bluuebunny/crossref_metadata_2025 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

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

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

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

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

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

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

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

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.
Abstract 6390: Chromosome arm aneuploidies as genetic vulnerabilities of triple-negative breast cancer
Authors: Dandage Rohan , Schwartz Michael , Karam Lynn , Hart Traver , Kuzmin Elena
Published: June 2022

Triple-negative breast cancer (TNBC) is a subtype of breast cancer which lacks the expression of key biomarkers, namely Estrogen and Progesterone receptors and HER2. Therefore, it lacks targeted therapies, resulting in the worst prognosis compared to other breast cancer subtypes. Chromosome 4p (chr4p) loss is recurrent in TNBC, correlates with poor prognosis, is an early event in tumor evolution, and confers a proliferative advantage onto cells. Here, we propose that chr4p loss can be leveraged as a genetic vulnerability of TNBC by identifying its specific synthetic lethal (SL) genetic interactions. An SL interaction occurs when the inactivation of individual genes is tolerated, however, the combined inactivation of the corresponding genes leads to a loss of cellular viability. SL interactions offer promising avenues for precision oncology therapeutic strategies of TNBC. We leveraged the publicly available CRISPR-mediated genome-wide gene inactivation screens (DepMap project) dataset to computationally predict pairwise SL interactions that are specific to individual genes within chr4p, as well as complex SL genetic interactions involving multiple genes spanning large segments of chr4p and the entire chromosome arm. We developed regression models to compare the inactivation effect of a given SL partner gene, between cell lines that harbor chr4p loss and those characterized by a copy-neutral status of chr4p, while effectively accounting for potential confounding effects. We integrated the putative SL interactions identified from these regression models with those identified using other methods, drugZ and MAGECK, therefore, resulting in a robust set of putative SL interactions. We have prioritized the SL interactions for their subsequent experimental validation in TNBC PDX-derived 2D and 3D cell models, which we will carry out using CRISPR-based gene editing. The prediction model and collective set of SL candidates identified in this study is a unique resource for the development of SL-based therapeutic strategies for TNBC and other cancers harboring chr4p loss.

Citation Format: Rohan Dandage, Michael Schwartz, Lynn Karam, Traver Hart, Elena Kuzmin. Chromosome arm aneuploidies as genetic vulnerabilities of triple-negative breast 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 6390.
Abstract 6391: Scalable colorectal cancer (CRC) diagnosis model with arterial and unenhanced phases of KRAS mutational status using Apache Spark
Authors: Adewunmi Mary Adetutu
Published: June 2022

The exponential increase in Medical data and Computer-aided diagnostic tools has made it easier for Machine Learning (ML) Researchers to extract valuable insights from medical images resulting in better patient outcomes during Medical Summarization. According to current statistics, 8.8 million people died every year due to cancer death. It is therefore imperative to use these available image datasets for better diagnosis, increased patient outcomes and recovery from deadly diseases. As a result, this study proposes a Colorectal Cancer classification model for detecting KRAS mutation status of Patients through their Radiomic images using the transfer learning of a deep learning pipeline on Apache Spark. The datasets will be curated from National Cancer Institute (NCI), features extraction or classification of Computed tomography(CT) images into CT- based handcrafted radiomic signatures with a Convolutional Neural Network(CNN) using the Apache Spark framework, the system uploads the segmented scanning images to the High Distributed File System (HDFS), Confidence Interval(CI), Area Under Curve(AUC), Precision and Recall for Validation cohort, Docker for app deployment and containerization of the model developed for reproducibility, transfer learning and model reuse. The result would be a real-time model using Deep learning pipelines with Apache Spark and Keras Tensor flow for KRAS Mutation detection using Colorectal Cancer images. In conclusion, this research project would produce a scalable and reproducible model for the faster diagnosis of Colorectal Cancer through the KRAS mutation status of CRC Patients.

Citation Format: Mary Adetutu Adewunmi. Scalable colorectal cancer (CRC) diagnosis model with arterial and unenhanced phases of KRAS mutational status using Apache Spark [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 6391.
Abstract 647: Association between adrenal-restrictive HSD3B1 inheritance and hormone independent subtypes of endometrial and breast cancer
Authors: McManus Jeffrey M. , Vargas Roberto , Bazeley Peter , Sabharwal Navin , Sharifi Nima
Published: June 2022

Breast and endometrial cancer are both malignancies that can be classified into subtypes based on their hormone dependence, such as estrogen receptor (ER)-positive vs. ER-negative breast cancer and low-grade vs. high-grade endometrial cancer. The gene HSD3B1 encodes the steroid metabolism enzyme 3β-hydroxysteroid dehydrogenase 1, which catalyzes a critical step in conversion of adrenal precursor steroids to potent androgens and estrogens. Associations between the missense-encoding germline variant rs1047303 in HSD3B1 have been described to affect outcomes of castration-resistant prostate cancer and associated with ER positivity in postmenopausal breast cancer. The adrenal-permissive 1245C allele enables greater production of androgens and estrogens from adrenal precursors, whereas the adrenal-restrictive 1245A allele limits this reaction pathway. Using data from The Cancer Genome Atlas’s TCGA-UCEC and TCGA-BRCA projects, we interrogated whether the adrenal-restrictive rs1047303 genotype was associated with less hormone-driven genomic clusters of endometrial and breast cancer. We determined the genotype frequencies for different tumor subtypes, as classified by the genomic analysis of Sanchez-Vega, et al. (2018). The adrenal-restrictive HSD3B1(1245A) allele was associated with the copy number (CN) high cluster of endometrial cancer (OR 1.59, 95% CI 1.12-2.26, p = 0.0098), notably composed of serous and high-grade endometrioid endometrial cancers which are less estrogen-dependent than low-grade endometrioid tumors. Similarly, the adrenal-restrictive HSD3B1(1245A) was associated with the basal subtype of breast cancer (OR 1.54 [1.13-2.09], p = 0.0057), a subtype that is largely ER-negative and is known to share genomic features with serous-type endometrial cancers. Lastly, we interrogated overall survival of endometrial cancer patients in the UK Biobank and found that patients homozygous for the adrenal-restrictive HSD3B1(1245A) had worse overall survival (HR 1.39 [1.16-1.68], p = 0.00049) and cancer-specific survival (HR 1.39 [1.14-1.70], p = 0.0013), a finding consistent with the A allele being enriched in the more aggressive CN-high subtype of endometrial cancer. Taken together, these findings suggest a role for adrenal-restrictive vs. adrenal-permissive steroidogenesis, by way of rs1047303 genotype, in the development of and/or outcomes from at least three different commonly hormone-associated types of cancer: prostate, breast, and endometrial.

Citation Format: Jeffrey M. McManus, Roberto Vargas, Peter Bazeley, Navin Sabharwal, Nima Sharifi. Association between adrenal-restrictive HSD3B1 inheritance and hormone independent subtypes of endometrial and breast 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 647.
Abstract 667: Inhibition of protein-protein interaction between STING and TRIM29 is a new approach to enhance anti-tumor immune response
Authors: Park Seung Bum , Cho Wansang , Chan Won Sol , choi young il , Ahn Sungoh , Lee Dong-Sup
Published: June 2022

Introduction: STimulator of INterferon Genes (STING) has been reported to be essential for antitumor immunity by inducing type I IFN expression, resulting in activation of both innate and adaptive immunity. Thus, pharmacological activation of STING pathway is actively being tested in many clinical studies. However, the clinical efficacy was not that clear, probably due to the limited expression of STING protein, which is tightly regulated by TRIM29 (E3 ligase)-mediated protein degradation pathway. Here, we present a novel small-molecule drug candidate with strong antitumor response in murine syngeneic model by upregulating cellular levels of STING through the inhibition of protein-protein interaction.

Methods: The inhibitors of STING-TRIM29 interaction were identified using luciferase-complementation high-throughput screening system. Target protein of the inhibitor was identified in cellular thermostability shift assay. Their immunological activity was tested in Raw264.7 and A431 cells as well as CT26 murine syngeneic model.

Results: The lead compound, SB24011, bound to STING and upregulated cellular levels of STING by inhibiting ubiquitin-mediated degradation. Together with STING agonist, SB24011 synergistically enhanced the inflammatory cytokine expression in cell lines as well as in CT26 syngeneic animal model. Intratumoral injection of SB24011 showed strong abscopal activity as single agent as well as synergistic antitumor responses with anti-PD1 antibody in CT26 syngeneic animal model.

Conclusions: We identified novel small-molecule drug candidate, SB24011, which prevents the degradation of both murine and human STING proteins, resulting in enhanced immune responses together with STING agonist, cGAMP. SB24011 showed strong abscopal antitumor activity as single agent and synergistic antitumor activity as combination agent with other IO agents in CT26 syngeneic models.

Citation Format: Seung Bum Park, Wansang Cho, Won Sol Chan, young il choi, Sungoh Ahn, Dong-Sup Lee. Inhibition of protein-protein interaction between STING and TRIM29 is a new approach to enhance anti-tumor immune response [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 667.
Abstract 675: Facilitating G-quadruplex formation in the KRAS promoter with polypurine reverse Hoogsteen oligonucleotides
Authors: Psaras Alexandra Maria , Valiuska Simonas , Noe Veronique , Ciudad Carlos J. , Brooks Tracy A.
Published: June 2022

KRAS is a GTPase involved in the proliferation signaling of a number of growth factors, such as epidermal growth factor. The protein is mutated or upregulated in an array of cancers, including pancreatic and ovarian cancers, where it correlates with more aggressive and chemoresistant disease. Within the promoter of KRAS lies a G-rich region capable of forming the higher order non-canonical DNA structure, the G-quadruplex (G4). Stabilization of G4 formation in the mid-region of the KRAS promoter downregulates transcription and facilitates decreased growth and chemo-sensitization of pancreatic and ovarian cancer cells with aberrant KRAS signaling. Our collaborative team designed Polypurine Reverse Hoogsteen (PPRH) hairpin oligonucleotides that establish Watson Crick bonds with the pyrimidine strand within the KRAS-mid-G4-forming region to enhance G4 formation, downregulate KRAS transcription and mediate an anti-proliferative effect. In particular, two PPRHs were designed to interact with the 3’ and 5’ ends of the G4-forming region, along with two correlating W:C, and one scramble, controls. The binding of these oligonucleotides to the G4-forming region was verified by gel electrophoresis, and the cell activity was examined in vitro both by luciferase and in pancreatic and ovarian cancer cells. Using a luciferase construct driven by the KRAS promoter, only the PPRHs, and none of the control oligonucleotides, significantly decreased activity. Cytotoxicity experiments in KRAS dependent and independent cell lines demonstrated that PPRHs, but not control oligonucleotides, selectively suppressed proliferation only in the dependent pancreatic and ovarian cancer cell lines. Correlation of the cytotoxic effects of these potential therapeutic PPRHs with transcriptional downregulation is under investigation. PPRH also demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound in KRAS-dependent pancreatic cells, and sensitization to standard chemotherapeutic regimens is also being studied. These designed PPRH oligonucleotides selectively stabilize G4 formation within the KRAS mid promoter region, and represent an innovative approach both for G4-stabilization and KRAS modulation with potential for development into novel therapeutics.

Citation Format: Alexandra Maria Psaras, Simonas Valiuska, Veronique Noe, Carlos J. Ciudad, Tracy A. Brooks. Facilitating G-quadruplex formation in the KRAS promoter with polypurine reverse Hoogsteen oligonucleotides [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 675.
Abstract 694: E-slice: A novel 3D culture platform for precision medicine
Authors: Gopalan Archana , Gallup Thomas D. , Wood Stephanie , Maldonado Jose , Margain Corina , Esnaola Nestor F. , Kim Min P. , Kopetz E. Scott , Yun Kyuson
Published: June 2022

The need for personalized medicine in oncology is widely accepted but translating this important concept into clinical practice has been challenging. Currently, the dominant platform for precision medicine utilizes genomics/sequencing-based assays to measure the expression and/or mutational profiles and then infer patient responses to therapies based on previous knowledge; however, this approach benefits less than 10-15% of patients with profiled tumors. Recognizing the inherent limitations of these inference-based methods, functional assays (e.g., organoids and PDX models) have been developed; however, these approaches also have significant limitations including high cost and time required to establish the models, low “take rates”, and destruction of the native tumor microenvironment (TME). To overcome these challenges, EMPIRI uses a novel 3D ex vivo tumor slice culture method (E-slices) that enables rapid, personalized drug sensitivity testing in intact patient tumor tissues. Major differentiators of the E-slice platform from other ex vivo methods include the use of chemically defined, serum-free medium, longitudinal viability measurements from the same tissue, tracking of dynamic responses to treatment over 2-3 weeks, and retention of the native TME and tissue architecture, unlike other approaches. In addition, E-slices can be generated from any solid tumor tested thus far (breast, lung, colorectal, pancreas, brain, head 7 neck, and others) from patient tumors directly and PDX and genetically engineered mouse models. In addition, because E-slices retain tumor-infiltrating immune cells in their native microenvironment and spatial topography of all cell types in the endogenous configuration, it sustains immune cell survival and proliferation and measures immunotherapy responses ex vivo. The E-slice method is compatible with biopsies as well as surgical samples. Importantly, it has been shown to accurately predict individual patient treatment responses to chemotherapies and targeted therapies in 4-12 days, paving the way for evidence-based personalized treatment selections in a clinically actionable time frame. In summary, we present a novel ex vivo 3D human tumor tissue drug sensitivity platform that faithfully replicates the patient tumor tissues and provides personalized treatment responses in a clinically actionable time frame.

Citation Format: Archana Gopalan, Thomas D. Gallup, Stephanie Wood, Jose Maldonado, Corina Margain, Nestor F. Esnaola, Min P. Kim, E. Scott Kopetz, Kyuson Yun. E-slice: A novel 3D culture platform for precision medicine [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 694.
Abstract 715: Targeting the mTOR pathway for the prevention of er-negative breast cancer
Authors: Mazumdar Abhijit , Hill Jamal , Tahaney William , Ma Yanxia , Contreras Alejandro , Sei Shizuko , Mohammed Altaf , Brown Powel
Published: June 2022

Background: Triple-Negative breast cancer (TNBC) is an aggressive cancer that lacks expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. These cancers have a poor prognosis and are treated predominantly with chemotherapy. Therefore, we are working to find preventive therapies for these potentially lethal cancers. Dysregulation of PI3K-mTOR pathway is commonly associated with TNBC and other ER-negative breast cancers. We hypothesized that targeting mTOR may prevent development of ER-negative breast cancers and in this study tested whether the mTOR inhibitor, everolimus, prevents cancer in several mouse models of ER-negative breast cancer.

Methods: MMTV-erbB2 mice were purchased from the Jackson Laboratory. C3(1)/SV40TAg and BRCA1co/co;MMTV-Cre;p53+/- were generated through breeding. P53-null and p53-mutant mammary gland mice were generated by transplanting p53-null or p53-mutant mammary glands into cleared fat pads of p53-wild type mice. The mice were treated with either control or everolimus (5mg/kg), administered by oral gavage daily (5X/week). All mice develop palpable mammary tumors within 8-12 months. Mice were observed daily for tumor formation and drug toxicity. The percentage of tumor free mice was recorded, and time to tumor development was visualized using Kaplan-Meier curves and analyzed using the generalized Wilcoxon test.

Results: Treatment with everolimus significantly increased median survival of all mouse models. Everolimus treatment was also able to completely prevent mammary tumor formation in 27% of C3(1)/SV40Tag mice and 44% p53-mutant mammary gland mice. In p53-null mammary gland mice, 46% of control mice had developed tumors after 420 days, compared to 7% of everolimus treated mice. Long term treatment of everolimus was associated with mild toxicity that includes slight weight loss (<10%) and skin changes (matted hair, skin erythema).

Conclusions: Everolimus significantly delayed mammary tumorigenesis in all five breast cancer mouse models. Our results suggest that everolimus is a promising cancer preventive drug for ER-negative tumors, and that further studies of everolimus in combination with other targeted therapies are warranted. In the future, clinical trials of the everolimus should be considered for the prevention of breast cancer in high-risk patients. Supported by NCI PREVENT Cancer Preclinical Drug Development Program (HHSN-2612015000-18I (PB).

Citation Format: Abhijit Mazumdar, Jamal Hill, William Tahaney, Yanxia Ma, Alejandro Contreras, Shizuko Sei, Altaf Mohammed, Powel Brown. Targeting the mTOR pathway for the prevention of er-negative breast 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 715.
Abstract 740: Obesity promotes liver pro-oxidative and DNA damage in mammary tumor of Zucker rats
Authors: Hakkak Reza , Melnyk Stepan , Korourian Soheila
Published: June 2022

Obesity is a major health problem in the US and world. Obesity is associated with the risk of cardiovascular disease, type 2 diabetes, and certain types of cancers such as breast cancer. Our laboratory reported that obesity increased mammary tumor model using obese Zucker rat model. However, the effects of obesity in the liver in regard to oxidative stress and DNA reaction is less known. In present study, we used a DMBA induced-mammary tumor model to further investigate the effects of obesity on creation of pro-oxidative environment in liver and oxidative DNA reaction. Lean and obese female rats were at 50 days of age received 7,12-dimethylbenz(α)anthracene (DMBA) and were sacrificed 20 weeks later. HPLC-ECD, HPLC-UV and LC-MS were used. Obesity capable significantly modify methylation status in liver caused higher (p < 0.001) SAM/SAH ratio (know as “methylation ratio”) and increased (p < 0.05) global DNA methylation. Also, obesity decrease GSH/GSSG ratio (known as “oxidative stress ratio”) with p < 0.0001 inducing higher oxidative stress and as results, increase presence both oxidized guanosine (8-OH-Guanosine) with p < 0.001 and oxidized 5metylCytosine (5hmCytosine) p<0.04 in liver DNA. 8-OH-Guanosine/5hmCytosine ratio was not significantly affected. Obesity caused significant oxidative stress in liver, causing oxidative DNA damage and change of DNA methylation pattern. These changes may contribute to the development of fatty liver in breast cancer models.

Citation Format: Reza Hakkak, Stepan Melnyk, Soheila Korourian. Obesity promotes liver pro-oxidative and DNA damage in mammary tumor of Zucker rats [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 740.
Abstract 796: Dual inhibition of NHEJ and MMEJ induces synthetic lethality in TP53 mutant cancers
Authors: Patterson-Fortin Jeffrey , Bose Arindam , Tsai Wei-Chih , Grochala Carter , Nguyen Huy , Zhou Jia , Parmar Kalindi , Lazaro Jean-Bernard , Liu Joyce , McQueen Kelsey , Shapiro Geoffrey I. , Kozono David , D'Andrea Alan D.
Published: June 2022

DNA repair pathway inhibitors are a new class of anti-cancer drugs that are advancing in clinical trials. While inhibitors of targets in the Non-Homologous End Joining (NHEJ) DNA repair pathway, such as DNA-dependent protein kinase (DNA-PK), are available for clinical use, it remains unclear which cancers are vulnerable to these agents. In a genome-wide CRISPR knockout screen with the DNA-PK inhibitor M3814, we identify loss of POLQ, encoding polymerase theta, and other genes in the microhomology-mediated end-joining (MMEJ) pathway as key predictors of sensitivity to DNA-PK inhibition, whereas loss of TP53 conferred resistance to DNA-PK inhibition. Inhibition of DNA-PK led to increased DNA double strand break end-resection, increased expression of polymerase theta, and activation of MMEJ repair. Combined DNA-PK inhibition by M3814 and polymerase theta inhibition by novobiocin resulted in synthetic lethality mediated by the accumulation of resected DNA and apoptosis. Significantly, this drug combination efficiently killed TP53-deficient human patient-derived xenografts and the corresponding tumor organoids. Taken together, our results provide a rationale for the combination of an inhibitor of DNA-PK mediated NHEJ and an inhibitor of polymerase theta mediated MMEJ in an anti-cancer trial. If DNA-PK is inhibited, cancers develop a hyper-dependence on MMEJ and an upregulation of DNA double strand break end resection and polymerase theta expression. Similarly, P53-deficiency which confers resistance to DNA-PK inhibition, also leads to a hyper-dependence on MMEJ and an upregulation of polymerase theta expression. Thus, a combination of DNA-PK and polymerase theta inhibitors may provide a precision treatment strategy for TP53-mutant solid tumors, known to account for 50% of newly diagnosed cancers.

Citation Format: Jeffrey Patterson-Fortin, Arindam Bose, Wei-Chih Tsai, Carter Grochala, Huy Nguyen, Jia Zhou, Kalindi Parmar, Jean-Bernard Lazaro, Joyce Liu, Kelsey McQueen, Geoffrey I. Shapiro, David Kozono, Alan D. D'Andrea. Dual inhibition of NHEJ and MMEJ induces synthetic lethality in TP53 mutant cancers [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 796.
Abstract 856: Coiled-coil domain containing 3 suppresses breast cancer growth by protecting p53 from proteasome-mediated degradation
Authors: Lee Hyemin , Li Caiyue , Jung Ji Hoon , Zhang Yiwei , Wang Jieqiong , Liu Chang , Segall Allyson M. , Sheffmaker Roger L. , Zeng Shelya X. , Lu Hua
Published: June 2022

Coiled-coil domain containing 3 (CCDC3) was previously shown to regulate liver lipid metabolism as a secretory protein. Our recent studies revealed an intracellular role of CCDC3 as a tumor suppressor in breast cancer (BrC). Bioinformatics datasets analysis showed that CCDC3 is under-expressed in BrCs, while its higher levels are correlated with higher overall survival and lower relapse of cancer patients. CCDC3 is positively correlated with p53 and its target genes. Ectopic CCDC3 markedly suppressed proliferation, colony formation, and xenograft tumor growth by augmenting p53 activity in BrC cells. In contrast, knockdown of endogenous CCDC3 using CRISPR led to reduction of p53 level and activity, consequently promoting the proliferation of BrC cells. Mechanistically, CCDC3 bound to the C-termini of p53 and MDM2, consequently stabilizing p53 in the nucleus and impairing MDM2 recruitment of p53 to the 26S proteosome without inhibiting p53 ubiquitination. Also, knockdown of CCDC3 caused the resistance of BrC cells to 5-FU to some degrees by alleviating p53 response to this drug. p53 induced CCDC3 expression by binding to its promoter specifically in BrC cells. Our results unveil a unique mechanism underlying CCDC3 activation of p53 in a positive feedback fashion to suppress BrC growth and suggest that CCDC3 deficiency could be a factor for drug resistance of BrCs.

Citation Format: Hyemin Lee, Caiyue Li, Ji Hoon Jung, Yiwei Zhang, Jieqiong Wang, Chang Liu, Allyson M. Segall, Roger L. Sheffmaker, Shelya X. Zeng, Hua Lu. Coiled-coil domain containing 3 suppresses breast cancer growth by protecting p53 from proteasome-mediated degradation [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 856.