bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6926: Tracing clonal evolution and immune evasion in glioblastoma progression
Authors: Ceresa Davide , Garcia Mena Sebastian A. , Piaggio Francesca , Irene Appolloni , Marubbi Daniela , Malatesta Paolo
Published: March 2024

Glioblastoma continues to be a daunting obstacle in oncology research, with its early-stage progression being particularly enigmatic. In a recent published work, we traced the clonal dynamics of glioblastoma evolution by the simultaneous transfer of PDGFB and genetic barcodes into mouse brains. We observed a continuous clonal loss during the acquisition of a malignant phenotype, underlined by the modulation of the levels of c-Myc expression and their functional targets. In this study, we delve deeper into the evolution of glioblastoma by transplanting multiclonal, early-stage glioma cells into multiple immunodeficient NOD-SCID mice. This experimental design allowed us to track the clonal dynamics over serial transplants, revealing how early-stage glioma clones acquire immune-evasive features capable of initiating tertiary tumors in immune-competent hosts. Through barcode sequencing and single-cell RNA sequencing of early-stage gliomas, coupled with bulk RNA sequencing of secondary and tertiary gliomas, we dissected the clonal and transcriptomic landscape of these tumors. We observed that, of the various clones composing primary tumors, just a few of them are predominating in subsequent passages. Furthermore, the clonal composition of secondary tumors derived from the same primary glioma showed partial overlap, indicating a partial predetermination in the development of immune-evasive behavior. Our intra- and inter-clonal transcriptomic analysis across various stages of tumor progression is shedding light on how new functional traits can emerge in gliomas. Further analysis of our data will determine whether it is the result of the expansion of clones already possessing these traits, or if they stem from functional adaptations within the clones themselves. Our insights further reinforce the concept of clonal competition, highlighting the significant influence of immune-system interactions in driving these competitive dynamics.

Citation Format: Davide Ceresa, Sebastian A. Garcia Mena, Francesca Piaggio, Appolloni Irene, Daniela Marubbi, Paolo Malatesta. Tracing clonal evolution and immune evasion in glioblastoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6926.
Abstract 6933: Decoding the archetypes and eco-hubs of triple-negative breast cancer in responses to chemotherapy
Authors: Yan Yun , Lin Yiyun , Kumar Tapsi , Bai Shanshan , Li Jianzhuo , Tran Tuan , Hu Min , Ravenberg Elizabeth , Rauch Maia , Clayborn Alyson , Thompson Alastair , Lim Bora , Huo Lei , Moulder Stacy , Yam Clinton , Navin Nicholas
Published: March 2024

Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer commonly treated with neoadjuvant chemotherapy (NAC). While about 50% of patients achieve a pathological complete response (pCR), the remainder often develop resistance and metastasis, leading poor survivals. Currently, the factors linking pre-treatment cancer cells and the tumor microenvironment (TME) to NAC response are unclear. To investigate this question, we conducted single-cell RNA sequencing on fresh pre-treatment core biopsy samples collected from patients in the ARTEMIS clinical trial. We identified 13 gene expression meta-programs of cancer cells and about 50 cell states of immune and stromal cell types. Based on the cancer cells alone, we identified four TNBC archetypes at patient-level: luminal secretory-associated (LSA), basal-associated (BA), immunoreactive (IR), and luminal androgen receptor (LAR). Notably, the archetype BA and IR were associated with non-pCR and pCR, respectively. Additionally, we found the TNBC ecosystem was composed of eight eco-hubs, reflecting the co-occurrence patterns of cancer and TME cell states. These eco-hubs revealed different cell community across archetypes and NAC outcomes. For example, an eco-hub with a co-occurrence of interferon response-related cancer cells and immune cells was prevalent in the archetype IR and pCR patients. To facilitate a clinical application, we further developed a 13-gene-based machine learning model to predict NAC response. In summary, these results provide new insights into the cellular determinants of TNBC biology and chemotherapeutic response.

Citation Format: Yun Yan, Yiyun Lin, Tapsi Kumar, Shanshan Bai, Jianzhuo Li, Tuan Tran, Min Hu, Elizabeth Ravenberg, Maia Rauch, Alyson Clayborn, Alastair Thompson, Bora Lim, Lei Huo, Stacy Moulder, Clinton Yam, Nicholas Navin. Decoding the archetypes and eco-hubs of triple-negative breast cancer in responses to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6933.
Abstract 6937: High throughput single cell simultaneous DNA and RNA sequencing identified cell-of-origin of breast cancer
Authors: Wang Kaile , Ye Rui , Bai Shanshan , Xiao Zhenna , Yano Lei , Li Jianzhuo , Lin Yiyun , Sei Emi , Lin Steven , Thompson Alastair , Krishnamurthy Savitri , Navin Nicholas
Published: March 2024

Understanding the relationship between genotype and phenotype in breast cancer cells has been challenging at single cell resolution, mainly because existing high-throughput methods are limited to measuring a single modality and data must be integrated indirectly via computational methods. To address this challenge, we developed wellDR-seq, a high-throughput single cell method that can simultaneously measure the single cell whole genome and transcriptome directly from thousands of single cells in parallel. Using this method, we profiled 17,427 single cells in 6 different ER+ breast cancer patients with either premalignant disease (Ductal-carcinoma-in-situ) or invasive ductal carcinoma (IDC). From these data we identified the epithelial cell-of-origin in 3 cases, showing that ER+ breast cancer cells originated from Luminal Hormone Responsive (LumHR) in the normal breast tissues. We also found that autosomal somatic copy number aberrations were exclusively present in LumSec (< 2%) and LumHR epithelial cells, while chrX gain or loss also occurred in stromal cells (eg fibroblast and endothelial) but a low frequency (<2%) in our datasets. Additionally, our data show the impact of subclonal copy number profiles on gene expression programs, which reflects both gene dosage effects in CNA regions and more complex deregulation in copy-neutral regions. wellDR-seq offers a powerful tool for large-scale single cell genome and transcriptome simultaneous sequencing across different clinical samples, opens new avenue to investigate genotype and phenotypes interactions, identify tumor cells origins, reveal somatic copy number and mutation events, quantify the gene dosage effect and discerning differential genes expression between tumor subclones.

Citation Format: Kaile Wang, Rui Ye, Shanshan Bai, Zhenna Xiao, Lei Yano, Jianzhuo Li, Yiyun Lin, Emi Sei, Steven Lin, Alastair Thompson, Savitri Krishnamurthy, Nicholas Navin. High throughput single cell simultaneous DNA and RNA sequencing identified cell-of-origin of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6937.
Abstract 6952: The role of interleukin-11 (IL11) in cancer-associated cachexia (CAC)
Authors: Drinkwater Kimberly , Meyers Ashlyn , Kargari Samin , Crisan Natalie , Mody Aaron , Shim Minsub
Published: March 2024

Cancer-associated cachexia (CAC), characterized by loss of skeletal muscle and involuntary body weight loss, is responsible for increased chemotherapy toxicity, reduced quality of life, and impaired survival in cancer patients. While interleukin-11 (IL11), one of the IL6 family cytokines, was originally thought to be involved in hematopoiesis, studies increasingly suggest its involvement in tumorigenesis including chemoresistance, metastasis, and survival of cancer cells. However, the precise role of IL11 in CAC has not been determined. Our lab has previously shown that IL11 treatment suppressed the myogenic differentiation of C2C12 myoblasts, suggesting that IL11 may interfere with differentiation and subsequent regeneration of muscle. In this study, we investigated the role of IL11 in cancer-induced muscle wasting using cell culture and mouse models. The mRNA expression of IL11 was highly up-regulated in C26 and Lewis lung carcinoma (LLC) cells, two well-known cachectic mouse cancer cell lines, while its expression was low in a non-cachectic MC38 mouse colon cancer cell line. Knockdown of IL11 in C26 cells using two independent lentiviral shRNAs significantly reduced the mRNA levels of the cytokines involved in CAC including GDF15, LIF, and VEGFA, while the mRNA level of IL6 was not significantly altered. Additionally, IL11 knockdown significantly reduced the mRNA and protein levels of ATF4, CHOP, and TRIB3, suggesting that IL11 may be responsible for the activation of unfolded protein response, an adaptive mechanism for growth, survival, and angiogenesis of cancer. Knockdown of IL11 in C26 and LLC cell lines also significantly reduced the volume of tumors in syngeneic mouse models, while over-expression of IL11 in MC38 cells increased tumor size. Moreover, the knockdown of IL11 in cachectic cancer cells partially prevented the loss of muscle and body weight in tumor-bearing mice, although the amelioration of muscle loss by IL11 knockdown may be associated with the decreased tumor burden. Together, these results indicate that IL11 may contribute to muscle wasting and provide novel insight into the previously unestablished role of IL11 in CAC.

Citation Format: Kimberly Drinkwater, Ashlyn Meyers, Samin Kargari, Natalie Crisan, Aaron Mody, Minsub Shim. The role of interleukin-11 (IL11) in cancer-associated cachexia (CAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6952.
Abstract 6955: Activation of ERK pathway by mutant Ras oncoproteins derived from THP-1 cell exosomes
Authors: Hwang Joseph B. , Marquardt Laura , Sait Osama , Gilliam Brooke , Xiao Qiang
Published: March 2024

The study of exosomes has attracted worldwide research due to its ability to modulate cellular activities, especially in the progression of cancer. Exosomes are membrane vesicles that can deliver lipids, proteins, and nucleic acids from one cell to another through membrane fusion, providing a unique form of cell-to-cell communication. It has been shown that KRAS G12V oncoprotein has been detected inside various pancreatic cancer cell derived exosomes. Our goal for this study was to examine the effect of KRAS G12V oncoprotein, detected inside exosomes isolated from THP-1 cell line supernatant, to activate MAPK pathway in MCF-7 breast cancer and AsPC-1 pancreatic cancer cells. To this end, we developed MILLIPLEX® immunoassays to detect cell signaling proteins as well as the exosome markers (CD9, CD63 and CD81), using Luminex® xMAP® technology which allows multiplex detection of proteins in a small volume of sample. Exosomes were isolated from THP-1 derived cell supernatant, using Qiagen exoEasy Maxi kit according to the kit protocol. KRAS G12V oncoprotein was detected in isolated THP-1 exosomes. As a control, THP-1 exosomes were enriched with exosome markers such as CD9, CD63 and CD81. After 48-hour incubation of AsPC-1 cells with KRAS G12V containing THP-1 exosomes, significant increase in KRAS G12V levels were detected in AsPC-1 cells. More importantly, this increase in KRAS G12V levels corresponded with an increase in phosphorylated ERK signaling, but not JNK, p38, NFκB, Akt, STAT3 and STAT3 pathways. In contrast, 48-hour incubation of MCF-7 cells with KRAS G12V containing THP-1 exosomes had no effect on any signaling pathways. Using MILLIPLEX® multiplex immunoassays, our data supports research findings that exosomes contain mitogenic signaling components that may enhance progression of cancer.

Citation Format: Joseph B. Hwang, Laura Marquardt, Osama Sait, Brooke Gilliam, Qiang Xiao. Activation of ERK pathway by mutant Ras oncoproteins derived from THP-1 cell exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6955.
Abstract 6961: The cell adhesion molecule ninjurin 2 modulates tumorigenesis, inflammatory response and metabolism via modulating and pyroptosis
Authors: Zhang Jin , Zhang Weici , Chen Xinbin
Published: March 2024

The nerve injury induced protein 2 (NINJ2) belongs to a family of homophilic adhesion molecules and was initially found to be involved in nerve regeneration. However, the role of NINJ2 in other cellular process are not well studied. Here, by generating a Ninj2-deficient mouse model, we showed that mice deficient in Ninj2 have a short lifespan and are prone to spontaneous tumors, systemic inflammation and metabolic defects. To better understand the metabolic traits that contributes to these phenotypes, we performed comprehensive carbohydrate and lipid metabolic analyses. Indeed, carbohydrate metabolic analyses indicated that NINJ2-deficiency led to defects in monosaccharide metabolism along with accumulation of multiple disaccharides and sugar alcohols. Similarly, lipidomic analyses indicated that Ninj2 deficiency altered patterns of several lipids including triglycerides, phospholipids and ceramides. To identify a cellular process that associate with these metabolic defects, we examined the role of Ninj2 in pyroptosis, a programmed cell death that links cancer, inflammation and metabolic disorders. Indeed, we found that loss of NINJ2 promotes pyroptosis by activating the NLRP3 inflammasome. Taken together, these data reveal a critical role of NINJ2 in tumorigenesis, inflammatory response, and metabolism via modulating pyroptosis.

Citation Format: Jin Zhang, Weici Zhang, Xinbin Chen. The cell adhesion molecule ninjurin 2 modulates tumorigenesis, inflammatory response and metabolism via modulating and pyroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6961.
Abstract 6968: Extracellular vesicles as carriers of signaling factors in cancer
Authors: Schubert Antonia , Winkler Nadine , Mongkolsittisilp Ajaree , Schulz Matthias , Voloshanenko Oksana , Schaffrinski Meike , Kranz Dominique , Nienhaus Karin , Jäger Dirk , Trümper Lorenz , Büntzel Judith , Binder Claudia , Nienhaus Gerd Ulrich , Boutros Michael
Published: March 2024

Extracellular vesicles (EVs) are small, membrane-bound structures released by cells. They contain various biomolecules and have emerged as mediators of cellular signaling. EVs of various origins can be isolated from body fluids and hold potential as novel biomarkers. However, their clinical implementation as biomarkers is hampered by complex isolation procedures, limited expertise in routine labs, and a lack of mechanistic understanding of signal transfer, particularly in vivo.

To better understand how EVs carry oncogenic signaling factors, we performed mechanistic investigations into the role of EVs in WNT signaling as a representative cancer-associated signaling pathway, as well as ex vivo validation experiments analyzing patient-derived EVs.

Hydrophobic WNT ligands can use EVs to travel the extracellular space. However, little is known about the quantities of WNT secretion onto different EV fractions and potentially other vehicles. To explore WNT secretory routes and potential functional differences, we generated fluorescently tagged marker constructs of the non-canonical ligand WNT5a, which has context-dependent tumor-suppressing or -promoting functions in cancer. In additional in vitro experiments, we tested how the WNT signaling activation state and cancer-specific WNT mutations, such as CRISPR/Cas9-induced APC truncations, quantitatively and qualitatively affect EV shedding in colorectal cancer cell lines. Further functional testing of EV-bound vs. non-EV WNT ligands is ongoing.

For proof-of-concept analyses and to test EVs as biomarkers, we set up the liquid biopsy study EValuate. The project aims for cross-entity analysis of EV profiles and correlation of results to cancer presence, stage, and survival. For quality control and before pipeline implementation, we tested various EV isolation methods and compared different sample pre-processing steps.

By combining multidisciplinary expertise in biophysics, molecular biology, and basic and clinical cancer research, we aim to gain a better mechanistic understanding of EV-mediated signal transfer and facilitate the integration of EVs as diagnostic and prognostic biomarkers into clinical studies.

Citation Format: Antonia Schubert, Nadine Winkler, Ajaree Mongkolsittisilp, Matthias Schulz, Oksana Voloshanenko, Meike Schaffrinski, Dominique Kranz, Karin Nienhaus, Dirk Jäger, Lorenz Trümper, Judith Büntzel, Claudia Binder, Gerd Ulrich Nienhaus, Michael Boutros. Extracellular vesicles as carriers of signaling factors in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6968.
Abstract 6973: Investigates the role of exosomes in dasatinib treated HT29 colon cancer cells
Authors: Koo Hui Min , Lu Yi-Wen , Hou Xiang-Ling , Chao Wei-Ting
Published: March 2024

The mortality rate due to metastasis in cancer is as high as 90%. The underlying mechanisms have been studied, however, the process of metastasis is complicated, current therapies still cannot inhibit metastasis efficiently. In recent years, studies have mentioned that extracellular vesicles play an important role in cancer cell metastasis and tumor microenvironment. Extracellular vesicles can be divided into microvesicles, apoptotic bodies and exosomes, which can carry different substances into the tumor microenvironment. Especially exosomes, whose diameter is around 30-160nm, can carry DNA, RNA, miRNA, membrane proteins, lipids and other intracellular molecules into the cell through endocytosis for regulation. The process of metastasis and the drug resistance were also been demonstrated can be regulated by exosomes. At present, the physiological functions of exosomes are not clear. HT29 colon cancer cells previously discovered in our laboratory can have different responses to dasatinib treatment, and cells can be isolated to dasatinib selected cells(DSTS) and resistant cells(DSTR). In this study, the role of exosome upon dasatinib treatment in HT29 cell is investigated. In the experiment, exosomes from HT29, DSTS, and DSTR were isolated by ExoQuick-TC. Transmission electron microscope was used to observe the difference in exosome size. Exosomes from different cells were treated in HT29 cells, and differences at the protein and mRNA levels were observed using western blot and reverse transcription-polymerase chain reaction(RT-PCR). In my results, the diameter of exosomes in DSTS cells was smaller than that in DSTR and HT29 cells. The protein expression levels of Cavoelin-1 and Clathrin in HT29 were also increased in the treatment of exosome that from three types of cells. In addition, the DSTR derived-exosomes can induce the expression of pFAK and Clathrin mRNA in HT29 cells. The preliminary results suggest that DSTR derived-exosomes may affect cell migration. This study provides the clues that through understanding exosomes that secreted from different cells, cancer diagnosis and treatment can be conducted according to their characteristics, so as to reduce the possibility of cell metastasis precisely.

Citation Format: Hui Min Koo, Yi-Wen Lu, Xiang-Ling Hou, Wei-Ting Chao. Investigates the role of exosomes in dasatinib treated HT29 colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6973.
Abstract 6979: Uncovering the BRAF interactome to identify novel therapeutic targets for BRAF driven melanoma and lung cancer
Authors: OToole Kayla T.
Published: March 2024

BRAF protein kinases act downstream of RAS-GTP and upstream of MEK>ERK MAP Kinase signaling. Notably, mutational activation of BRAF is a driver of approximately 8% of all cancers, with a particular prevalence in lung cancer, thyroid cancer, and melanoma. Importantly, melanoma is responsible for the largest number of skin cancer deaths per year. Patients whose melanoma is driven by mutationally-activated BRAF account for ~50% of cutaneous melanoma cases. The most common mutational alteration is BRAFT1799A, which encodes the BRAFV600E oncoprotein kinase. Combined inhibition of BRAFV600E plus MEK1/2 has been approved for the treatment of BRAFV600E-driven melanoma and lung cancer. Although patients with mutationally-activated BRAF often have a significant response to BRAFV600E-targeted therapeutics, many patients develop lethal drug-resistant disease. The BRAFV600E oncoprotein kinase is proposed to be a monomer, yet many questions remain regarding the biochemistry of these signaling interactions. Thus, we aim to better understand the molecular mechanisms driving BRAFV600E-mediated melanoma and lung cancer cell growth. Preliminary data using gel filtration chromatography and immunoblotting analysis indicates that BRAFV600E is detected in a large multiprotein complex. Interestingly, the addition of a BRAFV600E inhibitor, vemurafenib, shifted the elution of complexes with BRAFV600E into lower molecular weight fractions. Based on these preliminary results, we characterized the components of these complexes to advance our understanding of how mutationally activated BRAF exists in melanoma cells; and thus, provide future insight into how we can better therapeutically target BRAFV600E. Moreover, in order to further elucidate BRAF interactions, we employed a novel form of proximity labeling, TurboID, to unbiasedly identify novel BRAF interactors and better understand how drug treatments change the BRAF interactome. The top ten statistically significant interactors are being investigated to understand how BRAF complexes are formed within the cell, how the complexes are influenced by drug treatments, and how important the individual components are in the formation of complexes with BRAF. Here, we investigate an interaction between BRAFV600E and the tumor suppressor, TP53. Moreover, elucidating the molecular architecture of BRAFV600E signaling complexes could highlight new therapeutic options for patients who display either primary or acquired resistance to inhibitors of BRAFV600E signaling.

Citation Format: Kayla T. OToole. Uncovering the BRAF interactome to identify novel therapeutic targets for BRAF driven melanoma and lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6979.
Abstract 7016: Plastic epigenome in youth: Insights from open chromatin in stem cells
Authors: Liu Yu-Yu , Takeuchi Chihiro , Yamashita Satoshi , Ushijima Toshikazu
Published: March 2024

The presence of plasticity in the epigenomes of young individuals is suggested by the aggressive phenotypes observed in cancers among adolescent and young adults (AYAs), including gastric cancers. To demonstrate the presence of this plasticity and identify the underlying epigenomic characteristics in gastric stem cells during youth, we examined organoids established from murine stomach tissue and conducted single-cell RNA-seq and ATAC-seq using murine antrum-pyloric tissues at ages of 3 weeks and 50 weeks, corresponding to human infancy and middle-age adult, respectively.

Organoids established from infant mice showed higher dependence to niche factors, and stronger response to cytokine signals. Ligand-receptor analysis using single-cell RNA-seq data of tissues showed that infant epithelial stem cells and stromal cells had stronger interactions. Both supported the plastic nature of infant gastric epithelial cells.

Single-cell ATAC-seq enabled us to identify epithelial stem cell population in infant and middle-aged mice, and 71 and 118 chromatin peaks were specific to infant and adult stem cells, respectively. Motif enrichment analysis showed an enrichment of binding sites for bZIP family transcription factors in infants, and an enrichment of binding sites of a chromatin remodeling factor and a family of transcription factors in adults. Additionally, comparison between differentiated epithelial cells, marked by the pit cell marker Muc5ac, and stem cells, marked by Lgr5, showed more pronounced differences: 46,772 peaks specific to pit cells and 39,076 peaks specific to stem cells. Interestingly, regions specific to pit cells were enriched in binding sites of the chromatin remodeling factor and the family of transcription factors, identical to the open regions specific to adult stem cells. This suggested that the chromatin accessibility profile of aged stem cells approaches to that of differentiated cells, leading to a loss of stemness. The single-cell RNA-seq data also revealed an increase in interactions between differentiated epithelial cells and fibroblasts in aged mice, as opposed to infant, suggesting the emergence of aberrant interactions in aged stomach tissue.

Collectively, our data suggested that chromatin accessibility of gastric stem cells undergo an epigenomic shift during aging, approaching to the profiles of differentiated cells and losing their plasticity, and that a specific chromatin remodeling factor and a family of transcription factors could be involved in the process.

Citation Format: Yu-Yu Liu, Chihiro Takeuchi, Satoshi Yamashita, Toshikazu Ushijima. Plastic epigenome in youth: Insights from open chromatin in stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7016.
Abstract 7024: Development of quality control methods for reliable NGS measurement of methylation in cfDNA
Authors: Willey James C. , Crawford Erin L. , Craig Daniel J. , Morrison Thomas M. , He Hua-Jun , Herman James G.
Published: March 2024

Background and Purpose: Reliable measurement of circulating free genomic DNA (cfDNA) methylation patterns promises a means for cancer early detection as well as treatment response. Use of Next Generation Sequencing (NGS) methods to measure the cfDNA methylome across a large number of potentially methylated CpG sites may enable earlier detection by overcoming stochastic sampling associated with the small specimens obtainable by a blood draw. Wide-spread adoption of these methods for clinical diagnostic applications will be facilitated through improved quality controls that increase reliability. The goal of this study is to assess the performance of the SNAQ-SEQ™ method as a means to establish a limit of detection (LOD) for measurement of methylation at each targeted CpG site. For this purpose, we used test materials that were developed by the National Institute for Standards and Technology (NIST) to mimic methylated cfDNA.

Methods: NIST test materials were formulated by physically shearing cell line gDNA then subjecting it to size separation to mimic a size distribution similar to that observed in cfDNA, then mixing sheared DNA in its “native” state of methylation with sheared DNA that was in vitro methylated. Non-sheared NIST test materials also were assessed in these studies. Synthetic spike-in internal standards (IS) were combined with NIST test materials and the combined samples were bisulfite converted, then subjected to PCR-amplicon library preparation. Libraries were sequenced on an Illumina MiSeq instrument and bioinformatic analysis was conducted using the Qiagen CLC Workbench.

Results and Conclusions: In initial testing of an off-the-shelf IS for a region of the SOX2 gene, average methylation (preserved C) across 14 targeted CpG sites was 16.4% (0.5%-50%) in the “native” NIST non-sheared sample. This was consistent with the 11.6% methylation across all of SOX2 measured by the NIST. In contrast, in IS corresponding to the targeted SOX2 region the average preserved C was 0.32% (0.03%-0.5%). Use of synthetic internal standards enabled determination of LOD for each individual methylated site, controlling for all known sources of technical error, including inefficient bisulfite conversion, nucleotide substitution error, and alignment error. Studies are now underway that use the NIST test materials to measure methylation at five targets known to be altered in cfDNA from lung cancer subjects, SOX2, CDO1, TAC1, SOX17, and HOXA7.

Citation Format: James C. Willey, Erin L. Crawford, Daniel J. Craig, Thomas M. Morrison, Hua-Jun He, James G. Herman. Development of quality control methods for reliable NGS measurement of methylation in cfDNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7024.
Abstract 7058: NSD2 overexpression drives multiple myeloma dependence on adenylate kinase 2 by diverting one-carbon metabolism to the epigenome
Authors: Sobh Amin , Encinas Elena , Patel Alisha , Surapaneni Greeshma , Kaestner Charlotte , Poullard Janai , Clerio Monica , Vasan Karthik , Freeman Tzipporah , Lv Dongwen , Dupere-Richer Daphne , Riva Alberto , Barwick Benjamin , Zhou Daohong , Boise Lawrence , Mitsiades Constantine , Kim Baek , Bennett Richard , Chandel Navdeep , Licht Jonathan
Published: March 2024

Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the oncogenic histone methyltransferase NSD2. Genome-wide CRISPR screens in isogenic MM cells with high and low NSD2 expression identified adenylate kinase 2 (AK2), an enzyme critical for transport of ATP from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased NADP(H) critical for conversion of ribonucleotides to deoxyribonucleosides by the enzyme ribonucleotide reductase (RNR). Consequently, metabolomic studies revealed depletion of deoxynucleotide triphosphates (dNTPs) in AK2-deficient t(4;14) MM cells, which resulted in replication stress, DNA damage and apoptosis. Exogenous supplementation with deoxynucleosides rescued AK2 depleted MM cells confirming the role of AK2 in dNTP homeostasis. In addition, NSD2 overexpression drives a large genome-wide increase in chromatin methylation, which depletes the methyl donor S-adenosylmethionine (SAM). Accordingly, we found that high NSD2 expression in MM cells disrupts creatine biosynthesis, a process dependent on SAM. The creatine/phosphocreatine system represents an alternative means of transporting high energy phosphate from the mitochondria. Hence, creatine supplementation restored NADP(H) levels and rescued AK2-deficient t(4;14) MM cells indicating that the hypersensitivity of these cells to AK2 depletion is driven by creatine deficiency due to NSD2 overexpression. Furthermore, AK2 inhibition impaired ATP-dependent protein folding increasing sensitivity of t(4;14) MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM.

Citation Format: Amin Sobh, Elena Encinas, Alisha Patel, Greeshma Surapaneni, Charlotte Kaestner, Janai Poullard, Monica Clerio, Karthik Vasan, Tzipporah Freeman, Dongwen Lv, Daphne Dupere-Richer, Alberto Riva, Benjamin Barwick, Daohong Zhou, Lawrence Boise, Constantine Mitsiades, Baek Kim, Richard Bennett, Navdeep Chandel, Jonathan Licht. NSD2 overexpression drives multiple myeloma dependence on adenylate kinase 2 by diverting one-carbon metabolism to the epigenome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7058.
Abstract 7065: AMBRA1 coordinates mitochondrial function to regulate cancer stemness and tumorigenesis in hepatocellular carcinoma
Authors: Liu Yan , Xie Yunong , He Yimiao , Ma Stephanie , Tong Man
Published: March 2024

Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer, with emerging evidence highlighting the role of cancer stem cells (CSCs) in its progression. AMBRA1 has been implicated to exert both tumor-promoting and tumor-suppressive roles through regulating cell cycle and mitophagy in various solid tumors. However, the functional roles of AMBRA1 in modulating cancer stemness and tumorigenesis in HCC remain elusive. Initial analysis of The Cancer Genome Atlas data revealed a significant inverse correlation between AMBRA1 expression and stemness indices (TSS) in HCC patients, with low AMBRA1 levels associated with an upregulated oxidative phosphorylation (OXPHOS) gene signature. To elucidate the functional consequences of AMBRA1 modulation in liver tumor development and stemness maintenance, we exploited both hydrodynamic mouse models and human HCC cell lines. Our findings indicate that hepatic AMBRA1 deletion enhances cancer stemness and tumorigenesis both in vivo and in vitro. Moreover, HCC cells with reduced AMBRA1 expression demonstrated heightened mitochondrial activity, characterized by increased oxygen consumption, respiration, mitochondrial membrane potential, and intracellular ATP levels. Apart from OXPHOS, we also observed a concomitant up-regulation and enrichment of MYC targets in TSS-high and AMBRA1-low HCC patients, pointing towards a deregulated c-MYC signaling to promote cancer stemness under AMBRA1 regulation in HCC. Collectively, our results demonstrate that AMBRA1 loss enhances mitochondrial function to support hepatic tumorigenesis in HCC.

Citation Format: Yan Liu, Yunong Xie, Yimiao He, Stephanie Ma, Man Tong. AMBRA1 coordinates mitochondrial function to regulate cancer stemness and tumorigenesis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7065.
Abstract 7069: NEK2 is frequently upregulated in esophageal adenocarcinoma associating with poor prognosis
Authors: Chen Lei , Lu Heng , Ballout Farah , Hu Tianling , Chen Zheng , Peng Dunfa
Published: March 2024

Esophageal adenocarcinoma (EAC) has become a significant clinical challenge due to rapid increase in the incidence during the past four decades in the USA and Western countries. However, the prognosis of EAC patients is still poor with 5-year overall survival rate about 20%. There is an urgent need to identify novel therapeutic targets associated with EAC biology. NEK2 is the first identified human NEK (Never in mitosis A (NIMA) related kinase) gene among the 11 serine/threonine kinases family. Aberrant NEK2 expression is reported in a variety of human cancers and plays important roles in tumorigenesis, progression, and drug-resistance. However, the expression of the NEK2 in EAC and its precancerous condition (Barrett’s esophagus, BE) has not been investigated. Using bioinformatic approaches, we first analyzed the TCGA and 9 GEO databases (a total of 10 databases in which eight contain EAC and 6 contain BE) and found that NEK2 was significantly upregulated in EAC as compared to normal esophagus in 7 of the 8 databases. Meanwhile, genomic alterations of NEK2 are only found in 1.2% of EAC. We validated the overexpression of NEK2 using qRT-PCR, western blotting, immunohistochemistry in EAC cell lines and in primary EAC tissues. Functionally, we demonstrated that overexpression of NEK2 in EAC cells promoted tumor cell growth. Targeting NEK2 in EAC cells significantly inhibited EAC tumor growth. Our data suggests that frequent overexpression of NEK2 plays an important role in EAC.

Citation Format: Lei Chen, Heng Lu, Farah Ballout, Tianling Hu, Zheng Chen, Dunfa Peng. NEK2 is frequently upregulated in esophageal adenocarcinoma associating with poor prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7069.
Abstract 7071: Pharmacological targeting of SUMOylation leads to cBAF complex stabilization and disruption of the synovial sarcoma signature
Authors: Floros Konstantinos V. , Li Jinxiu , Roberts Jane L. , Kurupi Richard , Fairchild Carter K. , Hu Bin , Xing Yanli , Dalton Krista M. , Boikos Sosipatros A. , Stamatouli Angeliki M. , Dozmorov Mikhail G. , Koblinski Jennifer E. , Jones Kevin B. , Radhakrishnan Senthil K. , Faber Anthony C.
Published: March 2024

Synovial Sarcoma (SS) is driven by the SS18-SSX translocation event and occurs often in people under 30. In general, SS is refractory to chemotherapy and other therapeutic approaches. SS18-SSX does not directly bind to DNA but instead alters BAF complexes away from a wild-type SS18-containing cBAF complex towards a SS18-SSX- containing ncBAF complex to drive a SS-specific transcription program. Small molecule targeted therapies are expanding beyond the kinome, providing additional tools to treat cancers that are not yet amenable to targeted therapies. Here we demonstrate that SS18-SSX activates the SUMOylation program and SS are sensitive to the small molecule SAE1/2 inhibitor, subasumstat (TAK-981). Mechanistically, we demonstrate TAK-981 de-SUMOylates the cBAF/PBAF complex member, SMARCE1, leading to stabilization and restoration of cBAF complexes. This phenotypic shift leads to a DNA damage and cell death response, resulting in tumor inhibition across mouse models of SS. As such, TAK-981 combined with standard-of-care chemotherapy enhances SOC-induced DNA damage, leading to tumor regressions. Targeting the SUMOylation pathway in SS restores cBAF complexes, positioning it as a clinical candidate to treat this refractory cancer.

Citation Format: Konstantinos V. Floros, Jinxiu Li, Jane L. Roberts, Richard Kurupi, Carter K. Fairchild, Bin Hu, Yanli Xing, Krista M. Dalton, Sosipatros A. Boikos, Angeliki M. Stamatouli, Mikhail G. Dozmorov, Jennifer E. Koblinski, Kevin B. Jones, Senthil K. Radhakrishnan, Anthony C. Faber. Pharmacological targeting of SUMOylation leads to cBAF complex stabilization and disruption of the synovial sarcoma signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7071.
Abstract 7075: Global epitranscriptomic and transcriptomic footprint revealed upregulation of methylated single minded 2-small variant (meSIM2-SV) in colorectal cancer (CRC) by NTMT1(METTL11A) via m6A-YTHDF1-dependent manner
Authors: Rai Sudhir Kumar , Gong Ting , Gao Zitong , Qin Yujia , Huo Matthew , Nakastu Ken , Yang Hua , Fu Yuanyuan , Wu Lang , Jijiwa Mayumi , Nasu Masaki , Chen Shaoqiu , Huang Gang , Fei Peiwen , Deng Youping
Published: March 2024

Establishing global footprint of altered m6A mRNA is a unique method to identify the new prognostics biomarkers. Altered N6-methyladenosine(m6A) RNA methylation regulates cancer progression with uncharacterized mechanism. Here, we present novel link between m6A modification on 5’-UTR SIM2-SV contribution in colorectal cancer progression. MeRIP and RNA-seq data from 68 fresh frozen CRC samples revealed 13,843 altered m6A methylated peaks and found highest in chromosomes 1, 2, and 3. Analysis uncovered 119 overlapping genes and clusters into 32 up-regulated genes with 56 up-hyper peaks, 8 up-regulated genes with 9 up-hypo peaks, 12 down-regulated genes with 14 down-hyper peaks, and 28 down-regulated genes with 40 down-hypo peaks. A significant increase in the SIM2 m6A and RNA level led us to perform correlation analysis between the SIM2SV and m6A RNA regulators. SIM2SV was significantly correlated with YTHDF1 m6A-readers and m6A-NTMT1 writer, suggesting important roles of SIM2SV in CRC oncogenicity. m6A-sequencing identified m6A enrichment in 5’-UTR of SIM2SV gene which further verified. siNTMT1 and siYTHDF1 manipulation in HCT116 and RKO cell lines verified a decrease in SIM2SV gene/protein expression, and m6A level. Knockdown effects also showed decrease in cell proliferation, clonogenicity and induced cell cycle arrest in the G2/M phase for NTMT1 and YTHDF1 versus SIM2 Knockdown showed cell cycle arrest in S/G2 phase. Our investigation revealed the role of new writer NTMT1 for methylated SIM2SV gene expression in colorectal cancer proliferation via m6A-YTHDF1(as “decoder”) dependent manner. Further investigation on m6A modification specifically on SIM2 mRNA revealed the essential roles of its writer NTMT1 and its reader YTHDF1 in CRC development, evidently supporting the importance of the unique landscape of m6A epigenetic modification in colorectal neoplasm, which not only advance our understanding of CRC pathobiology but also provides a new set of molecular targets for building up effective tools to fight CRC.

Citation Format: Sudhir Kumar Rai, Ting Gong, Zitong Gao, Yujia Qin, Matthew Huo, Ken Nakastu, Hua Yang, Yuanyuan Fu, Lang Wu, Mayumi Jijiwa, Masaki Nasu, Shaoqiu Chen, Gang Huang, Peiwen Fei, Youping Deng. Global epitranscriptomic and transcriptomic footprint revealed upregulation of methylated single minded 2-small variant (meSIM2-SV) in colorectal cancer (CRC) by NTMT1(METTL11A) via m6A-YTHDF1-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7075.
Abstract 7102: The central dogma of hepatocellular carcinoma: Genomic, transcriptomic, and proteomic changes
Authors: MA Hoi Tang , YU Chun Yin , NG Lau Yan
Published: March 2024

Cancer is a disease condition characterized by remarkable heterogeneity, wherein numerous molecular features display considerable disparities even with the same patient cohorts, leading to varied treatment outcomes. The primary factor contributing to the substantial differences observed across cancers stems from unique combinations of genetic mutations. Epigenetic alterations further contribute to differential gene expression and splicing, thereby enhancing transcriptomic diversity. The protein products originating from the cancer transcriptome constitute the specific intracellular and extracellular environment, consequently exerting supplementary effects on epigenetic, transcriptomic, and proteomic changes. The complex interplay and mutual influence between genetic aberrations, transcriptomes, and proteomes significantly contribute to shaping the extensive spectrum of molecular characteristics observed in cancer. With label-free mass spectrometry, RNA-sequencing, and low-pass whole genome sequencing, the quantification of the proteome, transcriptome, and genetic abbreviation of the cancer can be evaluated. With the mouse hydrodynamic tail vein injection (HDVI) models of hepatocellular carcinoma (HCC), different subtypes of HCC derived from different genetic abbreviations can be generated. With the proteome, transcriptome, and genetic abbreviation data of the better-defined HCC from HDVI models and HCC patients, the patient-derived HCC can be classified according to the origin of the genetic abbreviation used in the generation of the HDVI-derived HCC. Furthermore, the proteome of patient-derived plasma will be analyzed, and the signature change unique to HCC classified by different origins of genetic abbreviation will provide an abstract of the proteome, transcriptome, and genetic abbreviation of the HCC tissue. The transcriptomics and proteomics data of HDVI mice produced from five distinct sets of genetic abbreviations have been measured. The proteome and transcriptome of tissue, as well as the proteome of plasma, from a small cohort of HCC patients were studied and classified using this resource to illuminate the fundamental landscape of HCC. The final results will be presented and discussed at the meeting.

Citation Format: Hoi Tang MA, Chun Yin YU, Lau Yan NG. The central dogma of hepatocellular carcinoma: Genomic, transcriptomic, and proteomic changes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7102.
Abstract 7112: Preclinical profile of novel and potent small molecule inhibitors of PARG
Authors: Patra Sukanya , Pothuganti Manoj , Kummari Githavani , Sankeshi Venu , Kapaka Jyothipriya , Karche Navnath , Mekala Ramamohan , Viswanadha Srikant
Published: March 2024

Poly(ADP-ribose) glycohydrolase (PARG) is primary enzyme involved in the dePARylation process of DNA repair, specifically in the single strand break repair (SSBRs). PARG is a macrodomain protein with exo- and endo-glycohydrolase activity that liberates free ADP-ribose and PAR chains, respectively. It interacts with various Poly(ADP-ribose) polymerase (PARP) proteins to facilitate appropriate and timely DNA repair. The balance between PARP and PARG activity is essential for efficient DDR. Inhibition of PARG lead to altered DNA repair in cancer cells. We have identified highly potent PARG inhibitors with picomolar IC50 in. PARG is the primary enzyme for dePARylation and accounts for 90% of dePARylation activity. Treatment of HCC-1806 cells with PARG inhibitors showed dose dependent increase of PARylation signature, with sub micromolar IC50. Compounds also demonstrate significant functional, anti-proliferative activity in ovarian and breast cancer cell lines besides a favourable ADME profile. Further pharmacokinetic and pharmacodynamic characterization of the identified hits is currently in progress.

Citation Format: Sukanya Patra, Manoj Pothuganti, Githavani Kummari, Venu Sankeshi, Jyothipriya Kapaka, Navnath Karche, Ramamohan Mekala, Srikant Viswanadha. Preclinical profile of novel and potent small molecule inhibitors of PARG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7112.