bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 2031: The PERK arm of the unfolded protein response induces CXCL8 and CCL20 through C/EBPδ
Authors: Poria Dipak Kumar , Sheshadri Namratha , Sharan Shikha , Sterneck Esta 
Published: July 2021

Abstract
Cancer cells often experience endoplasmic reticulum (ER) stress due to either activated oncogenes, microenvironmental conditions or therapeutic interventions. ER stress initiates an adaptive unfolded protein response (UPR) program, mediated by PERK, IRE1 and ATF6 transmembrane sensors, which alleviates the stress and promotes cells survival. However, if unmitigated, the UPR proceeds to initiation of cell death. Cancer cells often usurp the UPR pathways that contribute to cell survival and metastasis through intra- and intercellular mechanisms. Intracellular mechanisms of cellular adaptation are well-characterized; however, the intercellular consequences of ER stress are less understood. Here, we show that in cell lines representing melanoma, breast and prostate cancer, the transcription factor C/EBPδ (CEBPD) is an early ER stress response factor that is induced by the PERK arm of the UPR. CEBPD induction occurs in part through activation of PERK-mediated non-canonical JAK/STAT3 signaling. Transcriptional profiling in MDA-MB-435 melanoma cells revealed that C/EBPδ supports the expression of genes involved in cell intrinsic adaptations such as chaperones, components of ER associated degradation, and apoptosis inhibitors. In addition, C/EBPδ promotes expression of the inflammatory mediators CXCL8 and CCL20, which are known to modulate the tumor microenvironment through the receptors present on various host cells. Depletion of CEBPD by siRNA or inhibition of PERK kinase reduced CXCL8 and CCL20 transcripts and secreted proteins upon ER stress induction by thapsigargin. Bioinformatic analysis and the ENCODE ChIP-seq data suggest direct interaction of C/EBPδ with CXCL8 and CCL20 promoter/enhancer, indicative of direct transcriptional activation of the genes. Indeed, ectopic expression of C/EBPδ, but not a mutant lacking the transactivation domain, was sufficient to induced CXCL8 and CCL20 transcription in HEK293 cells in the absence of ER stress. Consistent with these results, conditioned media from ER-stressed MDA-MB-435 cells induced the gene expression of proinflammatory cytokines IL-6, IL-12, TNFα and IL-1RA in HL-60 promyelocytic cells while suppressing IL-10 expression. Taken together, these data show that the PERK arm of the UPR activates JAK/STAT3 pathway to induce expression of C/EBPδ and thereby enhance secretion of immunomodulators and transmit UPR-induced inflammatory signals to immune cells in the tumor microenvironment. This pathway may contribute to the overall outcome of the UPR on cancer cell survival, metastasis and response to anticancer therapies.
Citation Format: Dipak Kumar Poria, Namratha Sheshadri, Shikha Sharan, Esta Sterneck. The PERK arm of the unfolded protein response induces CXCL8 and CCL20 through C/EBPδ [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 2031.
Abstract 2032: Vitamin B12 augments Ca2+ dependent cytotoxicity of diethyldithiocarbamate through excessive ER-vacuolization, oxidative stress and mitochondrial depolarization in MCF-7 cells in culture
Authors: Solovieva Marina E. 
Published: July 2021

Abstract
Coordinated organization of intracellular organelles into functional networks is vital for cells. Mitochondrial and endoplasmic reticulum (ER) interaction in regulation of cell Ca2+ homeostasis is crucial for cell death and survival. Function of these intracellular organelles involved in two-way regulation of the status of major cell metabolites including protein, lipid exchange and intracellular Ca2+ homeostasis. Disulfiram and its derivatives recently attracted attention as a potential anticancer treatment (Kona et al., 2011). Combined use of hydroxycobalamin (vitamin B12b) and diethyldithiocarbamate (DDTC) demonstrated synergistic effect (combination index=0.25) and enhanced the cytotoxic effect of DDTC in number of transformed cell lines in culture (Solovieva et al., 2020). In the present work using MCF-7, human breast cancer cells in culture we demonstrate that combined action of DDTC and B12b resulted in induction of UPRER manifested by dilation of ER cisterns, increased level of polyubiquitinated proteins, and increased expression of CHOP. Confocal microscopy revealed disorganization of mitochondrial network and ER and treatment of these cells with B12b/DDTC and accompanied by mitochondrial depolarization (~50% after 6 h of exposure to drugs). Treatments also induced changes in morphology of mitochondrial network, and within 6 h of exposure, mitochondria demonstrated large amplitude swelling, characteristic feature of mitochondrial permeability transition pore opening. Drug exposure induced mitochondrial oxidative stress and increase in superoxide anion level (monitored with MitoSOX) and increased content of mitochondrial Ca2+ (xRhod-1) which almost doubled following treatment with B12b/DDTC. Short-time exposure of MCF-7 cells to mixture of drugs in Ca2+-free incubation media (2.5 mM EGTA) resulted in generation of significantly smaller size of vacuoles as compared with control. Similarly, pre-loading of cells with intracellular Ca2+ chelator (BAPTA, 5 µM, 30 min, 37oC) or inhibition of IP3 receptor(s) with 2-APB, delayed the drug-induced vacuolization. Taken together, our data indicate that the mechanism of B12b/DDTC-induced cytotoxicity in human MCF-7 cancer cell lines involves mitochondrial oxidative stress, activation of MPT pore and Ca2+ overload.
Support of Funding Information: Russian Science Foundation № 19-75-20145
Citation Format: Marina E. Solovieva. Vitamin B12 augments Ca2+ dependent cytotoxicity of diethyldithiocarbamate through excessive ER-vacuolization, oxidative stress and mitochondrial depolarization in MCF-7 cells in culture [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 2032.
Abstract 2036: Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications
Authors: Song Kai , Minami Jenna , Ridgley Trevor , Huang Arthur , Jayaraman Rachana , Crosson William , Salazar Jesus , Pazol Eli , Niroshi Senaratne , Nagesh Rao , Paraiso Kim , Graeber Thomas 
Published: July 2021

Abstract
Cancer cells display two modes of focal amplifications (FA): extrachromosomal double minutes (DMs/ecDNAs) and intrachromosomal homogenously staining regions (HSRs). Understanding the plasticity of these two modes is critical for preventing targeted therapy resistance. We developed a combined BRAF plus MEK inhibitor resistance melanoma model that bears high BRAF amplifications through both ecDNA and HSR modes, and investigated FA dynamics in the context of drug resistance plasticity. Cells harboring FAs displayed mode switching between ecDNAs and HSRs, from both de novo genetic changes and selection of pre-existing subpopulations. We found that copy number plasticity is not exclusive to ecDNAs. Single cell-derived clones with HSRs also exhibit BRAF copy number and corresponding HSR length plasticity that allows them to respond to dose reduction and recover from drug addiction. In addition, upon kinase inhibitor escalation, we observed reproducible selection for cells with BRAF kinase domain duplications residing on ecDNAs. Whole genome sequencing of sublines with different FA formats allowed fine reconstructions of BRAF amplicon and revealed conserved structures during the transitions due to drug dose manipulations. The amplification boundaries in our cell line model were found consistent with those in clinically observed cases of combined BRAF/MAPK inhibitor resistance. RNA-seq and CRISPR screening performed on parental, ecDNA and HSR sublines revealed distinct biology and vulnerabilities associated with each kind of FA. Vulnerabilities for genes involved in ecDNA maintenance in DNA repair pathways identified in our model are consistent with previous reports, such as a role for DNA-dependent protein kinase (DNA-PK/PRKDC). Our screen reveals many new targets. In sum, the karyotypic plasticity of FAs allows cancer cells to respond to drug dose challenges through a myriad of mechanisms, including increases or decreases in DMs, shortening of HSRs, and acquisition of secondary resistance mechanisms. The cellular vulnerabilities identified in our study provide roadmaps for therapeutically targeting each kind of FA mode.
Citation Format: Kai Song, Jenna Minami, Trevor Ridgley, Arthur Huang, Rachana Jayaraman, William Crosson, Jesus Salazar, Eli Pazol, Senaratne Niroshi, Rao Nagesh, Kim Paraiso, Thomas Graeber. Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications [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 2036.
Abstract 2040: Analysis of DNA damage repair gene alterations and their association with tumor mutation burden in 1427 hepatocellular carcinoma patients
Authors: Yang Mengmei , Xie Wenzhuan , Huang Mengli , Zhu Xinhua 
Published: July 2021

Abstract
Background: DNA damage repair (DDR) pathway have attracted much attention because they modulate cancer risk, progression and therapeutic response. DDR deficiency sensitize the tumor to poly ADP-ribose polymerase inhibitors (PARPi). Also alterations in DDR genes are associated with higher tumor mutation burden (TMB) and better response to immune checkpoint inhibitors (ICI). Here we conduct a large-scale genomic analysis to clarify the landscape of DDR gene mutations in hepatocellular carcinoma and the correlation between individual gene mutation and tumor mutation burden.
Methods: Tumor samples from 1427 patients with advanced hepatocellular carcinoma were assayed through next-generation sequencing with 381 cancer-related genes including 30 predefined DDR genes: MRE11A, RAD50, BARD1, BLM, BRCA1, BRCA2, BRIP1, PALB2, MLH1, MSH2, MSH6, PMS2, POLD1, ATM, ATR, CHEK1, CHEK2, FANCA, FANCC, FANCD2, FANCG, FANCF, FANCL, RAD51, CUL3, ERCC1, POLE, MUTYH, and PRKDC. TMB (mutations per megabase [mut/Mb]) was calculated based on nonsilent somatic mutations in coding regions. Wilcoxon test was used for the comparisons and statistical significance was set at p = 0.05.
Results: Somatic DDR gene alterations were found in 18.8% of the HCC patients. The most common mutational types were copy number variation (30.54%), frameshift (21.89%), and nonsense (20%). The most frequently altered DDR genes are ATM (2.6%) and BRCA2 (2.2%), followed by FANCA (0.9%), PALB2 (0.8%), and MLH1 (0.7%). As to different functional pathways of DDR, the gene alteration frequency from high to low is HRR (6.1%), DS (3.2%), FA (2.5%), MMR (2.2%), BER (0.6%), NHEJ (0.6%), and NER (0.3%). The TMB of patients carrying somatic DDR gene alterations was significantly higher than those without somatic DDR gene alterations (7.26 vs 6.45 mut/MB, p&lt;0.001). As to individual gene, alterations in RAD50, MLH1, MSH2, CHEK2, and FANCE were significantly correlated with higher tumor mutation burden while alterations in other DDR genes were not.
Conclusion: This is the largest study to clarify the landscape of DDR gene mutations in hepatocellular carcinoma. This study confirms that mutations in DDR genes are relatively common and are correlated with higher tumor mutation burden. This also indicates not all DDR genes are equal since mutations of certain individual genes were associated with higher TMB whereas others were not; further studies are needed to confirm this.
Citation Format: Mengmei Yang, Wenzhuan Xie, Mengli Huang, Xinhua Zhu. Analysis of DNA damage repair gene alterations and their association with tumor mutation burden in 1427 hepatocellular carcinoma 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 2040.
Abstract 2046: Binding of FANCD2 to SRSF1 splicing factor prevents genomic instability through R-loop regulation
Authors: Olazabal-Herrero Anne , Green Allison M. , Chen Xiaoyong , Sung Patrick , Manoj Pillai M. , Kupfer Gary M. 
Published: July 2021

Abstract
BACKGROUND. Fanconi Anemia (FA) is a rare disease characterized by congenital abnormalities, bone marrow failure and cancer susceptibility. The pivotal role of the FA pathway is DNA interstrand crosslink (ICL) repair, where FANCD2-FANCI complex monoubiquitination is the key regulatory step. Recent studies have linked defects in the FA pathway to R-loop metabolism and implicated R-loops as an endogenous source of genomic instability that contributes to the FA phenotype, although the mechanism remains elusive. Splicing factors, such as SRSF1 (ASF/SF2), have also been linked to R-loops, cancer, and myelodysplastic syndrome (MDS), so we hypothesized a link between SRSF1 and FA.
METHODS. Cells: FA-D2 mutant (PD20) and corrected (D2), HeLa. siRNA transfections were used to reduce SRSF1 and FANCD2 protein levels. Survival assays were performed to determine cell sensitivity to DNA damaging agents. In vitro immunoprecipitation was performed using SRSF1 purified from bacteria and FANCD2-FANCI purified from insect cells. R-loops were studied using the Damage At RNA Transcription (DART) assay and S9.6 immunofluorescence. RNA FISH was used to measure mRNA export.
RESULTS. In order to study the involvement of splicing factors in the FA pathway, we knocked down SRSF1 by using siRNA. SRSF1 knockdown leads to impaired FANCD2 monoubiquitination after DNA damage as well as diminished cell survival, thus creating a FA-like phenotype. In addition, immunoprecipitation in cells and in vitro showed direct interaction between FANCD2 and SRSF1. We found that FANCD2 and SRSF1 knockdown leads to increased R-loops visualized by S9.6 immunofluorescence. Besides, DART assay revealed colocalization of SRSF1 and FANCD2 with R-loops in highly transcribed genomic sites, known to be R-loop hotspots. Additionally, we found that SRSF1 interacts with the mRNA export factor NXF1 in a FANCD2-dependent fashion and through RNA. Interestingly, SRSF1-NXF1 interaction is enhanced upon DNA damage. Finally, we found that FANCD2-deficient cells present defects in mRNA export, suggesting that SRSF1 and FANCD2 could cooperate in the prevention of R-loop formation by exporting accumulated RNA in the nucleus.
CONCLUSION. Our findings suggest a novel non-canonical mechanism of FANCD2 via regulation of SRSF1 in R-loop prevention via facilitation of mRNA export, thus preventing genomic instability.
Citation Format: Anne Olazabal-Herrero, Allison M. Green, Xiaoyong Chen, Patrick Sung, Pillai M. Manoj, Gary M. Kupfer. Binding of FANCD2 to SRSF1 splicing factor prevents genomic instability through R-loop regulation [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 2046.
Abstract 2048: Property analysis of chromosomal instability-adapted cells using multi-omics approaches
Authors: Morita Tomoko Yamamori , Haeno Hiroshi , Makinoshima Hideki , Suzuki Ayako , Kobayashi Susumu S. , Ohashi Akihiro 
Published: July 2021

Abstract
Background: Chromosomal instability (CIN), which includes polyploidy and aneuploidy, is characterized as the pillar of cancer hallmarks. CIN is observed in &gt;90% solid tumors, being correlated with malignancy grades, poor prognosis, and drug resistance. However, it is still unclear yet how aneuploidy triggers cancer initiation/progression, or how aneuploidy gives negative impacts on cancer therapeutics. In this study, we established our original CIN cell line model, aiming to understand biological significance of cancer aneuploidy on the CIN-mediated stress adaptation.Method: A colorectal cancer cell, HCT116, was treated with an Aurora-B kinase inhibitor for 24 hours to transiently induce CIN in these cells, and then cultured in a drug-free medium for &gt;10 days to establish “CIN-adapted cells”. Cell growth was evaluated by intracellular ATP concentrations. RNAs and DNAs extracted from the cells were subjected to RNA-seq, Exome-seq, and ATAC-seq by the NovaSeq sequencing systems. The cell lysates were subjected to metabolomics analyses by GC-MS system. Bioinformatics analyses were performed using these multiple-omics data.Results: In 10 days after aneuploid formation, 90% aneuploid cells were killed, while 10% aneuploid cells were proliferative with a high growth rate equivalent to that of the parental euploid cells, which appeared to overcome the CIN-mediated antiproliferative pressure. The CIN-adapted cells revealed to be highly sensitive to the low-glucose and hypoxic condition, suggesting that the metabolic pathways would be modulated to adjust the cells to the aneuploid-mediated stress. Transcriptome and metabolome analyses also revealed the gene expressions and the metabolites for the lipid or amino acids metabolisms were significantly changed in the CIN-adapted cells. These findings suggest that the CIN-adapted cells appear to restructure their signaling networks of the lipid and amino acid metabolic pathways, which is expected to play important roles for the aneuploid cells to survive under the CIN-mediated stress. We perform drug screening to identify essential factors in the process leading to CIN-adapted, aiming for a comprehensive understanding of the molecular mechanism and signal networks.Conclusions: We comprehensively characterized the CIN-adapted cancer cell model by the multi-omics approaches. These findings will help us to deeply understand the biological significance of CIN in cancer cells, providing a new insight to novel cancer therapeutics targeting CIN.
Citation Format: Tomoko Yamamori Morita, Hiroshi Haeno, Hideki Makinoshima, Ayako Suzuki, Susumu S. Kobayashi, Akihiro Ohashi. Property analysis of chromosomal instability-adapted cells using multi-omics approaches [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 2048.
Abstract 2050: Comprehensive genomic instability characteristics analysis in Chinese patients with osteosarcoma
Authors: Wang Yonggang , Li Hongtao , Xia Jing , Chen Shiqing , Bai Yuezong 
Published: July 2021

Abstract
Background: Previous study indicated that &gt;80% of osteosarcomas (OSs) have genomic instability characteristics similar to BRCA1/2-deficient cancers. Such BRCA-like traits could manifest a deficiency in the DNA damage repair (DDR) pathway. Patients with alterations in DDR pathway was reported sensitive to platinum-based chemotherapy and PARP inhibitors. In order to seek more precision therapeutic opportunities, we explored the molecular profile of Chinese OS patients to identify whether the analogous phenomena exists as reported.
Material and Methods: 44 archival Chinese OS patients from 3D Medicines and a public dataset including 35 OS patients (download from cBioPortal), were collected and analyzed next-generation sequencing (NGS) results on tumor tissues for gene mutations, copy number alterations (CNAs) and fusions in 33 cancer-associated DDR genes. Simultaneously, 44 Chinese OS patients' tumor analysis generated homologous recombination deficiency (HRD) scores (defined as the unweighted sum of LOH, TAI and LST scores) through the 733 gene panel sequencing.
Results: Among 44 Chinese OS patients, 18 (40.9%) of OS had DDR pathway gene somatic alterations, and TP53 (31.8%) were the most frequently mapped gene. Other DDR somatic alterations were identified in BRCA2 (6.8%), BARD1 (4.5%), BRCA1 (2.3%), PMS2 (2.3%), FANCA (2.3%) and CHEK1 (2.3%). It was worth mention that all gene alterations were copy number reduction except TP53. Collectively, the frequencies of HRR and mismatch repair (MMR) alterations were 15.9% and 2.3%, respectively. HRD score were available in 18 patients, the median was 40.5 (range, 0-81). As to public dataset, 6 (17.1%) patients harbored somatic alterations in DDR genes. The variations were BRCA2 (5.7%), ATR (2.9%), BARD1 (2.9%), FANCC (2.9%), MLH1 (2.9%), MSH6 (2.9%) and PMS2 (2.9%). The HRR mutations frequency was 11% and MMR was 8.6%.
Conclusions: This is the first large-scale comprehensive genomic instability characteristics analysis for Chinese patients with OS. Chinese patients and public dataset patients covers many common features. The DDR gene alteration frequency and HRD score are higher in OS patients, the association of variations in DDR genes and HRD score could be considered substitutable in predicting platinum-based therapy and PARP inhibitor response in clinical practice.
Citation Format: Yonggang Wang, Hongtao Li, Jing Xia, Shiqing Chen, Yuezong Bai. Comprehensive genomic instability characteristics analysis in Chinese patients with osteosarcoma [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 2050.
Abstract 2060: Therapeutic approaches for CCNE1-amplified HR-proficient ovarian cancer
Authors: Bai Shoumei , Buckanovich Ronald J. 
Published: July 2021

Abstract
Poly (ADP-ribose) polymerase (PARP) inhibitors have shown great success in ovarian cancer patients that are homologous recombination deficient (HRD). However, approximately 50% of high grade serous ovarian cancer (HGSOC) are HR-proficient. HR-proficient Cancer cells evade lethal DNA damage by triggering HR pathways at the cost of genome stability and evolving of more aggressive cancer cells. Cyclin E1 (CCNE1) amplification is a major subset of known HR-proficient HGSOC. This subset of cancer responds poorly to essentially all available ovarian cancer therapies including PARP inhibitors. Our study showed that the mTOR, HR, and checkpoint pathways were enriched in CCNE1-amplifed ovarian cancer patients. Interactome mapping demonstrated that many of the interactive proteins in the mTOR pathway were also components of HR and DNA checkpoint pathways. mTOR inhibition resulted in downregulation of HR and DNA checkpoint proteins. Downregulation of ATR partly mirrored mTOR inhibition. mTOR inhibitors and ATR inhibitors synergized with PARP inhibitors in vitro and in vivo in CCNE1-amplified HGSOC tumors. This was associated with decrease in cancer stem-like cancer cells and loss of polyploid tumor cells, which are vital for evolving of more aggressive, therapy-resistant cancer cells. These studies will have potential impact on further preclinical validation and clinical applications to cure CCNE1-amplifed HRP ovarian cancer.
Citation Format: Shoumei Bai, Ronald J. Buckanovich. Therapeutic approaches for CCNE1-amplified HR-proficient 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 2060.