bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 6246: WNT signaling-mediated DKC1 expression drives stemness associated with colorectal cancer
Authors: Nigam Shivansh , Praveen Ayush , Ateeq Bushra 
Published: June 2022

Abstract
The emergence of subclonal mutations in high grade colorectal (CRC) tumors driving resistance against EGFR targeting is a major challenge, thus necessitating the discovery of alternative therapeutic strategies. Targeting aberrantly active WNT signaling provides an alternate route for CRC treatment. Here we show that the high DKC1 expression in CRC patients associates with advanced-stage disease regardless of RAS/RAF mutation status, indicating the potential of DKC1 as a pan-CRC therapeutic target. We show a direct regulation of DKC1 downstream of canonical WNT signaling and confirm it as one of the WNT targets. Mechanistically, DKC1 regulates several critical cellular processes such as telomere maintenance, pseudouridylation of ribosomal RNAs, ribosomal biogenesis, etc. However, its plausible association with cancer stemness remains largely unexplored. We generated stable DKC1 knockdown (DKC1-KD) cell lines, namely WiDr and Colo320HSR to investigate its role in cancer stemness and CRC progression. DKC1-KD cells show a significant reduction in the tumor sphere forming ability along with diminished cell surface expression of stemness markers. Using multiple publicly available gene expression datasets for CRC patients, we performed Gene Set Variation Analysis to characterize the association of DKC1 with upregulated gene signature of consensus molecular subtype 2 (CMS2) of CRC, which has been associated with the highest incidence rate and active WNT signaling. Further, Database for Annotation Visualization and Integrated Discovery tool-based analysis across these CRC patient samples striated based on DKC1 expression shows positive enrichment of biological processes related to cell cycle regulation. Consistently, we observed a G2/M phase cell cycle arrest upon silencing DKC1 in CRC cells. We also show that silencing DKC1 results in enhanced chemosensitivity with a remarkable reduction in lipid accumulation. Moreover, upon DKC1 silencing, an increase in epithelial marker E-cadherin and a decrease in mesenchymal marker N-cadherin expression was observed, suggesting its role in epithelial-to-mesenchymal transition (EMT) in CRC. Our findings demonstrate the role of DKC1 in regulating stemness, drug resistance, and EMT to drive CRC oncogenicity. Taken together, we highlight the role of DKC1 as the most suitable pan-CRC drug target by employing the potential of pharmacological inhibitors against WNT signaling.
Citation Format: Shivansh Nigam, Ayush Praveen, Bushra Ateeq. WNT signaling-mediated DKC1 expression drives stemness associated with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6246.
Abstract 625: Immunophenotyping of TCR and BCR clonotypes
Authors: Chenchik Alex , Makhanov Mikhail , Liu Tianbing , Kobzik Lester 
Published: June 2022

Abstract
TCR and BCR repertoire profiling holds great potential for understanding disease mechanisms and for development of new therapeutics in infectious diseases, autoimmunity and in immuno-oncology. However, this potential could be greatly improved by combining information about receptor clonotypes with immunophenotypes of T and B cells. To facilitate these studies, we developed a novel technology for combined profiling of all human TCR and BCR variable regions and phenotypic characterization of immune cells. The developed TCR/BCR immunophenotyping method involves multiplex RT-PCR amplification and sequencing of CDR3 regions of TCR and BCR genes and a set of the most informative T- and B-cell phenotyping genes. Bioinformatic analysis of NGS data allows profiling of TCR/BCR clonotypes, and identification of major immune cell subtypes and their activation status. Data will be presented showing how combined TCR/BCR clonotype analysis combined with targeted expression profiling of immune cells can be applied for large-scale discovery of novel cell typing and activation biomarkers in several immune-responsive model systems. Preliminary studies indicate the assay has unparalleled throughput, sensitivity, and improved cost-effectiveness for high-throughput immunity biomarker discovery applications.
Citation Format: Alex Chenchik, Mikhail Makhanov, Tianbing Liu, Lester Kobzik. Immunophenotyping of TCR and BCR clonotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 625.
Abstract 6255: CRISPRa-mediated induction of apoptosis in therapy-resistant glioblastoma cells via derepression of pro-apoptotic genes
Authors: Yilmaz Ebru , Kayabolen Alisan , Atalar Semra Cemre , Yigit Buket , Ulukan Burge , Zeybel Mujdat , Bagci-Onder Tugba 
Published: June 2022

Abstract
Induction of apoptosis in cancer cells via de-repressing dormant pro-apoptotic genes might be a successful therapeutic strategy. In this study, we utilized several dCas9-mediated CRISPR activation (CRISPRa) systems to activate two key pro-apoptotic genes DR4 and XAF1 in glioblastoma cells. CRISPRa is used to activate endogenous genes via either recruiting transcription factors or epigenetic modifiers to regulatory regions in the genome. Previously conducted studies showed that VPR, Suntag, and SAM systems are the most effective transcription factor-based systems for activating several genes in a variety of cell lines. However, epigenetic modifier-based systems that involve the recruitment of TET demethylases have more permanent effects. Therefore, we hypothesized that combinations of these systems may induce stronger activation. We systematically tested various CRISPRa systems and their combinations to activate XAF1 and DR4, which are known to be epigenetically silenced in drug-resistant glioblastoma cells. Inducing the expression of these genes, we aimed to observe apoptosis in glioblastoma cells either at a basal state or upon treatment with the pro-apoptotic ligand TRAIL. By transcriptomic alteration and cell viability analyses, we demonstrated that the combinatorial system including dCas9-TET1 and MS2-p65-hsf1 was the most efficient one for the activation of DR4 and XAF1 genes in both 293T and glioblastoma cells. Another system in which multiple TET1 enzymes were recruited to the gene promoter together with MS2-p65-hsf1 was found to be effective similarly, suggesting that the promoter demethylation is critical for the activation of these genes. Activation of these genes with the most efficient systems caused apoptosis and also sensitized glioblastoma cells to TRAIL treatment. Our preliminary work on measuring methylation levels of several CpG sites in the promoter regions of DR4 is in line with our observations of cell-based phenotypes. Together, our study indicates that the combination of transcription factor-based and demethylation-based CRISPR activation systems can induce pro-apoptotic gene expression in cancer cells and lead cancer cells to undergo apoptosis. Future applications of this methodology might involve the development of cancer-specific pro-apoptotic therapies.
[E.Y. and A.K. contributed equally to this work.]
Citation Format: Ebru Yilmaz, Alisan Kayabolen, Semra Cemre Atalar, Buket Yigit, Burge Ulukan, Mujdat Zeybel, Tugba Bagci-Onder. CRISPRa-mediated induction of apoptosis in therapy-resistant glioblastoma cells via derepression of pro-apoptotic genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6255.
Abstract 628: Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies
Authors: Glez-Vaz Javier , Azpilikueta Arantza , Olivera Irene , Cirella Assunta , Teijeira Álvaro , Ochoa Carmen , Álvarez Maite , Eguren Iñaki , Luri-Rey Carlos , Sanmamed Miguel F. , Melero Ignacio 
Published: June 2022

Abstract
Background: On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved Chimeric Antigen Receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and co-stimulation of T cells will facilitate clinical development of CD137 agonists in the clinic.
Methods: We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both of the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored upon treatment with agonist anti-CD137 mAbs. Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-Mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISAs and Luminex and flow cytometry was used to monitor CD137 surface expression.
Results: CD137 co-stimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 co-stimulation inside subcutaneous implanted Matrigel® plugs. CD137 signaling domain-containing CAR T cells readily released sCD137 upon antigen recognition in a manner favored by the CD137 signaling domain. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137.
Conclusion: sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic co-stimulatory activity elicited by agonist CD137-targeted agents.
Citation Format: Javier Glez-Vaz, Arantza Azpilikueta, Irene Olivera, Assunta Cirella, Álvaro Teijeira, Carmen Ochoa, Maite Álvarez, Iñaki Eguren, Carlos Luri-Rey, Miguel F. Sanmamed, Ignacio Melero. Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 628.
Abstract 6300: Cyprohepatdine, an anti-histamine and epigenetic modifier, reverses epigenetic silencing of the tumor suppressor IRF6 in urothelial carcinoma
Authors: Chan Michael W.Y. , Chen Pie-Che , Huang Wan-Hong , Chuang Yu-Ming , Lin Ru-Inn , Lin Hon-Yi , Jou Yeong-Chin , Shen Cheng-Huang 
Published: June 2022

Abstract
Urothelial carcinoma (UC) is the most common malignancy of the urinary system. We have previously demonstrated that cyproheptadine (CPH), an anti-histamine, inhibited UC tumor growth in vitro and in vivo. However, the exact mechanism of how CPH inhibited tumor growth is not fully understood. In this study, we performed RNA-Seq in control BFTC905 UC cells and cells treated with CPH. The results showed that several genes, including IRF6 (interferon regulator factor 6), showing differential upregulation after CPH treatment. Real-time RT-PCR confirmed the restoration of IRF6 expression in a panel of UC cell lines treated with CPH. Similar to 5azaDC, bisulphite pyrosequencing found that CPH could decrease promoter methylation of IRF6 in UC cell lines. Interestingly, CPH could also increase total mono-methylation of H3K4 and acetylation of H3K27. Functional studies found that ectopic expression of IRF6 suppressed UC tumor growth in vitro and in vivo. Clinical studies also found that high-grade UC patient tissue samples has significantly higher IRF6 methylation than low-grade tumor samples. Taken together, our results suggested that CPH may be a novel epigenetic modifier exhibiting both DNMTi and HDACi activities in UC. The therapeutic potential of CPH in the treatment of UC deserves further investigation.
Citation Format: Michael W.Y. Chan, Pie-Che Chen, Wan-Hong Huang, Yu-Ming Chuang, Ru-Inn Lin, Hon-Yi Lin, Yeong-Chin Jou, Cheng-Huang Shen. Cyprohepatdine, an anti-histamine and epigenetic modifier, reverses epigenetic silencing of the tumor suppressor IRF6 in urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6300.
Abstract 6328: GPR56 as a therapeutic target for the development of antibody-drug conjugates for the treatment of colorectal cancer
Authors: Francisco Liezl E. , Chatterjee Treena , Liang Zhengdong , Zhang Sheng , Carmon Kendra 
Published: June 2022

Abstract
Cancer stem cell (CSC) marker LGR5 is highly expressed in colorectal cancer (CRC) and has been considered as a therapeutic target for the treatment of CRC. Previously, we and others showed that CRC tumors were eliminated by treatment with LGR5-targeted antibody-drug conjugates (ADCs). However, a fraction of tumors eventually recurred, likely due to CSC plasticity or the ability of LGR5-positive CSCs to interconvert into more drug resistant LGR5-negative cancer cells. Through our efforts to identify underlying mechanisms of drug resistance and plasticity and develop therapies to eliminate CRC, we showed that GPR56 expression is increased with loss of LGR5 protein expression. GPR56 is a member of the adhesion G-protein coupled receptor (GPCR) family which mediates diverse functions, including cell adhesion and tumorigenesis. GPR56 is also upregulated in CRC and increased expression levels correlate with poor patient survival. We recently showed GPR56 can promote tumor growth and drug resistance of CRC cells through upregulation of multidrug resistance proteins. These findings identify GPR56 as a potential therapeutic target for the treatment of CRC. Presently, we are focused on the development and characterization of anti-GPR56 ADCs that selectively bind GPR56-high CRC cells to deliver potent cytotoxic drugs and destroy them. We reported the generation of unique anti-GPR56 monoclonal antibodies (mAbs) and are currently developing and optimizing GPR56-targeted ADCs conjugated with different drugs. Cell-based binding assays show that our anti-GPR56 mAb binds with high affinity and specificity to 293T cells overexpressing recombinant GPR56 or GPR56-high CRC cells, with no detectable binding to GPR56-negative cells. Using cytotoxicity assays, we evaluated the in vitro efficacy of anti-GPR56 ADCs against several CRC lines with different levels of GPR56 expression and showed that the ADC is only effective against CRC cells expressing GPR56. Moreover, we evaluated the therapeutic efficacy of our anti-GPR56-ADC against CRC cell line and patient-derived tumor xenograft models in vivo and demonstrated significant inhibition of tumor growth. Altogether, these findings suggest that novel GPR56-targeted ADCs may be a promising therapeutic strategy to overcome resistance for the improved treatment of CRC.
Citation Format: Liezl E. Francisco, Treena Chatterjee, Zhengdong Liang, Sheng Zhang, Kendra Carmon. GPR56 as a therapeutic target for the development of antibody-drug conjugates for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6328.
Abstract 6336: Regulation of tissue factor dependent procoagulant properties by CD44: Implication for metastasis of breast tumor cells
Authors: Villard Amélie V. , Genna Anthony , Gilles Christine 
Published: June 2022

Abstract
The aim here is to study the implication of the stem cell marker and fibrin receptor, CD44, in the regulation of tissue factor and its associated pro-coagulant and pro-metastatic properties in breast tumor cells. We examined this CD44/TF/coagulation axis in breast cancer EMT+ (MDA-MB-231, Hs578T), EMT- (MCF7, T47D) and EMT-inducible (EGF-treated MDA-MB-468, MDA-MB-468 harboring a doxycycline-inducible snail expression vector) cellular models. We used siRNA to inhibit CD44 expression and realized RT-qPCR, western blot, flow cytometry analyzes and promoter reporter assays to monitor regulations of our gene of interest. Furthermore, we realized in vitro clotting assays and TF activity assays to examine coagulant properties, and in vivo experimental metastasis assays to assess early metastatic potential. We first confirmed by WB, RT-qPCR and FACS analyzes that CD44 is over-expressed in our EMT+ and EMT-induced (EMTi) cellular models, concomitantly with TF expression. Using siRNAs, we showed that CD44 silencing in these same EMT+ and EMTi cellular models decreased both mRNA and protein levels of TF. Clotting assays and TF activity assays further showed that CD44 silencing decreased pro-coagulant properties of our EMT+ and EMTi cellular models in vitro and of their pro-metastatic properties in vivo. Examining further the molecular mechanisms of TF regulation by CD44, we employed truncated TF promoter reporter vectors to identify key transcription factors linking CD44 to TF transcription. These experiments revealed that a TF promoter region containing Sp1 binding sites is sufficient to mediate the diminution of TF promoter activity seen after CD44 silencing. Mutations in the Sp1 binding sites abrogated the activity of the TF reporter vector. Further pointing to Sp1 as a key transcription factor implicated in TF regulation by CD44, we observed that silencing CD44 inhibited Sp1 expression and that silencing Sp1 decreased TF mRNA levels. Our results thus demonstrate a key role of CD44 in regulating TF expression in EMT+ cancer cells, and point to Sp1 as a transcriptional mediator of this regulation. This modulation of TF expression by CD44 in EMT+ cells led to a concomitant regulation of pro-coagulant properties, and impacts metastatic potential.
Citation Format: Amélie V. Villard, Anthony Genna, Christine Gilles. Regulation of tissue factor dependent procoagulant properties by CD44: Implication for metastasis of breast tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6336.
Abstract 6380: Essential chemokine score as a potential surrogate for immune checkpoint therapy in pan cancer
Authors: Bao Xuhui , Xie Liyi 
Published: June 2022

Abstract
The inhibition of the immune checkpoint axis of programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) has achieved significant anti-tumor therapeutic success, though it is still inadequate to ameliorate the dismal outcomes of the majority of cancer patients. Therefore, it is urgent and of interest in scrutinizing predictive indicators to enable a precision anti-tumor strategy, predict the therapeutic response, and better comprehend the mechanisms of resistance to immunotherapies. To evaluate the vigorous potential of CXCL9/10/11 score as a single, universal, and reliable surrogate to predict the response to anti-PD-1 therapy in different cancer types. The analyses in this study used the cancer genomic data from 31 different solid cancers in the Cancer Genome Atlas (TCGA) to calculate the CXCL9/10/11 score, CD274 expression, tumor mutational burden (TMB), and tumor immune phenotypes (IPs). The difference of CD274 expression, TMB, and tumor IPs was then compared between CXCL9/10/11 scoreLow and scoreHigh groups for each cancer type. Furthermore, this score was examined in two cohorts of patients with different cancer types treated with anti-PD-1 therapy to predict the expression of CD274, regression/progression status, and progression-free survival (PFS) between the scoreLow and scoreHigh groups. CD274 expression, TMB, and most tumor IPs were significantly elevated in CXCL9/10/11 scoreHigh group than in the scoreLow group among 12 different TCGA cancer types including breast cancer, lung cancer, colon cancer, melanoma, bladder cancer, and endometrial cancers. In two validation cohorts of 49 patients with lung cancer, melanoma, and head and neck cancer, and 85 patients with metastatic melanoma treated by anti-PD-1 therapy, respectively, scoreHigh tumors exhibited significantly high expression of CD274 than scoreLow tumors (p = 0.0006 and p = 0.0023, respectively). Significantly more patients in the scoreHigh group achieved post-treatment cancer regression than those patients in the scoreLow group (p = 0.0200 and p = 0.0427, respectively). The patients in the scoreHigh group enjoyed a significantly longer PFS than those in the scoreLow group (p = 0.0179 and p = 0.0229, respectively). Our findings suggest that CXCL9/10/11 score can serve as a single, universal, and reliable surrogate corresponding to CD274 expression, TMB, and tumor IPs within one variable among many different cancer types, and exhibits a vigorous and attractive potential to predict the therapeutic response to anti-PD-1 therapy in patients.
Citation Format: Xuhui Bao, Liyi Xie. Essential chemokine score as a potential surrogate for immune checkpoint therapy in pan cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6380.