bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 20: HiBiT tagging system for high throughput chemical screening for chemotherapy
Authors: Uchida Yutaro , Matsushima Takahide , Kurimoto Ryota , Chiba Tomoki , Inutani Yuki , Asahara Hiroshi
Published: July 2021

Chemical screening is an essential procedure for finding novel drugs of cancer treatments. Especially, cell-based screenings are mostly used for discovery of novel compounds or drug repositioning of already-used drugs. Cell viability or cell proliferation are usually used for evaluation of effects of candidate novel compounds for cancer treatments. However, high throughput system for rapid evaluation of endogenous target protein expression changes has not been established so far. Therefore, an easy and quick method for detection of endogenous target protein expression changes has been strongly needed for drug discovery. Herein, we established a novel chemical compound screening system using the HiBiT-tagging system for this aim using PD-L1 as an example. HiBiT tag is a small peptide tag composed of only 11 amino acids forming a dimer with LgBiT, and functions as a Nanoluciferase. The shortness of the peptide tag is useful for insertion of tag using genome editing technology. Thereby, we established a rapid chemical screening method using HiBiT-tagged cell lines created by CRISPR-Cas9 system. With this method, we made it possible to detect endogenous target protein expression changes within 10 minutes by just adding lytic buffer and LgBiT. For validation of this method, we performed chemical screening of PD-L1 using a HiBiT-tagged lung adenocarcinoma cell line with 1,280 compounds. As a result, we identified microtubule inhibitors as compounds upregulating PD-L1, and validated its upregulation of PD-L1 with qPCR and western blotting. In conclusion, we succeeded in establishing the method for chemical compounds detecting endogenous target protein expression changes with HiBiT-tagging system. Furthermore, we validated this method by performing chemical screening with PD-L1, and succeeded in identifying microtubules inhibitors as up-regulators of PD-L1.

Citation Format: Yutaro Uchida, Takahide Matsushima, Ryota Kurimoto, Tomoki Chiba, Yuki Inutani, Hiroshi Asahara. HiBiT tagging system for high throughput chemical screening for chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 20.
Abstract 2022: Palmitate promotes breast cancer progression in vitro through induction of a senescent-like phenotype in fibroblasts
Authors: Harlow Brittany Susanne , Davalos Albert R. , Brenner Andrew J. , Jolly Christopher , Tiziani Stefano , Hursting Stephen D. , deGraffenried Linda A.
Published: July 2021

Background: Obesity confers a worse breast cancer prognosis, including an increased risk of recurrence and mortality. While the causative mechanisms have yet to be fully uncovered, emerging evidence implicates palmitate, increased in the obese state, in development of cellular senescence, an inflammatory state associated with breast tumorigenesis in preclinical models. However, studies are warranted to corroborate the impact of palmitate on induction of a cohesive senescent-like phenotype as well as the extent to which palmitate-induced senescence impacts breast tumorigenesis either in vitro or in vivo. This said, we hypothesize that palmitate exposure induces a senescent-like phenotype in fibroblasts, contributing to measures of breast cancer progression.

Methods: HCA2, IMR-90, and human mammary fibroblasts were exposed to bovine serum albumin or palmitate in media supplemented with 2% charcoal-stripped fetal bovine serum, after which the cells were measured through qPCR for expression of IL-1a, IL-6 and IL-8, some of the most prominent members of the senescence-associated secretory phenotype. Palmitate-exposed fibroblasts were also stained for senescence-associated beta-galactosidase and BrdU incorporation, well-established senescence markers. Experiments were then repeated with administration of eicosapentaenoic and docosahaexanoic acids to evaluate the potential of omega-3 fatty acids to limit the effects of palmitate on fibroblast senescence. Finally, we assessed the tumor-promoting potential of these palmitate-exposed fibroblasts by culturing MCF-7 and T47D breast cancer cells in their conditioned media and assessing changes in carcinogenic measures.

Results and Conclusions: Palmitate induced pro-inflammatory gene expression and SA-beta-gal positivity and decreased proliferation in fibroblasts, while omega-3 fatty acid supplementation reversed these effects. These palmitate-exposed fibroblasts also appeared to be of pathological impact, as exposure to their CM increased proliferation in breast cancer cells. These findings are important in that they support emerging evidence implicating obesity-associated factors in the exacerbation of breast cancer progression as well as indicate the potential of omega-3 fatty acids to improve outcome.

Citation Format: Brittany Susanne Harlow, Albert R. Davalos, Andrew J. Brenner, Christopher Jolly, Stefano Tiziani, Stephen D. Hursting, Linda A. deGraffenried. Palmitate promotes breast cancer progression in vitro through induction of a senescent-like phenotype in fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2022.
Abstract 2024: Studying the effects of interfering with doxorubicin-induced senescence in human colon cancer HCT116 cells
Authors: Alotaibi Moureq Rashed , Sobeia Homood As , Alohaydib Munirah , Alaqil Faten , Alhazzani Khalid , Alhoshani Ali , Saleh Tareq , Gewirtz David
Published: July 2021

Colorectal cancer (CRC) is type of cancer that growing in the colon or the rectum. Although CRC can be treated and managed through several means of therapy, the disease mortality is still high in Saudi Arabia. Upon treatment, cancer cells undergo different form of cell death or growth arrest such as apoptosis and senescence. Senescence is a state of proliferation arrest that is considered as a cause of drug resistance. The current study was designed to interfere with Doxorubicin-induced senescence in cancer cells, and shift the cytotoxicity of doxorubicin from proliferation arrest to apoptosis. β-galactosidase (β-gal) staining has demonstrated that Doxorubicin induces senescence in wild-type HCT116 but not in p21 knock-out cells. (DAPI)/TUNEL stating and Annexin V assay indicate that treatment with Doxorubicin promotes apoptosis in p21 knock-out HCT116 significantly more than in wild-type HCT116. β-galactosidase staining and 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranosid (C12FDG) demonstrated that combination of Doxorubicin with Sorafenib, Rapamycin, or Venetoclax has significantly reduced senescence and induced more apoptosis as shown by Propidium iodide (PI)/Annexin v assay and Caspase 3/7 Assay. PCR data analysis demonstrated that not all combination of Doxorubicin with other agents significantly decreased expression of senescence protein, however, the results of this study provide findings that interference with senescence pathways may shift cytotoxicity from senescence to apoptosis.

The authors extend their appreciation to the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia for partly funding this work through the project number (DRI-KSU-1273).

Citation Format: Moureq Rashed Alotaibi, Homood As Sobeia, Munirah Alohaydib, Faten Alaqil, Khalid Alhazzani, Ali Alhoshani, Tareq Saleh, David Gewirtz. Studying the effects of interfering with doxorubicin-induced senescence in human colon cancer HCT116 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2024.
Abstract 2029: The ER stress protein IRE1a regulates intracellular calcium, ROS, and the UV damage response through the IP3R inhibitor CIB1
Authors: Son Jeongin , Mogre Saie , Glick Adam
Published: July 2021

UV induced DNA damage is the primary driver of human skin cancer. UV can also cause endoplasmic reticulum (ER) stress, but the role of unfolded protein response (UPR) in the cellular response to UV exposure is not clear. Inositol-requiring enzyme 1α (IRE1α) is one of the key regulators of the UPR but its function in the UV response has not been resolved. Here we show that IRE1α, is transiently dephosphorylated and its RNase activity downregulated by UVB resulting in reduced XBP1 splicing. Keratinocytes with either IRE1α knockout or knockdown have elevated cytosolic calcium and ROS resulting in increased apoptosis after UV. Pharmacological inhibition of the ER inositol triphosphate receptor (IP3R) or phospholipase C (PLC) restored normal cytosolic calcium and ROS levels to cells lacking IRE1α. While loss of IRE1α did not alter the frequency of cells with UV-induced cyclo-pyrimidine dimers (CPD), it significantly reduced the percent of γH2AX positive keratinocytes 6 hours after UVB irradiation. This percent of CPD positive cells and the level of γH2AX 6 hours after UV suggesting a defect in the DNA repair response. Pharmacological inhibition of IRE1α RNase or Xbp1 knockdown did not phenocopy IRE1α loss, but overexpression of IRE1α in IRE1α knockdown cells restored γH2AX expression and reduced keratinocyte apoptosis. IRE1α overexpression also enhanced the survival of keratinocytes deficient in p53 function after UV irradiation. Additionally, mice with an epidermal deletion of IRE1α showed more rapid loss of UV-induced CPD positive keratinocytes compared to wildtype mice, primarily through accelerated sloughing of UV damaged cells from the skin. Knockdown of Calcium and integrin binding protein 1 (CIB1), which binds and inhibits the IP3R, elevated cytosolic calcium in keratinocytes and suppressed UV-induced γH2AX. Knockdown of Apoptosis signal-regulating kinase 1 (ASK1), which can interact with IRE1α through TRAF2 as well as CIB1, restored normal levels of UV-induced γH2AX to IRE1α knockdown keratinocytes. Together these results show that IRE1α is essential for maintaining normal cellular calcium and ROS levels and the UV-induced repair response, and suggest a model where IRE1a activation state drives CIB1 binding either to the IP3R or ASK1 to regulate ER calcium efflux, ROS and DNA repair responses following UV.

Citation Format: Jeongin Son, Saie Mogre, Adam Glick. The ER stress protein IRE1a regulates intracellular calcium, ROS, and the UV damage response through the IP3R inhibitor CIB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2029.
Abstract 2034: Patient-specific enhancer-gene networks for hundreds of primary cancer samples
Authors: Xu Duo , Forbes Andre Neil , Cohen Sandra , Palladino Ann , Karadimitriou Tatiana , Khurana Ekta
Published: July 2021

Epigenomic alterations at regulatory elements have been implicated in cancer tumorigenesis. As a hallmark for epigenetic changes, chromatin accessibility profiles are generated for cancer patients. However, not all accessible regions are active regulatory elements. We developed a computational model that uses machine learning to integrate ATAC-seq and RNA-seq data and is trained using 3D genomic data. Our model is able to classify accessible regions into active regulatory elements with links to target genes and poised elements with no links to target genes. We used multi-omics data including histone modification profiles from ENCODE to validate our results. Application of our model on chromatin accessibility and transcriptomic data from 400 cancer patients of 22 cancer types revealed novel cancer-type and -subtype-specific enhancer-gene connections for known cancer genes. In one example, we identified two enhancers that regulate the expression of ESR1 in only ER+ breast cancer samples but not in ER- samples. These enhancers explain the high expression of ESR1 in 93% of ER+samples. Functional validation using CRISPRi confirms that inhibition of these enhancers decreases the expression of ESR1 in ER+ samples. Overall, our model predicts the connections of active enhancers to their target genes in a patient-type specific manner revealing key regulatory loci that modulate the expression of key genes.

Citation Format: Duo Xu, Andre Neil Forbes, Sandra Cohen, Ann Palladino, Tatiana Karadimitriou, Ekta Khurana. Patient-specific enhancer-gene networks for hundreds of primary cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2034.
Abstract 2037: Targeting chromosome rearrangements in cancer with CRISPR
Authors: Yang Huibin , Suhas-Hulbatte Radhika , Ljungman Mats
Published: July 2021

Background: We have developed a novel precision and personalized cancer therapeutic approach to specifically target cancer cells using CRISPR technology. This approach is universal to all cancers and not dependent on the unique biology of the tumor. The innovation of this approach is that chromosome rearrangement junctions (CRJs), which typically exist in the hundreds in each tumor cell, are targeted by guide RNAs that bring together two parts of a dCas9-fused endonuclease, Fok1, leading to the induction of DNA double strand breaks (DSBs) at sites of the targeted CRJs in cancer cells specifically.

Materials and Methods: HCT116 colon cancer cells and UMUC-3 bladder cancer cells were engineered to express Fok1-dCas9 and two pairs of CRJ-targeting gRNA under doxycycline regulation. Induction of DSBs at CRJs were assessed by γH2AX foci and γH2AX ChIP-PCR and cell death was assessed by apoptosis (PARP1 & caspase cleavage and flow cytometry for sub-G1 DNA content and Annexin V staining) and clonogenic survival.

Results: Induction of Fok1-dCas9 lead to the induction of 1-2 DSBs per cell and promoted cell death when accompanied with the expression of pairs of CRJ-targeting gRNAs. However, no DSBs or cell death was observed with expression of Fok1-dCas9 alone or in combination with non-targeting gRNAs.

Conclusions: We have shown that we can direct two parts of a dCas9-fused Fok1 endonuclease to sites of CRJs in cancer cells using CRISPR gRNAs leading to the induction of DSBs and loss of cell survival specifically in cancer cells. Recent breakthroughs in CRISPR delivery using lipid nanoparticles will allow us to next explore the utility of our precision CRISPR approach in animal models. If successfully implemented, the impact of our universal approach targeting CRJs in cancer with CRISPR could be transformative for cancer therapy.

Citation Format: Huibin Yang, Radhika Suhas-Hulbatte, Mats Ljungman. Targeting chromosome rearrangements in cancer with CRISPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2037.
Abstract 2065: Mutations inhibiting KDM4B drive ALT telomere maintenance pathway in ATRX-mutated cancers
Authors: Wong Lee H. , Udugama Maheshi , Hii Linda , Garvie Andrew , Garvie Matthew , Vinod Benjamin , Chan Lyn , Mann Jeffrey , Collas Philippe , Voon Joanna
Published: July 2021

Alternative Lengthening of Telomeres (ALT) is a telomere maintenance pathway utilized in 15% of cancers. ALT cancers are strongly associated with inactivating mutations in ATRX; yet ATRX knockout (KO) alone is insufficient to trigger ALT, suggesting that additional cooperating factors are involved. We identify H3.3G34R and IDH1/2 mutations as two such factors in ATRX-mutated glioblastomas. Both mutations are capable of inactivating histone demethylases, and we identify KDM4B as the key demethylase which is inactivated in ALT. Mouse ES cells inactivated for ATRX, TP53, TERT and KDM4B (KDM4B KO or H3.3G34R) show characteristic features of ALT. Conversely, KDM4B over-expression in ALT cancer cells abrogates ALT-associated features. Our data demonstrate that inactivation of KDM4B, either through H3.3G34R or IDH1/2 mutations, acts in tandem with ATRX mutations to promote ALT in cancers.

Citation Format: Lee H. Wong, Maheshi Udugama, Linda Hii, Andrew Garvie, Matthew Garvie, Benjamin Vinod, Lyn Chan, Jeffrey Mann, Philippe Collas, Joanna Voon. Mutations inhibiting KDM4B drive ALT telomere maintenance pathway in ATRX-mutated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2065.
Abstract 2107: Transcription factor repertoire basis for epigenetic and consensus molecular subtypes of colorectal cancer
Authors: Bhandari Yuba , Powers Rachael , Parmar Sehej , Krishna Vinod , Bachman Kurtis E. , Baylin Steve , Easwaran Hariharan
Published: July 2021

Colorectal cancer (CRC) is classified into distinct subtypes based on genetic, epigenetic and/or transcriptional subtypes. The underlying mechanisms that drive these subtypes are unknown. By integrating whole genome DNA methylation, gene expression, chromatin patterns, and enhancer elements in CRCs, we show that expression of large numbers of transcriptional factors (TFs) is deregulated in CRCs and these expression patterns drive epigenetic and transcriptional subtypes of CRCs. The first key result emerging is a new understanding of important DNA methylation patterns central to classifying CRCs. Of these, different degrees of cancer associated promoter methylation have been recognized that suppress expression, and/or inducibility of important tumor suppressor genes. Stratified by high to low degrees of hypermethylation, this pattern is termed CpG-island promoter DNA methylation phenotypes or CIMP. We now tightly link CIMP to a cancer-specific hypermethylation at enhancer regulatory elements we term, CpG- enhancer methylation phenotype (CEMP). CIMP and CEMP are driven by decreased expression of multiple TFs with binding sites in these regulatory elements, and the involved TFs are frequently identical between the two regions. In contrast, a different set of TF factors are downregulated in non-CIMP tumors, again in conjunction with methylation patterns linking promoter and enhancer target sites for the involved TFs. Further, enhancer regulatory elements that are hypomethylated in cancer relative to normal tissue are associated with increased expression of another distinct set of TFs with target sites in the corresponding enhancers. Importantly, the TF expression patterns, and the above CIMP, CEMP, and hypomethylation patterns, are established early in the pre-malignant adenomas. Importantly, pan-cancer analyses showed that the above CIMP and CEMP subtypes occur across important cancer types in addition to CRC, again in parallel with marked downregulation of TFs that have target sites in the methylated regulatory elements. All of the above newly defined patterns also provide a basis for the transcriptome-based consensus molecular subtypes (CMS) of CRC. Overall, our studies may provide key understanding of how cancers, and their initiation and progression, are diseases which at the end of the day depend on abnormal patterns of TF expression, and how these relate to methylation alterations at target enhancer and promoter regions.

Citation Format: Yuba Bhandari, Rachael Powers, Sehej Parmar, Vinod Krishna, Kurtis E. Bachman, Steve Baylin, Hariharan Easwaran. Transcription factor repertoire basis for epigenetic and consensus molecular subtypes of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2107.
Abstract 2111: RAD6-mediated epigenetic reprogramming contributes to therapy-induced chemo-resistance in ovarian cancer
Authors: Omy Tasmin Rahman , Jonnalagadda Shirisha , Reedy Mark , Palle Komaraiah
Published: July 2021

Epithelial Ovarian Cancer (EOC) is the fifth leading cause of cancer-related deaths in women in the USA, gets diagnosed at an advanced stage (stage III-IV) with local or distant metastasis. The primary therapeutic regimen of EOC involves surgical removal of all the visible tumor mass followed by platinum-based chemotherapy either as a single agent or in combination with a taxane. Despite initial responses, over 70% of EOC patients develop recurrent disease and they are often resistant to available chemotherapeutic drugs, contributing to poor survival. This appalling situation emphasizes the critical need to identify the molecular mechanisms involving recurrence of these tumors' aggressive growth and developing therapeutic resistance. Our analysis from chemo naïve and recurrent tumors from the same patients' samples, and isogenic ovarian cancer cell lines data showed recurrent tumors and resistant cell lines several upregulated cancer stem cell markers including ALDH1A1, SOX2 and altered expression of DNA damage response and repair genes that are involved in the processing of platinum adducts and crosslink repair. Prominently, RAD6, an E2ubiquitin-conjugating enzyme, is significantly (> 6 folds) overexpressed in recurrent ovarian tumors and associated with aggressive tumor cell growth, stemness, chemoresistance to platinum drugs, and poor prognosis. Our studies validated that, chemotherapy induced upregulated RAD6 reprograms epigenetic milieu in tumor cells through ubiquitination of histone variants H2A, H2AX, and H2B, which further recruits additional epigenetic modifiers to these regions and regulates genes involved in DNArepair, cell survival, stemness, and chemoresistance. Additionally, our mechanistic studies demonstrate RAD6 dependent recruitment of several epigenetic modifiers such as histone methylases and demethylases to regions of ubiquitylated histones and their crosstalk. Collectively, our study presents novel chemotherapy induced epigenetic modulator contributing to therapeutic recurrence of EOC and could be an important therapeutic target to treat chemo-resistant EOC and improve the progression-free survival of patients suffering from this deadly disease.

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Citation Format: Tasmin Rahman Omy, Shirisha Jonnalagadda, Mark Reedy, Komaraiah Palle. RAD6-mediated epigenetic reprogramming contributes to therapy-induced chemo-resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2111.
Abstract 2168: Multi-regional RNA-Seq reveals pattern of RNA-editing in brain tumor
Authors: Yan Cheng , Wu Jun , Wang Dan , Su Lan , Yang Yufei
Published: July 2021

Introduction: RNA-editing is widespread across human genome. Previous studies have observed that RNA editing level differs significantly across multiple cancer types and may influence cancer development. Increased level of RNA editing has been found to correlate with enhanced tumorigenesis in some cancers but reduced tumorigenesis in others. In brain tumor, the RNA editing landscape has not been well characterized partially due to the difficulty of sample accessibility. We performed multi-regional RNA-Seq on cancerous, peritumoral and distant tissue collected from 40 brain cancer patients. Our aim is to depict the landscape of RNA editing and to comprehend the relevance of RNA editing with other molecular characteristics of brain cancer.

Methods: We collected 40 cancerous brain tissue sample (30 LGG and 10 GBM), each coupled with a peritumoral tissue and a distant tissue. These 120 samples were subjected to RNA-Seq sequencing. RNACocktail was applied for identification of RNA editing events. RADAR and ANNOVAR were used for identification and annotation of A-to-I editing sites. RNAEditingIndexer was used to estimate sample-wise editing level denoted as AEI. Pathway enrichment analysis was performed using GSEA. Statistical difference of molecular characteristics between sample groups was calculated using either Wilcoxon signed rank test or Kruskal-Wallis test.

Results: A total number of 21,105 and 5,634 informative editing events were identified in LGG and GBM respectively. As expected, most editing events were located within Alu element of intronic and 3'UTR region. In both LGG and GBM, RNA editing level showed decreasing trend from distant to peritumoral to cancerous samples. Compared with normal tissue, we found 5.12‰ up-regulated events and 16.92‰ down-regulated events in LGG samples. Similar pattern was found in GBM as well. We found two editing sites with significant editing differences (adjust p-value < 0.05) within coding region of GRIK2 and NEIL1 gene. In addition, numerous editing sites showed statistical correlation other molecular/pathological characteristics such as IDH1 mutation, existence of seizure, MGMT methylation and pathological classification.

At sample level, the same decreasing trend of RNA editing level was consistently observed from distant to cancerous tissue. Enrichment analysis showed that editing level was positively correlated with neural transmission and oncogenic related pathways. In addition, sample level editing was significant different between WHO as well as pathological classification.

Citation Format: Cheng Yan, Jun Wu, Dan Wang, Lan Su, Yufei Yang. Multi-regional RNA-Seq reveals pattern of RNA-editing in brain tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2168.
Abstract 2177: Tumor neoantigen profiling with validated patient-specific TCR characterization to improve neoepitope prediction
Authors: Pan Zheng , Dalmas Olivier , Peng Songming , Jacoby Kyle , Sennino Barbara , Ma Yan , Smith Chad , Momin Amin , Xu Allison , Heeringa Katharine , Johnston Jonathan , An Duo , Quach Boi , Lu William , Nguyen Diana , Conroy Andrew , Purandare Bhamini , Huang Eva , Stawiski Eric , Franzusoff Alex , Mandl Stefanie
Published: July 2021

PACT Pharma has developed an ultra-sensitive approach to validate predicted neoantigens and the cognate T cell receptors (neoTCRs) from tumor specific somatic mutations by capturing neoantigen-specific T cells from peripheral blood. This process is used in clinical trials (NCT03970382) of personalized neoTCR-T therapy for persons with solid cancers.

Using the state of the art prediction pipeline and screening process, more than one hundred twenty neoantigens were predicted from 5 type of solid cancers that were validated by characterizing cognate T cells and their close to 200 unique TCRs captured from the blood of the same individual. These validated neoepitopes of 8 to 11 amino acids represent broad HLA class I coverage with >30 alleles to date. Our analysis revealed that mutations can occur in all positions within the epitopes. Epitope immunogenicity is potentially affected by different mechanisms including mutation position, agretopicity, as well as HLA interacting positions and/or by interactions between mutated residues and its cognate TCRs. It was observed that these validated neoepitopes comprise broad ranges of predicted HLA binding affinities, stability, and neoantigen expression levels. The analysis presented here offers insights to enable machine learning to advance rules for epitope selection and prioritization that may be important for immunological approaches to address a broad range of diseases, including cancer.

Citation Format: Zheng Pan, Olivier Dalmas, Songming Peng, Kyle Jacoby, Barbara Sennino, Yan Ma, Chad Smith, Amin Momin, Allison Xu, Katharine Heeringa, Jonathan Johnston, Duo An, Boi Quach, William Lu, Diana Nguyen, Andrew Conroy, Bhamini Purandare, Eva Huang, Eric Stawiski, Alex Franzusoff, Stefanie Mandl. Tumor neoantigen profiling with validated patient-specific TCR characterization to improve neoepitope prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2177.
Abstract 219: Identifying genetic interactions resulting form diverse biological mechanisms to inform cancer drug development
Authors: Garcia-Quismondo Manuel , Elemento Olivier , Madhukar Neel , Gilvary Coryandar
Published: July 2021

Identifying gene interactions, such as synthetic lethal relationships, has led to actionable, clinical insights, enabling targeted oncology therapies. However, identifying these interactions has proven difficult, in large part due to the multiple mechanisms by which two genes can interact, such as occurring in the same pathways or conversely occurring in parallel pathways, but involved in the same biological functions. Therefore, experimentally testing all possible sources of gene interactions is costly and time consuming, however a robust computational approach would allow researchers to investigate these interactions efficiently. Here, we describe a machine learning approach that integrates multiple metrics, such as gene-gene pairwise expression correlations, similarity metrics for genetic biological functions, and pathway co-occurrence, to derive a global interaction score. Our model can simultaneously achieve significant predictive performance (Area under the receiver operator curve = 0.8), while also elucidating the underlying mechanism of predicted gene interactions, informing future experimental validation protocols. Additionally, we leveraged transcriptomic and genomic profiles of cancer patients to identify clinically actionable genetic interactions. Our tool provides a novel multi-faceted approach for the detection of gene interactions and can be used to identify biomarkers, novel oncology targets and overall enhance our understanding of biological mechanisms.

Citation Format: Manuel Garcia-Quismondo, Olivier Elemento, Neel Madhukar, Coryandar Gilvary. Identifying genetic interactions resulting form diverse biological mechanisms to inform cancer drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 219.
Abstract 2206: Investigation of DNA damage repair mutated characteristics and correlation with immunogenic biomarker in Chinese head and neck cancer patients
Authors: Chen Yong , Wang Yan , Zheng Yating , Xie Wenzhuan , Huang Mengli
Published: July 2021

Background: DNA damage repair (DDR) pathway have been reported in several cancer, and known as a predictive biomarker of platinum-based chemotherapy, PARP inhibitor therapy, radiotherapy, and immunotherapy. However, the predictive value and mutated characteristics of DDR in patients with head and neck cancer (HNC) is still uncertain. The purpose of this study is to assess the frequency of DDR mutations, and the correlation between DDR mutations and immunogenic biomarker such as tumor mutation burden (TMB), microsatellite instability (MSI) and PD-L1 expression in Chinese HNC patients.

Methods: Genomic profiling of DNA was performed through a next-generation sequencing (NGS) (panel on 381/733-gene) on tissue from Chinese patients with head and neck cancer between January 23, 2017 and April 10 2020. TMB was defined as total number of somatic non-synonymous mutations in coding region. MSI was evaluated by NGS of 500 known MSI loci. PD-L1 expression was determined by immunohistochemistry staining using Dako 22C3. Germline or somatic mutations of 35 DDR genes were classified as DDR gene mutations.

Result: Genetic mutation of 319 HNC patients were analyzed using NGS, of which 32 (10.03%) harbored alterations in DDR genes. The most frequently mutated DDR genes were BRCA2 (3.23%), followed by FANCA (2.02%), BRCA1 (1.61%), RAD51 (0.81%), RAD50 (0.81%), PMS2 (0.81%), and PLAB2 (0.81%). The frequency of KMT2C, CD274, FBXW7, APC, NOTCH1, PDCD1LG2, RB1, LRP1B, and JAK2 mutations was higher in DDR mutations (DDRmut) than in DDR wild-type (DDRwt) patients. PD-L1 status analysis was performed in 265 HNC patients, including 24 DDRmut patients and 241 DDRwt patients. But there was no significant difference on PD-L1 expression between DDRmut t and DDRwt (P=0.93). 310 HNC patients (including 31 DDRmut patients and 279 DDRwt patients) could be evaluated for TMB, the results showed that DDRmut (10.13Muts/Mb) was associated with higher TMB than DDRwt (5.65Muts/Mb) (P = 0.0031). Of the 308 HNC patients (including 31 DDRmut patients and 277 DDRwt), only 3 (0.97%) were diagnosed as MSI-H, including 2 (6.45%) patients with DDRmut and 1 (0.36%) patient with DDRwt.

Conclusions: The DDR pathway genes alteration characteristics was presented in Chinese HNC patients, and was associated with a higher TMB level. These findings provide a theoretical basis for future investigation of clinical therapy in DDR mutated Chinese HNC patients.

Citation Format: Yong Chen, Yan Wang, Yating Zheng, Wenzhuan Xie, Mengli Huang. Investigation of DNA damage repair mutated characteristics and correlation with immunogenic biomarker in Chinese head and neck cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2206.
Abstract 2227: Pan-cancer shedding patterns of tumor circulating cell free DNA
Authors: Navarro Fabio C. P. , Zhang Simo , Tan Mengyao , Abbott Charles , Northcott Josette , Lyle John , Bartha Gabor , Harris Jason , West John , Chen Richard , Boyle Sean Michael
Published: July 2021

Introduction: Tumor circulating cell-free DNA (ctDNA) comprises DNA molecules cast from tumors that reach and survive in a patient's bloodstream. Given its non-invasive nature, the liquid biopsy of ctDNA has remarkable potential for diagnosis, prognosis, disease progression tracking, and treatment monitoring. Nonetheless, little is known about which tumor features yield higher representation of ctDNA in blood or even which regions in the tumor genome are more inclined to shed and thus be observed as ctDNA.

Typically, studies of ctDNA have focused on a limited and well-established set of genes and recurrent variants. However, these limited gene panels may not capture the breadth of genetic alterations that reflect tumor biology. Their limited footprint hinders a comprehensive understanding of tumor heterogeneity, mechanisms of resistance, and DNA shedding patterns. To address these limitations, we developed a whole-exome scale cfDNA platform, NeXT Liquid Biopsy™, that enables sensitive detection and tracking of mutations in over twenty thousand genes from plasma samples.

Results: Here we profile over 50 tumor, normal, and plasma matched samples using NeXT Liquid Biopsy to investigate pan-cancer patterns of DNA shedding. We observe varying levels of ctDNA shedding in plasma, suggesting that tracking tumors with an exome-scale set of variants, as opposed to with a targeted panel, can benefit greatly from the higher sensitivity and granularity of our enhanced exome sequencing. Next, we sought to investigate the patterns of somatic variants representation in plasma. For this, we integrate shedding ratios with transcriptome and epigenome data from healthy paired tissues and examine variant shedding biases associated with transcription rate and histone modifications in associated nucleosomes.

Conclusion: We sequenced and analyzed one of the largest tumor, normal, and plasma cohorts to date with comprehensive coverage of somatic variants across all human genes. Our results suggest that tracking exome-scale somatic variants adds invaluable information to understand a tumor's biology. Moreover, our comprehensive coverage of the tumor genome can be used to unveil biases of tumor genome shedding patterns. We are able to investigate the shedding ratio across distinct genomic features deriving, to our knowledge, the first exome scale pan-cancer shedding resource of the human genome.

Citation Format: Fabio C. P. Navarro, Simo Zhang, Mengyao Tan, Charles Abbott, Josette Northcott, John Lyle, Gabor Bartha, Jason Harris, John West, Richard Chen, Sean Michael Boyle. Pan-cancer shedding patterns of tumor circulating cell free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2227.
Abstract 2253: Comparison of genomic alterations in neuroendocrine tumors originated from different primary sites
Authors: Tang Qiyun , Tian Ye , Li Xiaolin , Bai Jian'an , Liu Min , Hu Ping , Zhang Shanshan , Xu Zanmei , Qu Yueting , Wang Aodi
Published: July 2021

Background: Neuroendocrine tumors (NETs) are heterogeneous, relatively rare tumors derive from neuroendocrine system. Currently, limited therapeutic strategies are available for NETs. Next-generation sequencing (NGS) and target gene-based precision therapy have shown promising efficacy in many tumors, but fare in NETs. Several reports illustrated that the primary sites of NETs were significantly bound up with the clinical response and prognosis. However, characteristics of their genomic alterations are insufficiency nowadays. Methods: A cohort of 94 Chinese NET patients were recruited, and the FFPE tumor tissues and matching blood samples from different primary sites were collected. Targeted next generation sequencing (NGS) of 450 genes was performed in OrigiMed (Shanghai, China) with a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Results: In all patients, the top 5 most-frequently aberrant genes were TP53 (46.8%, 44/94), RB1 (23.4% 22/94), MEN1 (20.2%, 19/94), KRAS (11.7%, 11/94) and ATRX (9.6%, 9/94). The enrolled NETs contain 7 different primary sites, namely, 37 pancreatic NETs (PNETs), 16 hepatic NETs (HNETs), 11 gastric NETs (GNETs), 10 gallbladder NETs (GB-NETs), 8 esophageal NETs (ENETs), 7 colorectal NETs (CNETs), and 5 small intestine NETs (SI-NETs). Based on origin sites, TP53 is the most widely distributed mutant gene in all sites except for PNETs (Ranked 3rd), while RB1 mutations were significantly higher in ENETs (75.0%, 6/8), GB-NETs (60.0%, 6/10) and GNETs (36.4%, 4/11). Other higher -frequency mutated genes among different sites showed greatly dissimilarity (MEN1, ATRX and DAXX in PNETs; FAT3, MAX, MUC16, GATA4 and OBSCN in HNETs; APC, KRAS and PTEN in GNETs; TERT, ERBB2 and YES1 in GB-NETs; LRP1B and FBXW7 in ENETs; APC, ADGRA2 and FGFR1 in CNETs; NF1 in SI-NETs). Furthermore, signaling pathways involved in potential drug-target genes were analyzed. Results revealed 16.0% (15/94) patients with aberrations in PI3K/AKT/mTOR pathway, 11.7% (11/94) in Cell-Cycle pathway, 9.6% (9/94) in HRD pathway, 9.6% (9/94) in FGF pathway and 16.0% (15/94) in other cancer-related genes (ERBB2, VHL, MET etc.). Conclusions: This study reveals the genomic alteration characteristics of NETs from different primary sites. The profiles obtained in our study may provide evidences for further targeted therapy to improve outcomes of Chinese NETs.

Citation Format: Qiyun Tang, Ye Tian, Xiaolin Li, Jian'an Bai, Min Liu, Ping Hu, Shanshan Zhang, Zanmei Xu, Yueting Qu, Aodi Wang. Comparison of genomic alterations in neuroendocrine tumors originated from different primary sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2253.
Abstract 2255: The impact of primary immunodeficiency informed molecular landscape on the biology and prognosis of refractory malignancies
Authors: Tran Son , Murguia-Favela Luis , Narendran Aru
Published: July 2021

Introduction: Cancer is one of the most common causes of death in patients with primary immunodeficiencies (PIDs). The immune surveillance concept postulates that PIDs are an important contributor to cancer growth and outcomes. Although several immunodeficiency syndromes are known to be associated with malignancies in children and adults, currently, the molecular mechanisms that link immune functions to cancers are poorly understood. Here we describe distinct molecular characteristics in adult cancers with respect to PID-associated genes that impact survival outcomes in affected patients.

Methods: In this study, we integrated transcriptome data of 28 adult cancers from the public database The Cancer Genome Atlas (TCGA) with respective healthy tissues as control from the Genotype-Tissue Expression (GTEx) project. Unified datasets integrating GTEx healthy samples and TCGA cancer data were provided by the Recount2 protocol and differential expression analyses (DEA) were carried out using the integrated limma-voom and edgeR pipelines in the TCGAbiolinks R package. Gene ontology (GO) enrichment analyses were performed using the pathfindR R package. Mutational and clinical data of adult cancer patient samples in TCGA and the AACR Project GENIE were consolidated using cBioPortal and the maftools R package. PID-associated genes were curated from the latest registry by the European Society for Immunodeficiencies (ESID).

Results: Of 307 PID-associated genes, 151 (32.0%) up-regulated and 88 (18.6%) down-regulated genes were identified across the 28 unified TCGA-GTEx datasets. From the identified differentially expressed gene sets, GO analysis revealed enrichment in immunological pathways related to complement and coagulation cascades (27/28 datasets), systemic lupus erythematosus (25/28), in addition to Fanconi anemia (22/28). The greatest frequency of mutations in PID-associated genes from the consolidated TCGA and GENIE datasets were KRAS (14.83%), KMT2D (10.03%), TERT (8.99%), PRKDC (7.59%) and PTEN (6.18%) across all cancer types, with a total of 259 affected PID-associated genes. Survival analyses using Cox proportional-hazards and Kaplan-Meier models will assess the clinical implications of the identified molecular alterations in affected patients.

Conclusion: Our integrative approach aids in the elucidation of molecular mechanisms that bridge PIDs to tumour biology. These data may help identify important biomarkers and potential future targeted therapeutics.

Citation Format: Son Tran, Luis Murguia-Favela, Aru Narendran. The impact of primary immunodeficiency informed molecular landscape on the biology and prognosis of refractory malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2255.
Abstract 2256: A population-based mouse model for an experimental basal-like mammary cancer
Authors: French John Edgar , Pressel William , Albright Jody , VerHague Melissa , Hursting Stephen D.
Published: July 2021

The prevalence of obesity, an established risk and prognostic factor for basal-like breast cancer (BLBC), has risen dramatically in the US and many other countries over the past 4 decades. Obesity is also associated with increased resistance to several classes of cancer chemotherapeutic agents used for advanced BLBC. Unfortunately the mechanisms underlying chemotherapy resistance, and the inter-individual differences driving resistance, are poorly understood. Population-based experimental mouse models for determining inter-individual genetic differences in chemotherapy pharmacokinetics and treatment-related efficacy or resistance in mammary gland cancer are not well-defined. Our aim is to develop a population-based experimental model based on outcrosses between 30 different Collaborative Cross line females and hemizygous male B6.FVB-Tg(C3-1-TAg) (greater than N15) congenic mice to produce 30 Collaborative Cross (CC) recombinant inbred line intercross (CC RIX) mouse lines to investigate the interactive effects between diet-induced obesity (DIO) and genetic susceptibility to chemotherapy in a genetically heterogeneous mouse model of BLBC. We report here that thirty genetically-different CCRIL X hemizygous B6.FVB-Tg(C3-1-TAg) congenic female and male CC RIX mice observed longitudinally for occurrence of basal-like mammary tumors showed significant differences in both age at first mammary tumor observation and the fraction of females and males in each RIX bearing tumors. Litter size and the number of females and males of each RIX varied across the 30 CC RIX line. The study was well-powered, with at least X males and Y females for each CC RIX line evaluated longitudinally. We observed that 25 of 30 female CC RIX lines presented with BLMC, with distinct times-to-tumor with each CC RIX. The penetrance of the fraction of each CC RIX line (birth cohort) presenting with BLMC varied greatly in females (25-100%). Surprisingly, 11 of 30 male CC RIX lines presented with BLMC, with penetrance of BLMC varying in males between 10-75%. In conclusion, we present preliminary data to support development of an experimental mouse model for BLBC based on the random mating of 8 genetically-diverse homozygous inbred founder lines followed by in-breeding to greater than 95% homozygosity carrying 45 million single-nucleotide and structural variants. Studies are underway using these CC RIX lines to evaluate the genetic underpinnings of obesity-associated resistance to carboplatin in C3-TAg-driven mammary tumors.

Citation Format: John Edgar French, William Pressel, Jody Albright, Melissa VerHague, Stephen D. Hursting. A population-based mouse model for an experimental basal-like mammary cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2256.
Abstract 2281: Impact of DNA contaminants on next-generation sequencing data quality
Authors: Dubinsky Amy , Saunders Lauren
Published: July 2021

Nucleic acid purification and clean-up are critical steps for genomics applications, including next-generation sequencing (NGS). Variability in the quality of sample extraction methods can result in differential removal of inhibitors, or reaction contaminants, that diminish NGS experimental data quality. To maximize sample recovery and create more consistent results, efficient removal of inhibitors or contaminants is critical during nucleic acid isolation and purification. In this study, we examine the effect of three common tissue-specific nucleic acid contaminants: (1) collagen, (2) myoglobin, and (3) melanin, on NGS library prep and sequencing metrics. These contaminants are present in skin and muscle tissue and are known to cause inhibition of PCR reactions. In addition, we will extract DNA from both of these samples types and evaluate the performance of two commonly used DNA extraction technologies: (1) magnetic bead-based clean-up; and (2) silica-based columns, for their ability to remove contaminants from DNA extractions and the differential effect of these extraction methods on downstream NGS metrics. These findings demonstrate the importance of the choice of extraction chemistry on NGS metrics and read quality. We also demonstrate that magnetic bead-based- protocols offer a superior reliable, rapid, automatable method to produce high quality DNA and remove contaminants.

Citation Format: Amy Dubinsky, Lauren Saunders. Impact of DNA contaminants on next-generation sequencing data quality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2281.