase (SGK), which controls production of ceramide signaling lipids that control cell growth and size. Here, we present evidence that a similar TORC2 network controls cell growth and size in mammalian cells. In addition, we show that expression of oncogenic drivers in NIH3T3 cells causes defects in cell size, as well as defects in TORC2-dependent control of cell size. Cancer cells also show severe defects in TORC2-dependent control of cell size. Our working hypothesis is that the TORC2 network is downstream of multiple oncogenes, and that aberrant TORC2 signaling is a major cause of the nearly universal size defects observed in cancer cells. A better understanding of the mechanisms that control cell growth and size in normal cells, and how they go awry in cancer, could identify new targets for drugs to improve cancer therapies.

Citation Format: Jerry T. DeWitt, Douglas Kellogg. TORC2 dependent control of cell growth and size in cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1432.
Abstract 1439: NUDT5 is a critical regulator of triple-negative breast cancer growth
Authors: Qian Jing , Tahaney William , Ma Yanxia , Bollu Lakshmi , Mazumdar Abhijit , Hill Jamal , Zhang Yun , Brown Powel
Published: September 2020

Background: Triple-negative breast cancers (TNBC) represent 15% of all breast cancers, and are highly aggressive cancers with poor clinical outcome. We have previously conducted a whole phosphatase RNA microarray analysis of TNBCs compared to estrogen receptor (ER)-positive breast cancers. These studies identified phosphatases that were either over-expressed or under-expressed in TNBCs as compared to ER-positive breast cancers. One of the phosphatases highly expressed in TNBCs was NUDT5, which hydrolyzes 8-oxo-dGDP and ADP-ribose (ADPR). Recent studies have shown that NUDT5 regulates cell cycle progression, and increases cellular ATP in response to hormone stimulation in cancer cells. However, the biological function of NUDT5 in TNBCs is largely unknown.

Hypothesis: We hypothesized that NUDT5 is a critical phosphatase over-expressed in TNBCs that regulates the growth of these breast cancer cells.

Methods: We analyzed TNBC NUDT5 mRNA expression and clinical outcome using publicly available TCGA, METABRIC, and the CCLE datasets. Overall survival was evaluated using Kaplan-Meier curves and compared using log-rank analysis (* p<0.05). We next conducted siRNA knockdown studies using DharmaFECT transfection, and subsequently measured the effect on growth using automated cell counting. Results at day 7 were expressed as mean cell numbers +/- standard deviation, and analyzed using Student's t-tests.

Results: Analysis of NUDT5 mRNA expression demonstrated that NUDT5 is overexpressed in triple-negative breast cancers, specifically in the basal-like subtype of TNBC. Analysis of publically available breast cancer datasets showed that high NUDT5 expression is associated with poor overall survival of breast cancer patients. Knockdown of NUDT5 decreased TNBC growth, but did not decrease the growth of non-TNBC cells.

Conclusions: Our results demonstrate that NUDT5 is a critical regulator of TNBC growth, and raise the possibility of targeting this critical phosphatase for the treatment of TNBC tumors. Future studies will investigate the function of NUDT5 in regulating nucleotide pool metabolism in TNBCs. Understanding the role of NUDT5 in controlling TNBC growth will provide the basic science foundation for the future development of NUDT5 inhibitors for the treatment of women with these aggressive, potentially life-threatening cancers. This work was supported by Susan G Komen Promise Grant (PB), and the John Charles Cain Endowment (PB).

Citation Format: Jing Qian, William Tahaney, Yanxia Ma, Lakshmi Bollu, Abhijit Mazumdar, Jamal Hill, Yun Zhang, Powel Brown. NUDT5 is a critical regulator of triple-negative breast cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1439.