bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 670: Distinct biochemical properties of KRAS Q61H mutant render cancer cells resistant to SHP2 inhibitors
Authors: Gebregiworgis Teklab , Kano Yoshihito , Ohh Michael , Marshall Christopher B. , Ikura Mitsuhiko
Published: June 2022

Recent reports suggest that specific KRAS mutations are associated with either resistance or sensitivity of cancer cells to SHP2 inhibitors (SHP2i). However, a lack of understanding of the underlying mechanism(s) is hindering the clinical advancement of SHP2i therapy for cancer. Here we report that cancer cells that harbor KRAS Q61H mutation (found in about 5% of pancreatic ductal adenocarcinoma tumors) or HEK293 cells that over-express KRAS Q61H are resistant to SHP2i. Our biochemical studies supported by all atom molecular dynamic simulations show that the Q61H mutation impairs intrinsic GTP hydrolysis and impedes stimulation of the GTPase cycle by both SOS1 and RASA1, but has negligible impact on binding to BRAF-RBD. Similar to wild-type KRAS, the Q61H mutant can be phosphorylated by Src at Tyr-32 and Tyr-64, and both site scan be dephosphorylated by SHP2, although SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. In vitro, phosphorylation of KRAS Q61H increased intrinsic nucleotide exchange without affecting its insensitivity to SOS1 and RASA1. Phosphorylation of wild-type and SHP2i-sensitive mutants (e.g., G12V) confers resistance to SOS1/RASA1 activities and impairs binding to BRAF-RBD, thus the constitutive resistance to upstream regulation and uncompromised ability of phosphorylated KRAS Q61H to activate MAPK signaling are distinct properties of this mutant. Decoupling of KRAS Q61H from upstream signaling and impaired intrinsic nucleotide hydrolysis lead to a highly GTP-loaded pool that is insensitive to the suppressive effects of Src phosphorylation. While SHP2 plays multiple roles in stimulating RAS signaling, including promoting the GEF function of SOS1, reducing p120 GAP-mediated inactivation of KRAS, and reversing Src phosphorylation of KRAS, we revealed that none of these are required by KRAS Q61H. These insights provide a mechanistic understanding of oncogenic KRAS mutants that can guide clinical trials of SHP2 inhibitors for patients with pancreatic and other cancers bearing KRAS Q61H.

Citation Format: Teklab Gebregiworgis, Yoshihito Kano, Michael Ohh, Christopher B. Marshall, Mitsuhiko Ikura. Distinct biochemical properties of KRAS Q61H mutant render cancer cells resistant to SHP2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 670.
Abstract 673: The EMT activator ZEB1 initiates polarized secretion of pro-tumorigenic effector proteins to drive lung adenocarcinoma progression
Authors: Xiao Guan-Yu , Tan Xiaochao , Rodriguez Leticia , Liu Xin , Yu Jiang , Vasquez Mayra , Tran Hai , Russell William , Gibbons Don , Kurie Jonathan
Published: June 2022

There are few effective therapeutic options for metastatic lung adenocarcinomas (LUADs) that lack actionable mutations in receptor tyrosine kinases, and there is an urgent need to develop a better understanding of the biological basis of LUAD growth and metastasis. Our group has developed mouse models of LUAD metastasis and found that LUAD metastasis is epithelial-to-mesenchymal transition (EMT)-dependent. EMT causes cancer cells to switch their axis of polarity from apical-basal to front-rear, which orients organelles and actinbased cytoskeletal structures in ways that facilitate purposeful cancer cell motility and metastasis. The EMT-activating transcription factor ZEB1 silences microRNAs that target key inhibitors of the polarity axis switch. These data support the general belief that EMT-dependent metastasis is a cell-autonomous process. However, our new findings here show that ZEB1 coordinates the components of secretory trafficking machinery, Rab6A and Rab8A, via silencing miR-148a that target these effectors, to drive polarized secretory trafficking toward the leading edge of LUAD cells, to enhance surface exposure of MMP14 so that triggers ECM degradation, accelerates focal adhesion turnover and promotes cancer cell migration. Moreover, the ZEB1-driven secretory trafficking increases the secretions of cytokines and immunomodulation factors, to generate an immunosuppressive tumor microenvironment, and to promote LUAD metastasis. These findings advance a paradigm in which EMT drives LUAD metastasis through a cell non-autonomous mechanism. The novelty rests in our results that demonstrate a transcriptional governance of polarized vesicular transport, providing the potential to target ZEB1-driven secretory pathways for the purpose of blocking metastasis in LUAD.

Citation Format: Guan-Yu Xiao, Xiaochao Tan, Leticia Rodriguez, Xin Liu, Jiang Yu, Mayra Vasquez, Hai Tran, William Russell, Don Gibbons, Jonathan Kurie. The EMT activator ZEB1 initiates polarized secretion of pro-tumorigenic effector proteins to drive lung adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 673.
Abstract 74: Single-cell 3D spatial omics for tumor hypoxia
Authors: Wu Yi-Chien , Lee Steve Seung-Young
Published: June 2022

Tumor hypoxia arises from the abnormal vasculature in tumors which limit the oxygen supply to the rapidly growing cancer cells. To acclimate to this deprived microenvironment, cancer cells upregulate hypoxia-inducible factor 1-alpha (HIF-1) signaling pathway priming themselves towards more aggressive and heterogeneous phenotypes. Hence, tumor hypoxia becomes a major obstacle for effective cancer treatment. Deciphering hypoxia-driven tumor heterogeneity within the tumors is essential to understanding cancer biology and developing novel therapeutic strategies. A stronger focus should be directed towards the effect of hypoxia types and levels rather than just the absence or presence of hypoxia since hypoxia is heterogeneous in the three-dimensional (3D) tumor microenvironment (TME). There are acute and chronic types of tumor hypoxia, and the hypoxia levels are also variable, forming unpredictable and irregular 3D hypoxic areas. Although traditional techniques (i.e., single-cell RNA sequencing (scRNA-seq), immunohistochemistry (IHC), and immunofluorescence (IF)) and innovative spatial transcriptomics have been developed and applied for investigating tumor cell heterogeneity. Underlying issue of missing spatial information, limited numbers of the biomarker detection, underrepresentation of 3D tumor structure, or limited analyzed area onto the array chip size exist respectively in these methods.

The objective of this project is to develop a 3D spatial genomics approach with single-cell resolution, named sc3DSG, as an advanced spatial transcriptomic method to study hypoxia-driven cancer cell heterogeneity in 3D. Given that, I developed a 3D cellular fluorescence barcoding method by microscopic photobleaching in tumor macro-sections. The barcodes would maintain a fluorescent footprint of cells in different hypoxic areas upon tissue dissociation. Cells encoded with varied barcodes could be differentiated on flow cytometry and further sorted into separate groups for scRNA-seq. With the optimization with RNA preservation reagent in the workflow, sc3DSG achieves high-quality and high-yield RNA transcript for unbiased transcriptomic analysis. In summary, sc3DSG is an advanced spatial transcriptomic method including a sequential workflow of photobleaching barcoding, tissue dissociation, fluorescence-activated cell sorting (FACS), and scRNA-seq. Sc3DSG enables the study of tissue molecular features without losing spatial information and is a better fit for the study of complex 3D structure like vessels or tumor hypoxia. This approach will unravel hypoxia-driven bimolecular and cellular features by considering the degrees and types of tumor hypoxia.

Citation Format: Yi-Chien Wu, Steve Seung-Young Lee. Single-cell 3D spatial omics for tumor hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 74.
Abstract 748: Role of LINC01614 in papillary thyroid cancer
Authors: Quaranto Danielle , Carnazza Michelle , DeSouza Nicole , Dadafarin Sina , Moscatello Augustine , Tiwari Raj K. , Geliebter Jan
Published: June 2022

Papillary thyroid cancer (PTC) accounts for the vast majority of thyroid cancer cases (~80%), with a significantly higher prevalence in females, but much more aggressive and with poorer outcomes in males. Despite a high survival rate, disease recurrence and metastasis remain significant issues. Therefore, there is an unmet need for the elucidation and identification of molecular markers associated with PTC, to aid in identifying early-stage disease, as well as patient prognosis and therapeutic approaches. Long intergenic non-coding RNA (lincRNA) molecules comprise the largest subclass of RNA molecules within the human genome, yet the majority of their functions are yet to be identified. Evidence supports that these non-coding RNA molecules play a role in protein and RNA localization and scaffolding - implying a critical role in the fine tuning of gene expression. Recently, lincRNAs have been identified as major players in cancer development and progression. This deems lincRNAs as attractive, specific biomarkers and/or therapeutic targets, making them important aspects for further study. Data from our lab identified LINC01614 as being significantly upregulated (~10-fold increase) in PTC, when compared to normal thyroid tissue. There was also ~30-fold increase in LINC01614 in male PTC when compared to females, highlighting potential sex-related correlation for study. Thus, we are studying LINC01614 as a potential biomarker for diagnosis and prognosis, as well as a target for therapeutic intervention. LINC01614 was found to be upregulated in various thyroid cancer cell lines, with significant upregulation in the TPC1 (~5-fold; RET/PTC rearrangement) and K1 (~2-fold; BRAFV600E) cell lines when compared to the “normal”, immortalized thyroid cancer cell line (Nthy-ori-3-1). To decipher a phenotype for LINC01614 upregulation in PTC, we utilized the CRISPRi technique to decrease its expression in TPC1. The knockdown of LINC01614 resulted in a ~75% reduction in proliferation in TPC1 cells. Studies are underway to investigate the role of LINC01614 in migration, invasion, morphology, and tumorigenicity in TPC1 cells, as well as in the BRAFV600E cells, K1. Exploring the role that lincRNAs have in cancer phenotypes can lead to a plethora of different molecular markers for therapeutic intervention. The mechanisms by which these non-coding molecules regulate the expression of genes and other elements of the cellular milieu can serve as an open revenue for understanding how they impact PTC development and progression.

Citation Format: Danielle Quaranto, Michelle Carnazza, Nicole DeSouza, Sina Dadafarin, Augustine Moscatello, Raj K. Tiwari, Jan Geliebter. Role of LINC01614 in papillary thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 748.
Abstract 75: Highly multiplexed EGFR mutation detection from liquid biopsy samples using the 6-color Crystal Digital PCR™
Authors: Jovelet Cécile , Remy Myrtille , Pata-Merci Noémie , Vasseur Damien , Lacroix Ludovic , Labrador-Rached Claudia , Mallory Allison C.
Published: June 2022

Liquid biopsies are a minimally invasive sampling approach to overcome the heterogeneity of tumors and represent a valuable source of circulating tumor DNA (ctDNA) for oncological biomarker analysis. ctDNA measurements require a highly sensitive and reliable detection technology to quantify often low-level genetic aberrations within a high background of wild-type sequences. Digital PCR emerged as a powerful technology for the next-generation analysis of liquid biopsies. Stilla Technologies' 6-color naica® system is an ultrasensitive, easy-to-use digital PCR platform capable of simultaneously and precisely quantifying high numbers of biomarkers in a single reaction.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. Epidermal growth factor receptor (EGFR) is frequently mutated in NSCLC. A handful of anti-EGFR Tyrosine Kinase Inhibitor therapies are approved by the FDA; however, most patients develop resistance over time to these treatments. Both monitoring primary EGFR mutations to predict treatment response and precociously detecting resistance mutations to adapt treatments promptly through ctDNA analysis stand to improve the effectiveness of NSCLC patient management.

Stilla has developed a highly multiplexed 6-color Crystal Digital PCR EGFR kit detecting more than 90% of EGFR mutations described in NSCLC from ctDNA. The assay detects 32 common and rare somatic EGFR mutations in exons 18, 19, 20, and 21, including both activating and resistant mutations.

In this work, we evaluated the sensitivity, precision and specificity of the EGFR 6-color Crystal Digital PCR assay highly specific and sensitive for the detection of EGFR mutations, with a Limit of Detection in a high background of wild-type DNA ranging from 0.30 to 0.46 cp/μL (with observed MAFs ranging from 0.06 to 0.09%). Moreover, a high concordance was observed between those EGFR results obtained for NSCLC ctDNA samples using 6-color Crystal Digital PCR and other technologies. With a rapid time to results and straightforward ctDNA analysis workflow, Crystal Digital PCR on the naica system promises ultrasensitive, highly multiplexed 6-color mutation detection, maximizing the information obtained from precious samples.

Citation Format: Cécile Jovelet, Myrtille Remy, Noémie Pata-Merci, Damien Vasseur, Ludovic Lacroix, Claudia Labrador-Rached, Allison C. Mallory. Highly multiplexed EGFR mutation detection from liquid biopsy samples using the 6-color Crystal Digital PCR™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 75.
Abstract 768: Consensus subtypes of hepatocellular carcinoma associated with clinical outcomes
Authors: Lee Ju-Seog , Yim Sun Young , Lee Sung Hwan , Jeong Yun Seong
Published: June 2022

Background & Aims: While many studies revealed transcriptomic subtypes of hepatocellular carcinoma (HCC), they are not translated to the clinic yet due to lack of consensus. We aim to examine consensus of transcriptomic subtypes and uncover their clinical significance.

Methods: We integrated 15 previously established transcriptomic signatures for HCC to uncover consensus subtypes. We also developed and validated a robust predictor of consensus subtype with 100 genes (PICS100). Informatics and statistics approaches were applied to find clinical relevant association of genomic features. Patient derived xenograft (PDX) models were used for testing hypothesis from analysis of transcriptomic data.

Results: We identified 5 clinically and molecularly distinct consensus subtypes. STM (STeM) is characterized by high stem cell features, vascular invasion, and poor prognosis. CIN (Chromosomal INstability) has moderate stem cell features but high genomic instability and low immune activity. IMH (IMmune High) is characterized by high immune activity. BCM (Beta-Catenin with high Male predominance) is characterized by prominent β-catenin activation, low miRNA expression, hypomethylation, and high sensitivity to sorafenib. DLP (Differentiated and Low Proliferation) is differentiated with high HNF4A activity. We also identified potential serum biomarkers that can stratify patients into 5 subtypes.

Conclusions: Five HCC subtypes are associated with potential response to treatments and highly conserved in pre-clinical models, providing a framework for selecting the most appropriate models for preclinical studies of new drugs and potentially for future clinical trials.

Citation Format: Ju-Seog Lee, Sun Young Yim, Sung Hwan Lee, Yun Seong Jeong. Consensus subtypes of hepatocellular carcinoma associated with clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 768.
Abstract 784: Andrographolide altered inflammation pathways and cytokine expression in prostatecancer
Authors: Rosario Mitzy J. Santiago , Carde Gabriela Erazo , Vázquez María Sánchez , Cardona Valerie , Ferrer Magaly Martínez
Published: June 2022

Prostate cancer is the most diagnosed cancer in men in the USA. Andrographolide (AG) is a labdane diterpenoid compound isolated from the medicinal plant Andrographis paniculata. Previous studies in our laboratory showed that Andrographolide decreased prostate tumor size in mice, induced expression of DNA repair genes associated with double-strand break repair and altered mitochondrial function. The objective of this study is to determine the role of AG in inflammation in prostate cancer. Microarray analysis of tumors treated with Andrographolide 10 mg/kg and their vehicle were conducted. Tumors were developed using a xenograft model in which the prostates of SCID mice were injected with 22RV1, and mice were treated three times per week with Andrographolide 10 mg/kg. Tumor tissues were collected and snap frozen. Gene expression was identified and analyzed using the Affymetrix GeneChip® Human Gene 2.0 array. Using Ingenuity Pathway Analysis (IPA) we found that Andrographolide 10 mg/kg altered genes associated with inflammatory response such as ATM, DHFR, HIF-1-α, CDKAL1, DHFR, NOX1, C5, CCL4, COG6, SUPT20H, CA5B, TTC3. To determine changes of cytokine expression in PC3 prostate cancer cells treated with 25 μM Andrographolide, we used the Abcam Cytokine Antibody Array test®. PC3 prostate cancer cells treated with Andrographolide showed altered expression of cytokines such as IL-2, TNF-α, TNF-β, EGF, IGF-1, ENA-78, GM-CSF, GRO, GRO-α, I-309, and Angiogenin. These results showed that AG may alter the tumor microenvironment promoting the secretion of diverse cytokines that may inhibit tumor development. Understanding these biological pathways and networks is essential to determine possible targets of Andrographolide in prostate cancer.

Citation Format: Mitzy J. Santiago Rosario, Gabriela Erazo Carde, María Sánchez Vázquez, Valerie Cardona, Magaly Martínez Ferrer. Andrographolide altered inflammation pathways and cytokine expression in prostatecancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 784.
Abstract 787: RE1-silencing transcription factor (REST) controls neuroendocrine gene programs in pancreatic ductal adenocarcinoma
Authors: MacPherson Kevin Alexander , Joly Meghan M. , Allen-Petersen Brittany , Pelz Carl , Thoma Mary C. , Torkenczy Kristof , Adey Andrew , Liefwalker Daniel , Worth Patrick J. , Sears Rosalie C.
Published: June 2022

Neuroendocrine (NE) differentiation features contribute to intratumoral heterogeneity and aggressive biology in a subset of pancreatic ductal adenocarcinoma (PDAC) tumors associated with poor disease outcome. We recently showed that treating pancreatic cancer cells with gemcitabine, a standard-of-care chemotherapy for PDAC, increases expression of NE markers, suggesting that ductal to neuroendocrine lineage plasticity could play a role in drug resistance. NE features in prostate cancer have been previously associated with loss of the neuronal gene repressor RE1-Silencing Transcription Factor (REST); in the present study, we explore a potential role of REST in regulating NE differentiation in PDAC. Our experiments show that loss of REST in PDAC cells increases NE gene expression, gemcitabine resistance, and colony formation. RNA sequencing data from gemcitabine-treated PDAC cells were enriched for REST target genes, suggesting gemcitabine relieves REST-mediated repression. Chromatin immunoprecipitation experiments corroborated this by demonstrating that gemcitabine reduces REST binding to the NE genes SYP and SNAP25. In addition, single cell ATAC sequencing also uncovered a heterogeneous response to gemcitabine treatment, revealing two gemcitabine-driven cell states with differing REST motif accessibility. Our study indicates that REST controls NE gene programs in PDAC and that loss of REST promotes gemcitabine resistance and oncogenic growth. Mirroring the heterogeneity of PDAC tumors, a subset of PDAC cells treated with gemcitabine have reduced REST motif accessibility, which may reflect a therapy resistant NE-like subpopulation.

Citation Format: Kevin Alexander MacPherson, Meghan M. Joly, Brittany Allen-Petersen, Carl Pelz, Mary C. Thoma, Kristof Torkenczy, Andrew Adey, Daniel Liefwalker, Patrick J. Worth, Rosalie C. Sears. RE1-silencing transcription factor (REST) controls neuroendocrine gene programs in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 787.
Abstract 788: Prrx1 regulates acinar cell plasticity in Kras-driven pancreatic acinar-to-ductal metaplasia
Authors: Li Alina L. , Sugiura Kensuke , Suzuki Kensuke , Pitarresi Jason R. , Chiarella Anna M. , Efe Gizem , Chandwani Rohit , Rustgi Anil K.
Published: June 2022

Introduction: Acinar cells in the adult pancreas demonstrate cellular plasticity and undergo de-differentiation to a progenitor-like cell type with ductal characteristics after injury. This process, termed acinar-to-ductal metaplasia (ADM), is an important feature facilitating pancreas regeneration after injury. In the absence of oncogenic mutations, the ADM lesions resolve and reform the acinar compartment (adaptive ADM). However, in the presence of oncogenic Kras mutations (oncogenic ADM), acinar cells undergo neoplastic transformation after ADM and evolve to pancreatic intraepithelial neoplasia (PanIN), a well-known precursor of pancreatic ductal adenocarcinoma (PDAC). We have characterized the role of Paired-Related Homeobox1 (PRRX1) in adaptive ADM. We demonstrated through our novel conditional Prrx1 knock-out mouse model that loss of Prrx1 abrogated ADM formation. Here we explore the relationship between Prrx1 and mutant Kras on promoting ADM and a pro-ADM microenvironment.

Methods: We generated novel Pdx1-Cre;LSLKrasG12D/+;Prrx1fl/fl;Rosa26YFP/YFP (KCY Prrx1 KO) mice, in which mutant Kras is efficiently expressed and Prrx1 is deleted in a pancreas-specific manner. KCY Prrx1 WT and KO mice were sacrificed at 3 months and 5 months for histological analysis. Immunofluorescence (IF) staining for CK19, characterized for the rate of ADM formation and evaluated for F4/80 and SMA. Quantification of ADM regions, F4/80 and SMA was performed through automated cell-counting of immunofluorescence staining (IF). Dissociated acinar cell culture in collagen was utilized for the evaluation of ADM under ex vivo conditions.

Results: IF staining revealed that KCY Prrx1 KO mice had fewer ADM lesions compared to Prrx1 WT mice at 3 months. This difference became dramatically apparent at the 5 month timepoint. Additionally, lower areas of fibrosis were identified via H&E staining in KCY Prrx1 KO mice, which was accompanied with lower F4/80 and SMA positivity at both 3 months and 5 months. Ex vivo cultures also demonstrated significant reduction in ADM formation in the context of oncogenic Kras and loss of Prrx1.

Conclusions: PRRX1 can influence ADM formation in both a cell-intrinsic and cell-extrinsic manner in the presence of oncogenic KRAS. Our preliminary data suggest Prrx1 facilitates PDAC progression through PanIN formation. We will continue to investigate the mechanisms driving Prrx1-dependent ADM formation.

Citation Format: Alina L. Li, Kensuke Sugiura, Kensuke Suzuki, Jason R. Pitarresi, Anna M. Chiarella, Gizem Efe, Rohit Chandwani, Anil K. Rustgi. Prrx1 regulates acinar cell plasticity in Kras-driven pancreatic acinar-to-ductal metaplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 788.
Abstract 789: Elucidating transcriptional dynamics in neuroendocrine differentiation of advanced prostate cancer
Authors: Chen Chia-Chun , Song Kai , Tran Wendy , Obusan Matthew , Sugimoto Tyler , Sheu Katherine , Cheng Donghui , Varuzhanyan Grigor , Wang Liang , Ta Lisa , Mao Zhiyuan , Bangayan Nathanael , Park Jung-Wook , Graerber Thomas , Witte Owen
Published: June 2022

Small cell carcinomas of the lung, bladder, and prostate share similar transcription patterns and drug sensitivities. Due to their high cellular plasticity, these cancers often escape treatment through a trans-differentiation from adenocarcinoma to the neuroendocrine state. We previously developed a pan small cell cancer in vitro/in vivo model named PARCB that can recapitulate this transition from primary patient tissues. To understand which transcription factors may be important in this transition, we conducted bulk and single cell RNA sequencing over time. We identified a developmental trajectory that is shared among all samples and is defined by stage-specific transcription factors. We plan to interrogate the role that these transcription factors play in the PARCB transformation assay. We performed ATAC sequencing to investigate how these transcription factors regulate these transitional states. Our study will provide a basic understanding of the transcriptional changes that occur during neuroendocrine differentiation and provide new potential therapeutic targets for small cell cancers.

Citation Format: Chia-Chun Chen, Kai Song, Wendy Tran, Matthew Obusan, Tyler Sugimoto, Katherine Sheu, Donghui Cheng, Grigor Varuzhanyan, Liang Wang, Lisa Ta, Zhiyuan Mao, Nathanael Bangayan, Jung-Wook Park, Thomas Graerber, Owen Witte. Elucidating transcriptional dynamics in neuroendocrine differentiation of advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 789.
Abstract 816: Cyclin c suppresses pancreatic intraepithelial neoplasm progression and neuroendocrine neoplasm development in a murine Kras model
Authors: Cai Kathy Q. , Hanley Sara E. , Klein-Szanto Andres , Campbell Kerry S. , Strich Randy
Published: June 2022

Cyclin c is a component of the Cdk8 kinase module that plays both a positive and negative role in transcription. In addition, cellular damage induces cyclin c re-localization to the mitochondria where it stimulates fission and recruits Bax in preparation for apoptotic initiation. Previous studies revealed that cyclin c suppresses thyroid hyperplasia in a Pten knockout mouse model. In the present study, a role for cyclin c in suppressing pancreatic cancer progression was investigated in the Pdx1-Cre LSL-KrasG12D murine pancreatic cancer model. Within eight weeks, a 6-8 fold increase in the appearance of pancreatic intraepithelial neoplasia (PanIN) and acinar ductal metaplasia (ADM) lesions was observed in the KrasG12D+Ccnc-/- animals compared to KrasG12D alone. Surprisingly, high-grade neuroendocrine neoplasms (NEN) were also observed. These aggressive NEN showed mixed morphology with areas of acinar-like differentiation. High grade NEN exhibited pronounced proliferative index (Ki67), high EZH2 expression, and elevated Notch1, suggesting a connection between aberrant Notch signaling and NEN formation. Our previous studies identified a positive role for cyclin c in autophagy gene transcription. Similarly, cell lines derived from a KrasG12D+Ccnc-/- pancreas exhibited a reduction in autophagy compared to KrasG12D+Ccnc+/+ cells. Autophagic deficiency induces pancreatic cell stress and is associated with apoptosis. Consistent with this model, high caspase 3 expression was observed in early KrasG12D+Ccnc-/- NEN but not in KrasG12D+Ccnc+/+ tissues. Taken together, these results demonstrate a role for cyclin c in suppressing both progression of early PanIN and the development of high-grade NEN and suggest a model that cyclin c loss suppresses autophagy and increases apoptosis.

Acknowledgements: Support was provided by NCI CCSG grant CA06927 for the FCCC Histopathology and Cell Culture Facilities, the PA Department of Health CURE fund (KSC), the Boye Foundation (RS), and the New Jersey Health Foundation (RS).

Citation Format: Kathy Q. Cai, Sara E. Hanley, Andres Klein-Szanto, Kerry S. Campbell, Randy Strich. Cyclin c suppresses pancreatic intraepithelial neoplasm progression and neuroendocrine neoplasm development in a murine Kras model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 816.
Abstract 821: Dissecting the role of Stag2 in lung adenocarcinoma
Authors: Ashkin Emily L. , Cai Hongchen , Tang Yuning J. , Li Chuan , Chew Su Kit , Hung King , Belk Julia , Karmakar Saswati , Hebert Jess , Yousefi Maryam , Swanton Charles , Petrov Dmitri A. , Winslow Monte
Published: June 2022

Lung cancer is the most common cause of cancer-related deaths worldwide. Cancers are driven by genomic alterations that activate tumor-promoting oncogenes and inactivate tumor suppressor genes. While some of the most common driver genes have been extensively characterized, many tumor suppressor genes are less well studied. A better understanding of the mechanisms by which different tumor suppressor genes constrain tumorigenesis has the potential to uncover unappreciated biological processes that influence carcinogenesis. Recently, we performed an in vivo screen within a genetically engineered mouse model of oncogenic KRAS-driven lung cancer and found that STAG2 inactivation dramatically increases tumor growth. While STAG2 is mutated in ~4% of human lung adenocarcinomas, 20% of lung adenocarcinomas are low or negative for STAG2 protein. STAG2 is a subunit of one of the two major cohesin complexes, which are present in all cell types and have been implicated in many different cellular processes. STAG2 has not previously been recognized as a critical tumor suppressor in lung cancer and the mechanism through which its inactivation drives lung tumor growth is unknown. Immunohistochemistry, low-pass whole genome sequencing, and analysis of canonical target genes suggest that STAG2 inactivation is not associated with mechanisms previously implicated in other cancer types including chromosomal instability, increased DNA damage, or the activation of MEK/ERK or cGAS/STING signaling. Interesting, we have found that Stag2 is the only cohesin subunit that functions as a tumor suppressor in lung cancer in vivo. Chromatin accessibility profiling and gene expression analyses on Stag2-proficient and -deficient mouse lung adenocarcinoma cells have unveiled potential mediators and effects of STAG2 function. Ongoing genomic analyses of human and mouse lung cancers will demystify the relationship between major cohesin complexes, uncover the impact of STAG2 inactivation on cohesin binding genome wide, and investigate the role of Stag2 inactivation on the tumor microenvironment. We anticipate that our study will uncover the mechanisms of STAG2-mediated tumor suppression in lung cancer and provide novel insights into lung tumorigenesis.

Citation Format: Emily L. Ashkin, Hongchen Cai, Yuning J. Tang, Chuan Li, Su Kit Chew, King Hung, Julia Belk, Saswati Karmakar, Jess Hebert, Maryam Yousefi, Charles Swanton, Dmitri A. Petrov, Monte Winslow. Dissecting the role of Stag2 in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 821.
Abstract 835: Estrogen receptor expression and ER dependent breast tumor growth are dependent on translation initiation factor EIF4A
Authors: Boyer Jacob A. , Dorso Madeline A. , Amor Corina , Reiter Jason , Xu Jianing , de Stanchina Elisa , Wendel Hans-Guido , Chandarlapaty Sarat , Rosen Neal
Published: June 2022

The majority of human breast cancers are dependent on Estrogen Receptor Alpha (ER) and sensitive to its inhibition. In advanced, ER+ dependent breast cancers, resistance usually develops and is associated with insensitivity of the estrogen receptor to inhibition. Mutations that activate PI3K signaling occur in over 40% of ER-driven breast cancers. The PI3K pathway regulates cap-dependent protein translation by controlling mTOR complex I (mTORC1). Inhibitors of PI3K/mTOR are effective in this setting when given with anti-estrogens, but induce ER activity and expression. We now show that despite reducing global cap-dependent translation, PI3K/mTOR inhibition does not reduce ER translation or expression. Translation of ER instead depends on the translation initiation factor, EIF4A. Inhibitors of EIF4A significantly reduce the expression of WT and mutant ER, with attendant blockage of breast cancer model growth in vivo, including models driven by estrogen-independent ER fusions that are unaffected by estrogen receptor antagonists. The utility of EIF4A inhibition can be enhanced when combined with Fulvestrant, a degrader of ER. Combining inhibition of ER translation and induction of ER degradation causes synergistic deep and durable inhibition of ER expression and tumor growth. Inhibition of ER translation represents a new potent strategy for treating ER-dependent breast cancers with acquired resistance to current therapies.

Citation Format: Jacob A. Boyer, Madeline A. Dorso, Corina Amor, Jason Reiter, Jianing Xu, Elisa de Stanchina, Hans-Guido Wendel, Sarat Chandarlapaty, Neal Rosen. Estrogen receptor expression and ER dependent breast tumor growth are dependent on translation initiation factor EIF4A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 835.
Abstract 836: CRACD/CRAD, a tumor suppressor for gastric cancer development
Authors: Zou Gengyi , Huang Yuanjian , Ko Kyung Pil , Zhang Shengzhe , Kim Bong Jun , Zhang Jie , Jun Sohee , Jung Youn-Sang , Zheng Biyun , Park Jae-Il
Published: June 2022

Gastric cancer (GC) is the third most common cancer and the second most common cause of cancer death in Asia. The highest incidence rates are observed in Eastern Asia. To date, the comprehensive mechanism of GC initiation remains elusive. Here, we discovered CRACD (Capping Protein Inhibiting Regulator of Actin Dynamics/CRAD/KIAA1211) as a tumor suppressor, frequently inactivated in GC. To determine the pathologic roles of CRACD, we employed Cracd knock-out (KO) mice and gastric organoids (GOs). Intriguingly, Cracd KO mice and GOs displayed hyperplastic gastric epithelium. Mechanistically, CRACD is essential for stabilizing the cadherin-catenin-actin (CCA) complex. The loss of CRACD leads to the release and nuclear translocation of β-catenin for Wnt target gene transactivation. Indeed, the genetic ablation of Cracd hyperactivated Wnt/β-catenin signaling with the disruption of the CCA complex. The genes encoding the Receptor Tyrosine Kinase (RTK)-RAS signaling pathway and the TP53 are genetically altered in 60% and 50% of gastric adenocarcinomas, respectively. To define the genetic interaction of Cracd loss with the RTK-RAS and TP53 signaling, we established genetically engineered GOs models carrying Trp53 deletion and KrasG12D activation in combination with Cracd KO (CKP) or Cracd wild type (KP). Compared to KP, CKP GOs exhibited neoplasia, higher mucin deposition, and increased carcinoma embryonic antigen (CEA) expression, pathologically related to the poor prognosis in GC patients. Meanwhile, loss of Cracd significantly accelerated the growth of CKP GOs with increased stemness. Furthermore, the CKP cell line derived from GOs exhibited relatively poor prognosis features of GC than KP cells in the xenograft models, represented by boosted tumor size and weight, poor differentiation, hyperplasia, increased CEA, and mucin secretion. Together, we identified CRACD as a tumor suppressor, of which inactivation contributes to GC initiation and progression, which may be translated into the development of a biomarker-guided regimen for CRACD mutations-associated GC patients.

Citation Format: Gengyi Zou, Yuanjian Huang, Kyung Pil Ko, Shengzhe Zhang, Bong Jun Kim, Jie Zhang, Sohee Jun, Youn-Sang Jung, Biyun Zheng, Jae-Il Park. CRACD/CRAD, a tumor suppressor for gastric cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 836.
Abstract 840: PMEPA1 is an oncogene linked to TGF-β signaling in hepatocellular carcinoma
Authors: Piqué-Gili Marta , Andreu-Oller Carmen , Pinyol Roser , Bárcena-Varela Marina , Esteban-Fabró Roger , Peix Judit , Abril-Fornaguera Jordi , Lindblad Katherine E. , Torres-Martin Miguel , Sia Daniela , Lujambio Amaia , Llovet Josep M.
Published: June 2022

Background: Transforming growth factor β (TGFβ) has a dual role in cancer, including hepatocellular carcinoma (HCC). It acts as a tumor suppressor in early stages of hepatocarcinogenesis, but promotes epithelial-to-mesenchymal transition, angiogenesis and immunosuppression in advanced stages. PMEPA1 (prostate transmembrane protein androgen induced 1), a direct target gene of the TGFβ pathway that negatively regulates TGFβ signaling by interacting with SMAD proteins, has been shown to promote TGFβ oncogenic effects in other cancers and we decided to explore its role in HCC pathogenesis.

Methods: We analyzed transcriptomic, genomic, epigenomic and clinicopathological data of a discovery cohort of 228 HCCs and a validation cohort of 361 HCCs. PMEPA1 levels were quantified by qPCR in the discovery cohort. PMEPA1 overexpression was validated in 6 independent cohorts (n = 916) using microarray and RNAseq data. Genetically engineered mouse models were generated through hydrodynamic tail-vein injection. Equal amounts of plasmids overexpressing PMEPA1, TGFB1 and MYC were injected in mice to evaluate survival. Molecular and histopathological analyses of the murine tumors are currently ongoing.

Results: PMEPA1 was overexpressed in 18% of human HCCs [FC > 2; n = 203/1144], a feature associated with TGFβ signaling (p < 0.05). Overexpression of PMEPA1 was associated with gene body hypermethylation (p < 0.0001). HCCs displaying both TGFβ signaling and high PMEPA1 levels (11% of cases) were significantly enriched in signatures of immune exhaustion, late TGFβ activation, TME response to TGFβ and active stroma (p < 0.05) compared to HCCs with TGFβ signalling alone (8% of cases) or PMEPA1 high levels alone (11% of cases). In vivo, overexpression of MYC+PMEPA1 led to HCC development in 9/16 mice and showed a lower survival when compared to the control arm, overexpression of MYC alone (p = 0.014). On the contrary, only 1/12 mice in the MYC+PMEPA1+TGFβ1 condition developed HCC and none of the MYC+TGFβ1 mice.

Conclusion: PMEPA1 upregulation is linked to TGF-β activation and an aggressive phenotype in human HCC. Overexpression PMEPA1 in combination with MYC in vivo led to HCC development, showing for the first time the oncogenic role of PMEPA1 in this cancer.

Citation Format: Marta Piqué-Gili, Carmen Andreu-Oller, Roser Pinyol, Marina Bárcena-Varela, Roger Esteban-Fabró, Judit Peix, Jordi Abril-Fornaguera, Katherine E. Lindblad, Miguel Torres-Martin, Daniela Sia, Amaia Lujambio, Josep M. Llovet. PMEPA1 is an oncogene linked to TGF-β signaling in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 840.
Abstract 890: DNA deaminase APOBEC3B regulates theresponse to PARP inhibitors of epithelial ovarian cancer stem cells
Authors: isquith Jane , Pham Jessica , Phavong Robert , Rivera Maria , Fisch Kathleen , Jamieson Catriona , McHale Michael , Eskander Ramez , Jiang Qingfei
Published: June 2022

The most aggressive types of ovarian cancer is high-grade serous ovarian cancer (HGSOC), and the 5-year survival is less than 20%. Relapsed HGSOC patients often have enrichment of cancer stem cells (CSCs) with enhanced pro-survival and self-renewal capacity, suggesting a potential vulnerable population for effective targeted therapies with less toxicity. APOBEC3 proteins are a family of primate-specific DNA deaminases that catalyze cytidine to uracil (C-to-U) on single-strand DNA thereby introducing C-to-T/G point mutations. This important DNA modification is abundant in a wide array of cancer types including HGSOCs. However, the prevalence and significance of A3-induced DNA deamination in HGSOC CSC is currently unknown. We recently discovered that CSCs maintain a low A3-induced mutation burden. Using patient-sample derived spheroid culture and HGSOC cell lines, we found A3B was the only A3 members differentially expressed between CSC and non-CSC and the expression was significantly reduced in CSCs. In addition, inhibition of A3B leads to elevated frequency of CSCs and enhanced expression of the stemness factors. Important, our data suggest that DNA deaminase activity of A3B leads to induction of DNA damage thereby sensitizing HGSOC CSCs to PARPi. These novel insights suggest that DNA deamination plays a fundamental role in CSC biology and also raises the possibility that A3B activation may sensitize HGSOC CSCs to PARPi by promoting DNA damages.

Citation Format: Jane isquith, Jessica Pham, Robert Phavong, Maria Rivera, Kathleen Fisch, Catriona Jamieson, Michael McHale, Ramez Eskander, Qingfei Jiang. DNA deaminase APOBEC3B regulates theresponse to PARP inhibitors of epithelial ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 890.
Abstract 951: BNIP3L/NIX promotes breast cancer growth and metastasis by inducing epithelial-mesenchymal transition
Authors: Berardi Damian E. , Macleod Kay F.
Published: June 2022

Mitophagy is induced in response to physiological stresses, including hypoxia, as an adaptive response that decreases mitochondrial mass to promote efficient use of limiting nutrients. BNIP3L/NIX is a mitophagy cargo receptor that integrates into the outer mitochondrial membrane where it interacts with processed LC3 at growing phagophore membranes to promote mitophagy. Our preliminary data shows that elevated BNIP3L/NIX expression in basal-like breast cancers correlates with worse overall patient survival. However, the functional significance of BNIP3L/NIX for growth and progression of breast cancer has not been determined. We generated a CRISPR/Cas9 KO BNIP3L/NIX 4T1 mouse mammary tumor model to determine how loss of BNIP3L/NIX affected tumor progression and metastasis. We observed that loss of BNIP3L/NIX decreased tumor cell growth and importantly attenuated metastasis to the lungs. By performing RNA-seq analysis, we found that these striking findings were associated with a decrease in molecular signatures expression of hypoxia-inducible genes and epithelial-mesenchymal transition (EMT) genes, including Twist and Vimentin. In addition, expression of Vimentin was complete ablated in 4T1 KO BNIP3L/NIX tumors in vivo and 4T1 KO BNIP3L/NIX tumor cells had significantly reduced ability to migrate and invade in vitro. Furthermore, CRISPR/Cas9-mediated deletion of HIF1α a in the 4T1 cell model reduced Vimentin expression in a manner dependent on BNIP3L/NIX expression. These results indicate that BNIP3L/NIX acts as an oncogene downstream of HIF1α in breast cancer by promoting expression of key EMT genes, stimulating tumor cell proliferation and migration, and that targeting BNIP3L/NIX might prove an effective strategy in the clinic.

Citation Format: Damian E. Berardi, Kay F. Macleod. BNIP3L/NIX promotes breast cancer growth and metastasis by inducing epithelial-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 951.
Abstract 953: MEK/ERK induces the EMT transcription factor ZEB1 that is regulated by USP10 and promotes colorectal cancer metastasis that can be inhibited by BRAFV600E-directed therapy
Authors: Qin Bo , Sun Lei , Sinicrope Frank A.
Published: June 2022

Background: Constitutive MEK-ERK activation induced by BRAFV600E or RAS mutation promotes tumor metastasis in CRC that is mediated by transcription factors regulating epithelial-to-mesenchymal transition (EMT). We determined the role of ZEB1, an EMT master transcription factor, and its potential regulation by the ubiquitin specific peptidase 10 (USP10), in tumor metastasis mediated by MEK-ERK.

Materials and Methods: Isogenic RKO [BRAFV600E or wild-type (wt)] and Colo320 (BRAF/KRAS wt/wt) human CRC cells were utilized. Colo320 cells were transfected with mutant KRASG12D. In addition to parental cells, we generated USP10 shRNA expressing RKO and Colo320 cells. Cells were treated with the MEK1/2 inhibitor cobimetinib or BRAF inhibitor encorafenib. Cell migration was analyzed by wound healing and transwell assays. Protein/protein interaction was analyzed by immunoprecipitation. Protein ubiquitination level was determined by ubiquitination assay. In an orthotopic model, lung metastases in mice were generated by tail vein injection of RKO cells.

Results: We demonstrate MEK-ERK activation, induced by BRAFV600E, promotes CRC metastasis through inhibition of the USP10-ZEB1 axis. USP10 is shown to interact with ZEB1 and to remove its K27 linkage ubiquitination chain, thus increasing K48 ubiquitination of ZEB1 protein that results in proteosome-mediated degradation of ZEB1. MEK-ERK induced the phosphorylation of USP10 that is shown to attenuate the USP10-ZEB1 interaction and results in ZEB1 stabilization in RKO and Colo320 cell lines. Increased ZEB1 expression promotes tumor cell migration in vitro and metastasis in a murine xenograft model that could be suppressed by cobimetinib or BRAFV600E-directed therapy with encorafenib and cetuximab.

Conclusions: Targeting RAF/RAS-driven MEK/ERK signaling can suppress ZEB1 stabilization to inhibit tumor metastasis. Suppression of metastasis occurs by a novel mechanism of ZEB1 stabilization that is regulated by an atypical USP10 deubiquitinase activity.

Citation Format: Bo Qin, Lei Sun, Frank A. Sinicrope. MEK/ERK induces the EMT transcription factor ZEB1 that is regulated by USP10 and promotes colorectal cancer metastasis that can be inhibited by BRAFV600E-directed therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 953.