bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4178: Lysosomal storage dysfunction as a risk factor in pancreatic cancer
Authors: Kim Hyemin , Koh Youngil , Park Joo Kyung
Published: April 2023

Background: Lysosome is closely linked to autophagy activity which has an important role in pancreatic adenocarcinoma (PDAC). We investigated whether lysosome storage dysfunction (LSD) contributes to PDAC development.

Materials and Methods: Comparison of germline putative pathogenic variants (PPV) in genes involved in lysosome functions was performed between PDAC patients (N=418) and healthy controls (N=745) using next generation sequencing. Using mouse pancreas organoid, consequences of GALC downregulation in PDAC development was evaluated. Transcriptome data analysis of human PDAC organoid according to PPV status was followed.

Results: PPV in LSD related genes were enriched in PDAC patients compared to healthy controls (Log2OR = 1.65, P = 3.08  ×  10−3). For limited stage PDAC patients, PPV carriers were diagnosed with PDAC in younger age compared to non-carriers (mean age 61.5 vs. 65.1 years, p=0.038). In mouse pancreas organoid, Ki-67 index significantly increased as GALC was downregulated. Altered autophagy activity with increased autophagy flux following GALC dysfunction was observed in both human cancer cell line and mouse organoid. mTOR downregulation was noted in mouse pancreas organoid. RNA sequencing analysis of human PDAC organoid revealed metabolism alteration related to LSD.

Conclusion: Genetically defined lysosome dysfunction is frequently observed in young age onset PDACs. Lysosome dysfunction might contribute to PDAC development via altered metabolism and autophagy activity.

Key words: lysosomal storage disease (LSD), germline, pancreatic ductal adenocarcinoma (PDAC)

Citation Format: Hyemin Kim, Youngil Koh, Joo Kyung Park. Lysosomal storage dysfunction as a risk factor in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4178.
Abstract 4194: The FuSion Project of Pan-Cancer Early Screening in Chinese– An integrative study by Fudan University and Singlera
Authors: Suo Chen , Zhao Renjia , Jiang Yanfeng , Zhang Yunzhi , He Qiye , Su Zhixi , Liu Rui , Jin Li , Chen Xingdong
Published: April 2023

Background: Cancer is the leading cause of premature deaths. Screening in the population can effectively improve the prognosis by diagnosing cancer early. Previous studies have found that cancer biomarkers, for example, ctDNA methylation and protein biomarkers are clearly associated with the occurrence and development of cancer. To evaluate the performance of reported biomarkers for cancer early screening in real world, we launched an integrative study by Fudan University and SInglera for pan-cancer early detectiON (FuSion) project since 2021. Method: The FuSion Project (NCT05159544) is a prospective and multicenter cohort study of pan-cancer screening in Chinese population. We will construct a stepwise model of risk stratification for screening of multiple cancer types, including lung, esophagus, stomach, liver, pancreatic and colorectal cancers. A total of 50,137 individuals recruited in the Taizhou Longitudinal Study (TLS) between 2012 and 2021 are investigated. Eligible participants are aged 20-75 years and without history of cancer. They have completed a questionnaire and provided blood samples for baseline tests, which measure a total of about 50 indicators in the routine blood work, blood biochemistry, and serum cancer biomarkers, such as carbohydrate antigen 19-9 and carcinoembryonic antigen. Informed consent is obtained from all participants. We build risk stratification models based on risk factors collected in the questionnaire and from baseline test results for each cancer type, respectively. Then, 10,000 high-risk- and 5,000 low-risk individuals of cancers are identified by the cancer-specific risk stratification model. They will be screened by the PanSeerX assay using cancer-related DNA methylation markers, and medically followed-up for at least two years. Sensitivity, specificity, positive- and negative-predictive values of the screening models will be determined based on the true cancer incidences. Finally, we will validate the cancer screening strategies using 10,000 average-risk participants recruited from multiple centers by regular checkups and from other large-scale cohort studies.

Citation Format: Chen Suo, Renjia Zhao, Yanfeng Jiang, Yunzhi Zhang, Qiye He, Zhixi Su, Rui Liu, Li Jin, Xingdong Chen. The FuSion Project of Pan-Cancer Early Screening in Chinese– An integrative study by Fudan University and Singlera. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4194.
Abstract 4309: Early apoptotic measurements of patient-derived organoids predict patient response to therapy
Authors: McQueeney Kelley E. , Bhola Patrick , Hill Sarah J. , Letai Anthony
Published: April 2023

The use of imperfect models and ex vivo culture systems to try to predict patient drug response represents an enormous bottle neck in cancer treatment. Nonetheless, determining how effective an approved drug will be for an individual cancer patient, as well as identifying novel compounds that may be beneficial to a specific population often requires the use of primary tumor cells. Patient-derived organoids represent an intermediate between primary tumor cells, whose limited supply may hinder reliable drug testing, and cell lines, which often do not reflect what happens in vivo. Herein, we describe the development of a novel assay platform, termed 3D-DBP (3D dynamic BH3 profiling), to detect early apoptotic measurements in ovarian cancer patient-derived organoids and present evidence that this method can be used to predict patient response to therapy. We have optimized the use of patient-derived organoids from 16 individual tumors in a microscopy-based imaging assay. We image the BH3 peptide-induced release of cytochrome c from mitochondria, which indicates permeabilization of the outer mitochondrial membrane, in intact organoids. The less cytochrome c retained in each organoid, the more primed that organoid is for apoptosis. By comparing results of drug-treated and untreated cells, we can identify drugs that cause a significant increase in apoptotic priming in organoids. In the 16 patient-derived organoids investigated this 3D DBP technique was an effective means of predicting patient response to carboplatin therapy. In summary, we have not only created a means of visualizing drug response in intact organoids, but also have demonstrated its clinical utility.

Citation Format: Kelley E. McQueeney, Patrick Bhola, Sarah J. Hill, Anthony Letai. Early apoptotic measurements of patient-derived organoids predict patient response to therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4309.
Abstract 4440: Efficacy of Gen-1, an interleukin-12 immune gene therapy, at different dose frequencies
Authors: Sood Subeena , Boyer Jean D. , Kim Jessica , Matar Majed M. , Signer Olivia , Rice Jennifer S. , Smith Alanna M. , Borys Nicholas , Anwer Khursheed
Published: April 2023

Introduction: The purpose of this study was to investigate the effect of dosing frequency on the antitumor activity of intraperitoneal GEN-1, an interleukin-12 (IL-12) immune gene therapy in a mouse model of peritoneally disseminated ovarian cancer.

Procedures: Three GEN-1 dosing regimens were examined for efficacy in ID-8 tumor-bearing mice: weekly, every 2 weeks and every 3 weeks. 2.5 million cancer cells were bolus injected into 4 groups (B, C, and D) of 10 mice each. A control group (A) of 6 mice had PBS injected IP without tumor and an untreated control (E) of 15 mice were injected with 2.5 million cancer cells. Groups B, C, and D were injected with GEN-1 IP weekly, every 2 weeks, and every 3 weeks respectively. Six animals from each group B, C, and D were harvested for translational research (TR) after 5 weekly, 3 every 2-week and 2 every 3-week treatments respectively. The remaining 4 animals in each group were followed for weight change (tumor burden) and survival. Additionally, TR evaluated change in ascites T-cell, B-cell and myeloid cell populations.

Results: There was a gradual rise in tumor burden and mortality in all treatment groups with comparable rate between once every week and once every 2-week regimens. Once every 3-week regimen had relatively higher mortality rate and higher tumor burden. There were similar or higher increases in T-cell and B-cells with reduced treatment frequency with lesser increases in myeloid cell density with reduced treatment frequency.

Conclusions: Once every 2-week dosing of GEN-1 in human studies is warranted. Future combination studies of Gen-1 with immune checkpoint inhibitors will evaluate the safety and efficacy of this regimen.

Citation Format: Subeena Sood, Jean D. Boyer, Jessica Kim, Majed M. Matar, Olivia Signer, Jennifer S. Rice, Alanna M. Smith, Nicholas Borys, Khursheed Anwer. Efficacy of Gen-1, an interleukin-12 immune gene therapy, at different dose frequencies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4440.
Abstract 446: LONP1 inhibition induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells
Authors: Tcheng Matthew
Published: April 2023

Acute myeloid leukemia (AML) is a hematological malignancy characterized by the uncontrolled proliferation of abnormal myeloid precursors that fail to fully differentiate, resulting in the accumulation of malignant leukemic cells. High rates of nuclear-encoded protein synthesis, supporting increased proliferation rates, render leukemic cells reliant on increased proteolytic systems to clear misfolded proteins. Compared to non-transformed blood cells, AML cells and stem cells exhibit a unique metabolic phenotype characterized by increased mitochondrial mass and greater reliance on oxidative metabolism for survival and proliferation. To identify novel biological vulnerabilities in the mitochondrial proteome, we analyzed the DepMap (depmap.org) for essential mitochondrial genes in AML. Through this analysis, we identified the mitochondrial protease LONP1 as essential for a subset of AML and in the top 10% of essential genes for this disease. LONP1 is a nuclear-encoded AAA+ serine protease that resides in the mitochondrial matrix and maintains the mitochondrial proteome, mainly via the clearance of misfolded, unassembled or oxidatively-damaged proteins. However, its importance and function in AML has not been previously reported. Compared to normal hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets while LONP1 protein was increased >2 fold in tested AML primary samples (N=16). Using shRNA and CRISPR gRNA, LONP1 knockdown and knockout reduced the growth and viability of OCI-AML2, OCI-M2, and NB4 cells. Mechanistically, genetic knockdown or knockout of LONP1 increased levels of insoluble, aggregated mitochondrial proteins including ClpP, TUFM, and NDUFA9 as measured by immunoblotting of soluble and insoluble protein fractions as well as by the fluorescence of the protein aggregation dye PROTEOSTAT by confocal microscopy. The accumulation of insoluble mitochondrial protein aggregates through LONP1 inhibition exploits an increased reliance on chaperone and protease systems due to abnormally high protein synthesis. This novel therapeutic avenue targets a unique metabolic vulnerability to eliminate AML cells.

Citation Format: Matthew Tcheng. LONP1 inhibition induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 446.
Abstract 458: Targeting p27Kip1 to inhibit CDK4/6/2 as a novel therapeutic strategy to treat pancreatic ductal adenocarcinoma
Authors: Guido Carolina , Gottesman Susan RS , Blain Stacy W.
Published: April 2023

CDK4/6 inhibitors are powerful tools used clinically to treat metastatic, HR+ BC patients. While combined CDK4/6i treatment in combination with Estrogen modulation significantly extends Progression Free Survival (PFS) in metastatic HR+ breast cancer patients, those treated invariably develop aquired resistance, which results in only marginal improvement in Overall Survival (OS). The drug resistance seen in the presence of CDK4/6i, such as palbociclib, has been shown to be attributed to activation and compensation by CDK2. Pancreatic ductal adenocarcinoma (PDAC) is characterized by aberrant activation of the CyclinD-CDK4/6 signaling pathway and inactivation of the CDKN2A locus encoding the CDK4/6 inhibitor, p16INK4A, and was initially thought to be a good candidate for CDK4/6i use. Surprisingly, CDK4/6i do not show response in most PDAC lines and have not been used clinically in this tumor type. We hypothesized that PDAC may be intrinsically resistant to CDK4/6 inhibition due to activation of CDK2 and as such inhibition of both CDK4/6 and CDK2 may be required. We have developed a novel strategy to kill drug-resistant tumor cells using a therapeutic liposomal:peptide formulation, IpY.1, to inhibit the tyrosine phosphorylation of p27Kip1(CDKN1B), which in turn inhibits both CDK4/6 and CDK2. IpY.1 is specific for p27, and this reduces toxicity relative to that seen with the small molecule ATP-competitive CDK4/6i or CDK2i. IpY.1 blocks DNA replication in HR+ breast cancer (BC) cells and mouse models, but unlike CDK4/6i or CDK2i treatment, it induces ROS-dependent necroptosis to kill the tumor cells.

Palbociclib did not have a significative effect on ATP levels or proliferation of PDAC cell lines. In contrast, we found that IpY.1 was able to block proliferation and reduce viability in palbociclib-resistant PDAC lines, suggesting that dual CDK4/6 and CDK2 inhibition via p27 targeting was required. Some PDAC lines were “super sensitive” and responded with IC50 values ~10X less than seen with the ER+ BC lines, and we are currently screening for biomarkers of response. CDK2 activity was reduced in IpY.1 treated lines and markers of necroptosis were detected. Immunoblot analysis showed decreased pY88 phosphorylation of p27 (the target of IpY) demonstrating target engagement. We have demonstrated that unlike palbociclib, IpY.1 blocks proliferation and viability in all PDAC lines tested, suggesting that targeting p27 may be a viable approach to treat PDAC.

Citation Format: Carolina Guido, Susan RS Gottesman, Stacy W. Blain. Targeting p27Kip1 to inhibit CDK4/6/2 as a novel therapeutic strategy to treat pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 458.
Abstract 4605: Chronic VEGF-C signaling exacerbates the progression of non-alcoholic steatohepatitis and hepatocarcinoma through endothelial VEGFR2 and VEGFR3
Authors: Youn Seock-Won , Eng Jason W.-L. , Swaminathan Bhairavi , Teneqexhi Pamela , Vadakath Rahul , Kitajewski Jan K.
Published: April 2023

Obesity, aging, and metabolic diseases contribute to non-alcoholic fatty liver disease (NAFLD) that can progress to non-alcoholic steatohepatitis (NASH) due to ongoing inflammation and pathological angiogenesis. At present, NASH is a leading cause of cirrhosis, hepatocarcinoma cancer (HCC) and liver transplant within the United States. Pathological angiogenesis of liver sinusoidal endothelial cells (LSECs) is one of the hallmarks of liver injury that disrupt normal LSEC physiology. Therefore, strategies which target factors involved in LSEC dysregulation may improve the progression of hepatic diseases. Examination of a human tissue microarray identified increased levels of vascular endothelial growth factor-C (VEGF-C) in NASH livers compared with healthy individuals. Additionally, single nuclei RNA sequencing and immunofluorescent staining revealed that the receptors for VEGF-C, VEGFR2 and VEGFR3, are primarily expressed by the LSECs. Using murine models of NASH, we further investigated the role of VEGF-C during disease progression. Chronic over expression of VEGF-C by adeno-associated viral (AAV) infection in mice fed a western diet with carbon tetrachloride injections increased NASH progression compared with control AAV infected NASH mice. Interestingly, overexpression of VEGF-C C156S, a variant of VEGF-C which exclusively binds to VEGFR3, resulted in significantly increased hepatic steatosis, but only minimal fibrosis. To determine whether blockade of VEGF-C signaling would delay disease progression, we administered lenvatinib, a tyrosine kinase inhibitor which primarily blocks VEGFR2 and VEGFR3 at low doses, to NASH mice. We discovered that low dose lenvatinib resulted in significantly decreased amount of fibrosis, steatosis, and tumor formation. Analysis of vehicle treated and lenvatinib treated livers by single nuclei RNA sequencing uncovered significant changes in endothelial cell genes associated with extracellular interactions and inflammation, as well as hepatocyte genes involved in lipid synthesis and metabolism. Taken together, these findings indicate that chronic VEGF-C production in NASH plays a role in promoting liver fibrosis and steatosis as well as HCC development, and that blockade of the downstream receptors for VEGF-C, VEGFR2 and VEGFR3, may be a promising therapeutic strategy to mitigate disease severity.

Citation Format: Seock-Won Youn, Jason W.-L. Eng, Bhairavi Swaminathan, Pamela Teneqexhi, Rahul Vadakath, Jan K. Kitajewski. Chronic VEGF-C signaling exacerbates the progression of non-alcoholic steatohepatitis and hepatocarcinoma through endothelial VEGFR2 and VEGFR3. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4605.
Abstract 4612: Microphysiological vascularized solid liver tumor model for drug and cell therapy
Authors: Vasudevan Jyothsna , Vijayakumar Ragavi , Reales-Calderon Jose Antonio , Lam Maxine Sin , Ow Jin Rong , Aw Joey , Tan Damien , Adriani Giulia , Pavesi Andrea
Published: April 2023

Development of efficacious therapeutic strategies against solid tumors is limited by the lack of pre-clinical models that can reliably predict treatment outcomes in patients. This is primarily because models often do not accurately reflect the complexity of the tumor microenvironment (TME). The TME consists of vasculature, stromal cells and immune cells that can promote tumor resistance or prevent targeted drug and cell therapy. Here, we developed an in vitro vascularized liver tumor model in a microfluidic device to evaluate drug delivery and immunotherapy approaches. The model consists of a tumor spheroid surrounded by a perfusable self-organized vasculature bed with physiologically relevant permeability. Sorafenib (Nexavar) efficacy was evaluated by assessing tumor spheroid viability, quantified by the loss of fluorescence signal. The presence of vasculature showed enhanced cytotoxicity, highlighting the importance of tumor-vascular interactions for testing therapeutic efficacy. Engineered immune cells (chimeric antigen receptor (CAR) T-cell and transiently expressing T-cell receptors (TCR)) were introduced into the model to demonstrate immunotherapy validation. Our results showed that the engineered immune cells were more effective at infiltrating the tumor spheroid without a vasculature but were more cytotoxic in the presence of the vasculature. By removing the cultured microtissue from the device and performing digital spatial transcriptomics on standard paraffin-embedded sections, we identified known and novel genes involved in creating a pro-tumorigenic microenvironment. This platform can be utilized to model critical features of the in vivo TME, providing better predictability of drug responses, with potential applications towards personalized medicine.

Citation Format: Jyothsna Vasudevan, Ragavi Vijayakumar, Jose Antonio Reales-Calderon, Maxine Sin Lam, Jin Rong Ow, Joey Aw, Damien Tan, Giulia Adriani, Andrea Pavesi. Microphysiological vascularized solid liver tumor model for drug and cell therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4612.
Abstract 4649: Design and validation of ultrahigh-plex discovery panels for immuno-oncology and oncology
Authors: He Yan , Berry Shannon , Shang Olive , McLane Michael , Ihley Nicholas , Zheng Yi
Published: April 2023

Introduction Ultrahigh-plex single-cell spatial phenotyping is revolutionizing cancer research through deeper interrogation of cellular and protein-level co-expression, localization, and arrangements within cancer tissues. To enable in-depth characterization of the tumor microenvironment (TME), we have developed and validated pre-optimized antibody panels on cancer tissues to reveal key cell types and cellular architecture using the PhenoCycler™-Fusion system. Here, we present our methodology for the development and validation of multiplex spatial phenotyping panels for oncology and immuno-oncology.

Methods We designed panel modules such that each module answers specific biological questions about the TME and can be multiplexed together for a more comprehensive view of the structure and function of the tumor and TME. The resulting PhenoCode™ Discovery panel modules include markers that identify key immune cell types, immune activations and checkpoints, mediators of proliferation, and other hallmarks of cancer. Each antibody in the panel module was conjugated to an oligo barcode and stained on human formalin-fixed paraffin-embedded (FFPE) tissue. The slide was imaged on a PhenoCycler™-Fusion platform, where dye-labeled oligo reporters (complementary to the barcodes) are hybridized to the barcode to visualize the antibody. The results were compared serial sections run with a 3,3’-Diaminobenzidine (DAB) chromogenic immunohistochemistry assay to ensure specificity of the antibody was retained. Antibody concentration, exposure time, and corresponding dye were further optimized, verified, and validated as a whole module using tonsil and cancer tissues based on image analysis and intensity analysis, to achieve an ultrahigh-plex detection.

Results and Conclusions In this study, we showcased the rigorous process used for development and validation of PhenoCode™ Discovery panel modules and demonstrated how they can seamlessly work together to collect more data out of one tissue sample. The introduction of these pre-optimized antibody panel modules mark a pivotal point in the evolution of ultrahigh-plex spatial phenotyping. They not only simplify experimental design and assay set up, with panels that make it easier for researchers to answer important questions with this technology, but also save precious time and resources, while reducing overall costs.

Citation Format: Yan He, Shannon Berry, Olive Shang, Michael McLane, Nicholas Ihley, Yi Zheng. Design and validation of ultrahigh-plex discovery panels for immuno-oncology and oncology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4649.
Abstract 4652: A cellular and transcriptomic dissection of the human breast for studying mechanisms of cell and tissue function
Authors: Toro Katelyn Del , Sayaman Rosalyn W. , Thi Kate , Licon-Munoz Yamhilette , Hines William C.
Published: April 2023

A fundamental question in biology, central to our understanding of cancer and other pathologies, is determining how different cell types coordinate to form and maintain tissues. Recognizing the distinct features and capabilities of the cells that compose these tissues is critical. Unfortunately, the complexity of tissues often hinders our ability to distinguish between neighboring cell types and, in turn, scrutinize their transcriptomes and generate reliable and tractable cell models for studying their inherently different biologies. A lack of comprehensive methods to identify, isolate, and culture each cell type from many tissues have impeded progress. Here, we will describe such a method for the breadth of cell types composing the human breast. Furthermore, we have sequenced mRNAs from each purified population and investigated transcriptional patterns that reveal their distinguishing features. These analyses have exposed differentially expressed genes and enriched biological pathways that capture the essence of each cell type, along with transcripts that display intriguing expression patterns. These data, analytic tools, and transcriptional analyses form a rich resource whose exploration provides remarkable insights into the inner workings of the cell types composing the breast, thus furthering our understanding of the rules governing normal cell and tissue function.

Citation Format: Katelyn Del Toro, Rosalyn W. Sayaman, Kate Thi, Yamhilette Licon-Munoz, William C. Hines. A cellular and transcriptomic dissection of the human breast for studying mechanisms of cell and tissue function. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4652.
Abstract 4738: Dysregulated miR-371b-5p/CSDE1/RAC1 axis is involved in pathogenesis of triple-negative breast cancer
Authors: Kim Yesol , Ko Je Yeong , Lee Soo-Been , Jun Jaehee , Ahn Yejin , Min Jinui , Oh Chaewon , Park Jong Hoon
Published: April 2023

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer; however, pathogenesis of TNBC has not yet been fully understood. Here, we identified dysregulated microRNAs (miRNAs) by analyzing miRNA microarray of TNBC patients. MiR-371b-5p expression was reduced, and ectopic expression of miR-371b-5p significantly inhibited TNBC progression in vitro and in vivo. In addition, we found that expression of cold shock domain-containing protein E1 (CSDE1), a direct target gene of miR-371b-5p, was upregulated in TNBC cells, and inhibition of CSDE1 alleviated TNBC cell growth by decreasing RAC1 known as pro-metastatic gene. Mechanistically, CSDE1, phosphorylated C-terminal domain (p-CTD) of RNA polymerase II (RNAPII), and CDK7 form a complex, and downregulation of CSDE1 results in weak interaction between RNAPII p-CTD and CDK7, which leads to a decrease in RNAPII p-CTD expression to reduce RAC1 transcript levels in CSDE1-deficient TNBC cells. Our data revealed the oncogenic function of miR-371b/CSDE1 involved in Rac1 transcription regulation, thus providing a basis for the pathological mechanism of TNBC along with potential biomarkers for TNBC.

Citation Format: Yesol Kim, Je Yeong Ko, Soo-Been Lee, Jaehee Jun, Yejin Ahn, Jinui Min, Chaewon Oh, Jong Hoon Park. Dysregulated miR-371b-5p/CSDE1/RAC1 axis is involved in pathogenesis of triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4738.
Abstract 4746: Amelioration of hemoglobinopathies by targeted deletion of zinc finger domain within BCL11A gene using CRISPR-Cas9 technology
Authors: Hussain Irfan , Mumtaz Kainaat , Hassan Hammad , Mian Afsar Ali
Published: April 2023

Background: In humans, the β-like globin genes are encoded from a single locus comprising five globin genes (ε-, Gγ-, Aγ-, δ-, and β-globin in sequence) and their expression is under developmental control. The γ-globin genes (Gγ- and Aγ-) are expressed during fetal life and replaced by adult β-globin after birth. Mutations in the β-globin gene cause β-hemoglobinopathies such as sickle cell disease (SCD) and β-thalassemia. The clinical severity of SCD and β-thalassemia can be mitigated by elevated fetal hemoglobin (HbF) levels, which have been found in individuals with the benign hereditary persistence of fetal hemoglobin (HPFH) syndrome. Thus, reactivating the expression of γ-globin genes is an attractive treatment strategy for β-hemoglobinopathies. Reactivation of γ-globin expression by disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter, provides a novel approach for inducing fetal hemoglobin.

Methodology: The BCL11A binds a 5′-TGACCA-3′ element (spanning nucleotides −118 to −113) of globin genes using its three C-terminal Zinc fingers (Znf). The binding efficiency of Znf4, Znf5, and Znf6 was predicted by using in silico tools (SIFT, SNAP, PolyPhen-2, PANTHER, I-Mutant, PROVEAN, SNPs&GO, mCSM, and PhD-SNP), molecular dynamic simulation and homology modelling. K562 cells were electroporated with CRISPR-Cas9 targeting the BCL11A Znf4.

Results: The binding energy scores illustrate that deleting Znf5 and Znf6 decreased the binding affinity by ~3.7-fold, whereas deleting Znf4 decreased the affinity by > 70-fold. Using CRISPR-Cas9 genome-editing strategy, we deleted Znf4 (260bp genomic region) within the BCL11A in K562 cell lines. Znf4 deletion resulted in a readily detectable γ-globin increase with a preferential increase in G-gamma.

Conclusion: Altogether, our findings highlight the valuable insights for improving gene editing therapy strategies by either deleting Znf4 of BCL11A or the TTGACCA motif to disrupt the interaction between BCL11A and other interacting partners and the γ-globin gene promoter. Complete failure in the protein-protein interactions with functional partners and to the γ-globin gene promoter revealed that Znf4 is a suitable target for disrupting BCL11A-mediated hemoglobin switching.

Citation Format: Irfan Hussain, Kainaat Mumtaz, Hammad Hassan, Afsar Ali Mian. Amelioration of hemoglobinopathies by targeted deletion of zinc finger domain within BCL11A gene using CRISPR-Cas9 technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4746.
Abstract 4750: TWIST2 mediated chromatin remodeling promotes fusion-negative rhabdomyosarcoma
Authors: Shah Akansha M. , Guo Lei , Morales Maria Gabriella , Jaichander Priscilla , Chen Kenian , Huang Huocong , Hernandez Karla Cano , Xu Lin , Bassel-Duby Rhonda , Olson Eric N. , Liu Ning
Published: April 2023

Rhabdomyosarcoma (RMS) is a common soft tissue sarcoma in children that resembles the developing skeletal muscle. Unlike normal muscle cells, RMS cells fail to differentiate despite expression of the myogenic determination protein MYOD. The TWIST2 transcription factor is frequently overexpressed in fusion-negative RMS (FN-RMS). TWIST2 blocks differentiation by inhibiting MYOD activity in myoblasts, but its role in FN-RMS pathogenesis is incompletely understood. Here, we show that knockdown of TWIST2 enables FN-RMS cells to exit the cell cycle and undergo terminal myogenesis. TWIST2 knockdown also substantially reduces tumor growth in a xenograft model of FN-RMS. Mechanistically, TWIST2 controls H3K27 acetylation at distal enhancers by interacting with the chromatin remodelers SMARCA4 and CHD3 to activate growth-related and repress myogenesis-related target genes, respectively. These findings provide novel insights into the role of TWIST2 in maintaining an undifferentiated and tumorigenic state of FN-RMS and highlight the potential of suppressing TWIST2-regulated pathways to treat FN-RMS.

Citation Format: Akansha M. Shah, Lei Guo, Maria Gabriella Morales, Priscilla Jaichander, Kenian Chen, Huocong Huang, Karla Cano Hernandez, Lin Xu, Rhonda Bassel-Duby, Eric N. Olson, Ning Liu. TWIST2 mediated chromatin remodeling promotes fusion-negative rhabdomyosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4750.
Abstract 4755: Screening non-small cell lung cancer organoids with epigenetic probes
Authors: Jafarova Khadija , Prinos Panagiotis , Radulovich Nikolina , Koga Takamasa , Arrowsmith Cheryl , Liu Geoffrey , Tsao Ming Sound
Published: April 2023

Introduction: NSCLC is associated with high cancer-related mortality worldwide. Epigenetic modifications at the chromatin level have widely been linked to carcinogenesis. Epigenetic regulation not only contributes to the development of cancer, but it may also confer resistance to therapies by promoting the survival of clones that can overcome treatment-induced stress or give rise to complex tumor heterogeneity in response to cancer therapy. Investigating susceptibility resulting from epigenetic aberrations in tumor cells may reveal novel markers of therapies that target epigenetic aberrations. In this study, we treated NSCLC organoid models with a library of 41 epigenetic probes to identify epigenetic targets that affect tumor cell survival.

Methods: Epigenetic screen. NSCLC organoid models were established from resected patient tumors or patient-derived xenografts. Organoids were dissociated into single cells and plated in matrigel-coated 384-well plate. Each model was treated with a library of epigenetic probes (1uM) and a DMSO control over a period of 8 days. Library was provided by Structural Genomics Consortium (www.thesgc.org) and contained compounds targeting a variety of protein domains some of which had activity on acetylation, methylation, histone de-methylation etc. CellTiter-Glo assay was performed to measure cell survival.

Results: Among the epigenetic probe compounds tested on 26 models, LLY 283 (PRMT5 inhibitor) showed the most significant effect on inhibition of cell growth in 69% of organoid models

(18/26) with suppression of >=40% as compared to the DMSO control. To assess differential responses to treatment with LLY 283, four organoid models were treated with 21 different concentrations of PRMT5 inhibitor. Growth curves of a sensitive (XDO181) and a less sensitive (XDO4056) models were compared. For XDO181 and XDO4056, IC50 values were calculated to be 4.7 nM and 31.8 nM respectively, indicating that a less sensitive model is associated with a 7-fold increase in IC50 value compared to the sensitive model.

Conclusion: A screen using epigenetic compounds on NSCLC patient-derived organoids revealed sensitivity to PRMT5 inhibitor in 69% of the organoid models tested. Further investigation will explore the mechanisms that are associated with sensitivity to PRMT5 inhibition.

Citation Format: Khadija Jafarova, Panagiotis Prinos, Nikolina Radulovich, Takamasa Koga, Cheryl Arrowsmith, Geoffrey Liu, Ming Sound Tsao. Screening non-small cell lung cancer organoids with epigenetic probes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4755.
Abstract 4758: SETD4 transcription levels correlates with leukemic burden and SMYD2 transcription in acute lymphoblastic leukemia
Authors: Telles Luis Augusto Muniz , Sakamoto Luis Henrique , Assis Alan Jhones , Rabello Doralina de Amaral , Motoyama Andrea Barretto , Pittella-Silva Fabio
Published: April 2023

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy worldwide. It has a history of great rates of success in treatment, but adults and infants still share a dismal prognosis. This condition makes the development of new prognostic markers linked to effective therapeutic strategies a matter of pressing concern. While cancer has traditionally been viewed as a genetic disease derived from alterations in oncogenes and tumor suppressors, it is well known nowadays that epigenetic changes also play a fundamental role in tumorigenesis. The SET family of lysine methyltransferases (KMT) has been implicated in a number of cancers, and SETD4 is a member that is still poorly characterized. In the present study we used qPCR to analyze the expression pattern of SETD4 among 83 pediatric ALL patients and non-neoplastic bone marrow (BM) samples and investigated the correlation between SETD4 transcription changes with the leukemic burden in ALL patients during chemotherapy. We found that SETD4 transcription levels are significantly upregulated in BM samples derived from ALL patients compared to non-neoplastic BM (8,5-fold higher, p < 0,001) and its expression correlated with leukemic burden. Importantly, levels of SETD4 decreased in patients that responded to chemotherapy treatment. We further investigated whether SETD4 transcription levels associates with that of SMYD2, another KMT previously identified as a prognostic marker in ALL. Transcription levels of SETD4 and SMYD2 were examined on day 15th and 29th of chemotherapy. Surprisingly, a high level of correlation (Spearman r = 0.925, p < 0.01) between both genes was observed in both treatment time points. Finally, survival outcomes were worst in ALL patients with high levels of SETD4 transcription (log-rank test, p < 0,05), evidencing its dysregulated expression is associated with an unfavorable disease prognosis. Together, these results point to SETD4 as a useful prognostic marker, a possible tool to assess response to therapy and an attractive target for drug development.

Citation Format: Luis Augusto Muniz Telles, Luis Henrique Sakamoto, Alan Jhones Assis, Doralina de Amaral Rabello, Andrea Barretto Motoyama, Fabio Pittella-Silva. SETD4 transcription levels correlates with leukemic burden and SMYD2 transcription in acute lymphoblastic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4758.
Abstract 4759: Dissecting EP300 and CBP function in prostate cancer models
Authors: Mirpuri Kiran , Tewari Alok K. , Brown Myles A.
Published: April 2023

Prostate adenocarcincoma is dependent on androgen and its cellular receptor the androgen receptor (AR) for most of the natural history of the disease including progression to the advanced and lethal state. Many prostate cancer therapeutics target the androgen signaling pathway, although tumors invariably become resistant to these treatments. EP300 and CBP are protein paralogs that are co-regulators of AR-mediated gene transcription and are mutated at low frequency in prostate cancers. Expression of CBP and EP300 has been found to correlate with AR expression in advanced disease, and their inhibition can impact AR-mediated transcription in model systems. However, both proteins are not equally essential in prostate cancer cell lines. As drugs inhibiting the bromodomains of these proteins are in early clinical trials, a greater understanding of the common and unique functions of EP300 and CBP may inform the clinical application of these therapeutics. Using chemical inhibition or degradation of either each paralog or both, in combination with genetic depletion, we identify converging and diverging epigenomic and transcriptional programs regulated by each paralog both in the presence and absence of androgens. If validated in tumor samples from patients, these results may nominate biomarkers of response to EP300/CBP inhibitors.

Citation Format: Kiran Mirpuri, Alok K. Tewari, Myles A. Brown. Dissecting EP300 and CBP function in prostate cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4759.
Abstract 4760: KDM4A promotes NEPC progression through regulation of MYC expression
Authors: Mak Celia Sze Ling , Zhu Ming , Liang Xin , Wang Feng , Hoang Anh G. , Song Xinzhi , Shepherd Peter , Liang Derek , Suh Jessica , Park Jiwon , Zhang Miao , Metzger Eric , Schule Roland , Jain Abhinav K. , Karasik Ellen , Foster Barbara A. , Lee Min Gyu , Corn Paul , Logothetis Christopher J. , Aparicio Ana , Navone Nora , Troncoso Patricia , Zhang Jianhua , Lin Sue-Hwa , Wang Guocan
Published: April 2023

Despite advancements in treatment, prostate cancer (PCa) remains the second leading cause of death among men. Neuroendocrine prostate cancer (NEPC) represents one of the most lethal forms of PCa and lacks life-prolonging treatment. Here we identified histone lysine demethylase KDM4A as a driver in NEPC progression and an effective therapeutic target. KDM4A mRNA and protein are overexpressed in human and mouse NEPC compared to adenocarcinoma. Knockdown or knockout of KDM4A in NEPC cell lines suppressed cancer cell growth in vitro and in vivo. Importantly, the inactivation of Kdm4a in a genetically engineered mouse model of prostate cancer reduces tumor burden, reduces the incidence of NEPC, and prolongs overall survival. Mechanistically, KDM4A directly regulates the transcription of MYC, which is hyper-activated in human and mouse NEPC. Furthermore, a potent pan-KDM4 inhibitor QC6352 significantly reduces NEPC cell growth in vitro and in vivo. Taken together, we demonstrate that KDM4A promotes NEPC progression through regulation of MYC expression and targeting KDM4A can be an effective therapeutic strategy for NEPC.

Citation Format: Celia Sze Ling Mak, Ming Zhu, Xin Liang, Feng Wang, Anh G. Hoang, Xinzhi Song, Peter Shepherd, Derek Liang, Jessica Suh, Jiwon Park, Miao Zhang, Eric Metzger, Roland Schule, Abhinav K. Jain, Ellen Karasik, Barbara A. Foster, Min Gyu Lee, Paul Corn, Christopher J. Logothetis, Ana Aparicio, Nora Navone, Patricia Troncoso, Jianhua Zhang, Sue-Hwa Lin, Guocan Wang. KDM4A promotes NEPC progression through regulation of MYC expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4760.
Abstract 4788: Oxidative stress promotes colorectal cancer aggressiveness through transmembrane chloride intracellular channel 1 (tmCLIC1) antioxidant activity
Authors: Cianci Francesca , Verduci Ivan , Tacconi Carlotta , Cannavale Gaetano , Ranucci Matteo , Bertolini Giulia , Malatesta Paolo , Barbieri Federica , Fantin Alessandro , Roz Luca , Florio Tullio , Mazzanti Michele
Published: April 2023

In developed countries, colorectal cancer is the third leading cause of mortality. Despite significant advances in therapeutic approaches, metastases remain the primary concern in the treatment of colorectal cancer. Ion channels have an important role in the development of aggressive behavior, but the majority of them are involved in regular physiological activities. Chloride Intracellular Channel 1 (CLIC1) is a promising contender among the new possible biomarkers. CLIC1 is expressed as a cytosolic monomer by all cell types during physiological homeostasis. The main characteristic of this protein is that it can exist in both cytosolic and transmembrane forms (tmCLIC1), with the latter being involved in a variety of clinical conditions, including cancer. CLIC1 mRNA overexpression in colorectal cancer cells has been linked to a poor prognosis. The primary goal of this study is to understand the role of tmCLIC1 in the carcinogenesis, invasion, and migratory potential of colorectal cancer. We initially investigated the level of CLIC1 protein expression and the activity of its transmembrane form as an ion channel in numerous colorectal cancer cells at various stages of development. Our results show that tmCLIC1 protein expression and activity are related to cell aggressiveness, and that its role in cellular proliferation, migration, and invasion is critical. The role of tmCLIC1 activity in colorectal cancer was confirmed also in in vivo models. Experiments in zebrafish and mouse models have shown that silencing CLIC1 impairs primary tumor development and significantly reduces the generation of distal metastases. Furthermore, when the CLIC1 protein is missing, many distinct metastatic genes are downregulated, particularly those associated to oxidative stress, typical of cancer cells. As a matter of fact, CLIC1's peculiar activity and its interaction with glutathione make this protein critical for the redox equilibrium of colorectal cancer cells. Overall, the findings could be considered as a beginning point for elucidating a mechanism of action that could constitute a new milestone in anticancer research.

Citation Format: Francesca Cianci, Ivan Verduci, Carlotta Tacconi, Gaetano Cannavale, Matteo Ranucci, Giulia Bertolini, Paolo Malatesta, Federica Barbieri, Alessandro Fantin, Luca Roz, Tullio Florio, Michele Mazzanti. Oxidative stress promotes colorectal cancer aggressiveness through transmembrane chloride intracellular channel 1 (tmCLIC1) antioxidant activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4788.