bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A032: MUC1-C regulates chromatin remodeling and drives the CSC state in human cancers
Authors: Bhattacharya trayee , Fushimi Atsushi , Yamashita Nami , Liu Tao , Liu Song , Kufe Donald W.
Published: December 2022

The oncogenic MUC1-C protein evolved in mammals to protect epithelial barrier tissues from the external environment. Chronic activation of MUC1-C promotes lineage plasticity, epigenetic reprogramming, and carcinogenesis. MUC1-C also induces the Yamanaka Pluripotency factors. Chromatin remodeling is critical for the cancer stem cell (CSC) state. Our studies have demonstrated that MUC1-C regulates chromatin remodeling by (i) activating the EZH2/PRC2 complex leading to H3K27 trimethylation, and (ii) the BMI1/PRC1 complex with H2A ubiquitylation. In addition, MUC1-C regulates global changes in chromatin accessibility that are mediated at least in part by JUN/AP-1 and ARID1A/BAF in association with driving the CSC state. We also found that silencing MUC1-C suppresses bulk H3K4 trimethylation and H3K27 acetylation levels in diverse human cancer cells. The COMPASS family of H3K4 methyl transferases (HMT) methylate histone H3K4 and the histone acetyl transferase (HAT) p300/CBP family catalyzes H3K27 acetylation. Involvement of MUC1-C in the regulation of COMPASS has not been previously reported. This study demonstrates that MUC1-C induces COMPASS SET1A and WDR5 gene expression by NF-kB p65-mediated transactivation. WDR5 is required for SET1A complex HMT activity. We found that MUC1-C and WDR5 are necessary for expression of FOS, ATF3 and other AP-1 family members. In a positive feedback loop, MUC1-C, WDR5 and AP-1 were shown to be responsible for induction of genes encoding effectors, including TRAF1 and RELB, in the chronic NF-kB inflammatory response. We also found that MUC1-C, NF-kB, WDR5 and AP-1 are necessary for induction of the (i) KLF4 master regulator of the pluripotency network, and (ii) NOTCH1 effector of stemness. Mechanistically, MUC1-C/NF-kB complexes recruit SET1A/WDR5 and AP-1 to enhancer-like signatures in these target genes, increase the deposition of H3K4me3 with the opening of chromatin, and activate their transcription. These findings demonstrate that MUC1-C activates the COMPASS SET1A/WDR5 complex in integrating the induction of genes that promote chronic inflammation, pluripotency, and the CSC state.

Citation Format: trayee Bhattacharya, Atsushi Fushimi, Nami Yamashita, Tao Liu, Song Liu, Donald W. Kufe. MUC1-C regulates chromatin remodeling and drives the CSC state in human cancers. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A032.
Abstract B013: Suppression of TET1 expression down-regulates p16 and contributes to the resistance of SWI/SNF LOF tumors to PRC2 inhibition
Authors: Zhang Hailong
Published: December 2022

Polycomb repressive complex 2 (PRC2) drives the expansion of H3K27me3 epigenetic marker and suppress the expression of target genes including tumor suppressors. Activation mutations of EZH2, the catalytic subunit of the PRC2 complex, are thought to be the driver for certain malignancies such as diffused large B cell lymphoma (DLBCL). The SWI/SNF complex remodels nucleosomes to modulate transcription, they are generally considered as tumor suppressors and loss-of-function (LOF) mutations are frequently found across a wide variety of cancers. Many studies suggest that PRC2 is important for the proliferation of tumor cells with SWI/SNF LOF mutations, indeed, EZH2 inhibitor tazemetostat was approved by FDA for epithelioid sarcoma with SWI/SNF subunit SMARCB1 deletion. While not all SWI/SNF LOF tumors respond to PRC2 inhibition, we would like to know what subset of these tumors are sensitive to PRC2 inhibition. In this study, we have developed a potent and selective PRC2 inhibitor BR1763 which blocks the interaction of EED with tri-methylated histone H3 at Lys 27 (H3K27me3) and inhibits cellular H3K27me3 levels. By analyzing the gene expression patterns of BR1763 treated cells, we found that up-regulation of CDKN2A/p16 and down-regulation of E2F downstream gene are essential for cellular response to PRC2 inhibition. Further studies also confirmed that PRC2 inhibition caused P16 up-regulation and induced cell senescence for the sensitive cells. Importantly, suppression of TET1 expression contributes to the resistance to PRC2 inhibition in SWI/SNF LOF tumors, which may be caused by PcG hypermethylation on p16 promoters. TET1 expression, together with SWI/SNF LOF mutation status, can largely predict the sensitivity of tumor cells to PRC2 inhibition.

Citation Format: Hailong Zhang. Suppression of TET1 expression down-regulates p16 and contributes to the resistance of SWI/SNF LOF tumors to PRC2 inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B013.
Abstract B015: Versatile biochemical and genomic platforms for drug discovery in chromatin remodeling research
Authors: Husby Natalia L. , Sun Lu , Willis Hannah , Marunde Matthew R. , Meiners Matt J. , Gillespie Zachary , Watson Rachel , Gopinath Saarang , Burg Jonathan M. , Cheek Marcus A. , Novitzky Katherine , Venters Bryan J. , Sun Zu-Wen , Keogh Michael-Christopher
Published: December 2022

Chromatin remodeling, driven by ATP-dependent remodeling enzymes, plays key roles in regulating gene expression, genome replication and repair. Aberrant nucleosome organization can severely disrupt these important processes, driving various cancers and developmental disorders. Remarkably, nearly 20% of all human cancers contain mutations in subunits from the SWI/SNF family of chromatin remodeling complexes, making them attractive therapeutic targets and interests of basic research. However, studies on the remodeling enzymes (and their multi-subunit complexes) in vitro are challenging partially due to the requirement for nucleosome-based substrates. We have created the EpiDyne® nucleosome remodeling substrate portfolio for use in biochemical assays to examine nucleosome immobilizing activities of chromatin remodelers. Here we present the development of multiple remodeling assays on the EpiDyne® platform (EpiDyne®-FRET, EpiDyne®-PicoGreenTM and EpiDyne®-TR-FRET). These nonradioactive plate-based assays are easy to use, ready for high-throughput inhibitor screening, and can be customized for various remodeling enzymes that exhibit distinct preferences in nucleosome compositions. Furthermore, genome-wide mapping of chromatin remodelers is fundamental for understanding their functions in diseases and responses to inhibitors in cells, but is often obscured by unreliable ChIP-seq approaches. To this end, we have optimized the CUTANA™ CUT&RUN approach to efficiently capture localizations of all major classes of chromatin remodelers with high signal to background. As a complementary research tool to the EpiDyne® platform, CUT&RUN facilitates the epigenomic research on chromatin remodelers for drug discoveries.

Citation Format: Natalia L. Husby, Lu Sun, Hannah Willis, Matthew R. Marunde, Matt J. Meiners, Zachary Gillespie, Rachel Watson, Saarang Gopinath, Jonathan M. Burg, Marcus A. Cheek, Katherine Novitzky, Bryan J. Venters, Zu-Wen Sun, Michael-Christopher Keogh. Versatile biochemical and genomic platforms for drug discovery in chromatin remodeling research. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B015.
Abstract B028: CDK12/13 inhibition antagonizes resistance to KRASG12C inhibitors
Authors: Stern Yaakov E. , Ghosh Pompom , Walker-Mimms Hannah L. , Mosior John W. , Imbody Denis , Solanki Hitendra S. , Monastyrskyi Andrii , Duckett Derek R. , Haura Eric B.
Published: December 2022

Covalent inhibitors selectively targeting the KRASG12C mutation offer a promising new therapeutic opportunity for non-small cell lung cancer (NSCLC) patients, with partial or complete response observed in up to 40% of cancers harboring this lesion. As observed for other precision medicines targeting oncogenic drivers, resistance to these agents develops upon prolonged treatment. We have developed cell-based models of acquired resistance to the KRASG12C inhibitor sotorasib by serial passage of sotorasib-sensitive NSCLC cell lines in the presence of the drug. We have observed dynamic phosphorylation of the transcriptional elongation kinases CDK12 and CDK13 as well as hyperphosphorylation of a network of DNA damage response (DDR) proteins in sotorasib-resistant cell lines by phosphoproteomic profiling of resistant and sensitive cells. This is accompanied by elevated sensitivity to DDR pathway and CDK12/13 (e.g., SR-4835) inhibitors. Combined treatment of NSCLC cell lines with sotorasib and SR-4835 delays or prevents adaptation to sotorasib and the development of acquired resistance. Furthermore, gene expression profiling of SR-4835-treated cells reveals that CDK12/13 inhibition suppresses both DDR gene expression and metabolic pathways associated with resistance to sotorasib. This reflects synergy between SR-4835 and inhibitors targeting the DDR pathway in breast cancer cell lines and demonstrates that transcriptional elongation is a critical vulnerability for cells undergoing adaptation to KRASG12C inhibitors via DDR-dependent mechanisms.

Citation Format: Yaakov E. Stern, Pompom Ghosh, Hannah L. Walker-Mimms, John W. Mosior, Denis Imbody, Hitendra S. Solanki, Andrii Monastyrskyi, Derek R. Duckett, Eric B. Haura. CDK12/13 inhibition antagonizes resistance to KRASG12C inhibitors. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B028.
Abstract IA002: Structural basis of chromatin transcription
Authors: Farnung Lucas
Published: December 2022

In eukaryotes, RNA polymerase (Pol) II transcribes chromatin and must move past nucleosomes, often resulting in nucleosome displacement. How Pol II unwraps the DNA from nucleosomes to allow transcription and how DNA rewraps to retain nucleosomes has been unclear. I will present the 3.0 Å cryo–electron microscopy structure of a mammalian Pol II-DSIF-SPT6-PAF1c-TFIIS-nucleosome complex stalled 54 base pairs within the nucleosome. The structure provides a mechanistic basis for nucleosome retention during transcription elongation where upstream DNA emerging from the Pol II cleft has rewrapped the proximal side of the nucleosome. The structure uncovers a direct role for Pol II and transcription elongation factors in nucleosome retention and explains how nucleosomes are retained to prevent the disruption of chromatin structure across actively transcribed genes. Importantly, the structure also provides the mechanistic basis for the survival of histone marks during transcription.

Citation Format: Lucas Farnung. Structural basis of chromatin transcription. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA002.
Abstract IA004: Novel inhibitors of histone H2B deubiquitinating enzymes
Authors: Wolberger Cynthia
Published: December 2022

Histone H2B-K120 is dynamically ubiquitinated and deubiquitinated during transcription and is a hallmark of actively transcribed genes. The Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator contains a four-protein subcomplex called the deubiquitinating enzyme (DUB) module that removes ubiquitin from histone H2B-K120. The human DUB module contains the catalytic subunit ubiquitin-specific protease 22 (USP22), which is overexpressed in a number of cancers that are resistant to available therapies. We screened a massive combinatorial library of cyclic peptides and identified potent inhibitors of USP22. The top hit was highly specific for USP22 compared with a panel of 44 other human DUBs. Cells treated with peptide had increased levels of H2B monoubiquitination, demonstrating the ability of the cyclic peptides to enter human cells and inhibit H2B deubiquitination. These macrocycle inhibitors are, to our knowledge, the first reported inhibitors of USP22/SAGA DUB module and show promise for drug development.

Citation Format: Cynthia Wolberger. Novel inhibitors of histone H2B deubiquitinating enzymes. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA004.
Abstract IA007: Transforming chromatin: Oncohistone mutations in pediatric high-grade glioma
Authors: Budd Kaitlin , Kwon Chang-Hyuk , Kasper Lawryn H. , Roberts Christopher , Roach Jordan , Ocasio-Adorno Jennifer , Larson Jon D. , Baker Suzanne J.
Published: December 2022

Pediatric diffuse high-grade gliomas (HGGs) are a heterogeneous spectrum of disease with abysmal survival rates. Approximately half of diffuse high-grade gliomas in children arise in midline structures predominantly the brainstem, but also thalamus, cerebellum and spinal cord. Approximately 80% of these tumors harbor H3 K27M mutations, which result in dramatic depletion of the post-translational modification H3K27me3. Alternative mutations in diffuse midline gliomas can result in similar reduction of H3K27me3, leading to a redefined classification of this collection of tumors as diffuse midline glioma, H3 K27-altered. In contrast, distinct histone H3 mutations, H3.3 G34R/V, are found in approximately 30% of diffuse gliomas arising in the cerebral hemispheres of older adolescents and young adults, defining the tumor subgroup of diffuse hemispheric glioma, H3 G34-mutant. The striking spatiotemporal pattern of these histone mutations, termed oncohistones, indicates an intimate association between epigenetic dysregulation, brain development, and tumorigenesis. We will discuss use of genetically engineered and patient-derived models to investigate the contribution of oncohistone mutations to disrupted development, epigenetic dysregulation and gliomagenesis.

Citation Format: Kaitlin Budd, Chang-Hyuk Kwon, Lawryn H. Kasper, Christopher Roberts, Jordan Roach, Jennifer Ocasio-Adorno, Jon D. Larson, Suzanne J. Baker. Transforming chromatin: Oncohistone mutations in pediatric high-grade glioma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA007.
Abstract IA020: Therapeutic targeting of MLL complexes in cancer
Authors: Armstrong Scott A.
Published: December 2022

Inappropriate expression of genes such as the homeotic (HOX) genes is found in up to 40% of cases of Acute Myelogenous Leukemia (AML). Two well-defined genetic subsets, those with MLL-rearrangements and those with NPM1 mutations, are known to possess high-level expression of HOX and MEIS1 genes and have been shown to be dependent on continued expression of these genes. Recent studies have defined the chromatin-bound protein complexes that maintain this aberrant gene expression. Furthermore, multiple groups including ours have now developed small molecule inhibitors of chromatin-associated complexes. Some of these small molecules, including those targeting DOT1L, EZH2, and Bromodomains are at various stages of clinical development. Another approach to target chromatin associated complexes is via disruption of protein-protein interactions. One such protein-protein interaction that is being targeted is the interaction between the histone methyltransferase MLL1 and Menin, an approach that has also been shown to reverse leukemogenic HOX/MEIS1 gene expression. We have developed novel, potent and orally bioavailable MLL1-Menin inhibitors and have brought one of these small molecules to clinical assessment. Inhibition of the MLL1-Menin interaction in MLL-rearranged and NPM1 mutant AML cells leads to loss of Menin from chromatin, destabilization of the Menin protein and specific changes in chromatin state that include loss of the MLL1 complex at select loci. This results in selective reversal of leukemogenic gene expression, changes in occupancy of multiple other chromatin complexes, rapid cellular differentiation and cell death. Also, we have recently found this interaction to be critical in subtypes of Sarcoma opening up novel potential therapeutic opportunities. These molecules have remarkable preclinical activity in human disease models including patient derived xenograft (PDX) models, show minimal toxicity and are now in phase I/II clinical trials. Inhibition of protein-protein interactions represents a potentially effective approach to destabilize chromatin associated complexes for cancer therapy.

Citation Format: Scott A. Armstrong. Therapeutic targeting of MLL complexes in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA020.
Abstract PR003: Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma
Authors: Mabe Nathaniel W. , Huang Min , Schaefer Daniel A. , Dalton Guillermo N. , Digiovanni Giulia , Alexe Gabriela , Geraghty Anna C. , Khalid Delan , Mader Marius M. , Sheffer Michal , Linde Miles H. , Ly Nghi , Rotiroti Maria Caterina , Smith Benjamin A. H. , Wernig Marius , Bertozzi Carolyn R. , Monje Michelle , Mitsiades Constantine , Majeti Ravindra , Satpathy Ansuman T. , Stegmaier Kimberly , Majzner Robbie G.
Published: December 2022

Epigenetic dysregulation is frequently observed in the disease pathology of pediatric cancers, including neuroblastoma, the most common extracranial solid tumor in pediatric patients. Neuroblastoma tumors co-opt developmentally linked adrenergic or mesenchymal super-enhancer landscapes that rewire their transcriptional programs. Here, we describe that the lineage commitment to a mesenchymal epigenetic state is an important mechanism of resistance to anti-GD2 therapy through loss of GD2 antigen, a ganglioside glycolipid expressed on the cell surface. Low GD2 expression was significantly correlated with the mesenchymal state in a large panel of neuroblastoma cell lines and a forced adrenergic-to-mesenchymal transition conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2 expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Genome-wide CRISPR/Cas9 screening to identify regulators of GD2 in neuroblastoma revealed that the ablation of the polycomb repressive complex 2 (PRC2) significantly upregulates GD2 expression in GD2-low cells. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells into an adrenergic-like state, re-expressed ST8SIA1, and restored surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential PRC2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

Citation Format: Nathaniel W. Mabe, Min Huang, Daniel A. Schaefer, Guillermo N. Dalton, Giulia Digiovanni, Gabriela Alexe, Anna C. Geraghty, Delan Khalid, Marius M. Mader, Michal Sheffer, Miles H. Linde, Nghi Ly, Maria Caterina Rotiroti, Benjamin A. H. Smith, Marius Wernig, Carolyn R. Bertozzi, Michelle Monje, Constantine Mitsiades, Ravindra Majeti, Ansuman T. Satpathy, Kimberly Stegmaier, Robbie G. Majzner. Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR003.
Abstract PR008: An epigenetic memory of inflammation controls context-dependent lineage plasticity and KRAS-driven tumorigenesis in the pancreas
Authors: Falvo David J. , Grimont Adrien , Zumbo Paul , Yang Julie L. , Osterhoudt Alexa , Pan Grace , Rendeiro Andre F. , Wilkinson John Erby , Dundar Friederike , Elemento Olivier , Yantiss Rhonda K. , Betel Doron , Koche Richard , Chandwani Rohit
Published: December 2022

Tissue homeostasis depends on responses to environmental insults to restore cellular phenotype, microenvironment composition, and tissue architecture. Inflammation is essential to the disruption of homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. Herein we employ lineage-traced mouse models to delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and unveil the presence of an epigenetic memory of inflammation in the pancreatic acinar cell compartment. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. In vivo, this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that both persistent chromatin and transcriptional changes constituting memory are recalled with oncogenic stress. Together, our findings define a capacity for an environmental insult to control future cell-fate decisions in a context-dependent manner. The ability of epigenetic memory to potentiate tumor initiation both broadens the relationship between inflammation and cancer and raises the possibility that inducing epigenetic ‘amnesia’ of an inflammatory insult could be leveraged as a novel cancer prevention strategy.

Citation Format: David J. Falvo, Adrien Grimont, Paul Zumbo, Julie L. Yang, Alexa Osterhoudt, Grace Pan, Andre F. Rendeiro, John Erby Wilkinson, Friederike Dundar, Olivier Elemento, Rhonda K. Yantiss, Doron Betel, Richard Koche, Rohit Chandwani. An epigenetic memory of inflammation controls context-dependent lineage plasticity and KRAS-driven tumorigenesis in the pancreas. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR008.
Abstract A001: The development of a multilevel risk prediction model to identify risk of failure to complete CRC screening
Authors: Petrik Amanda F.
Published: December 2022

Context: The use of predictive analytics can help health systems target the right services to the right patients at the right time while improving population health. Multilevel data, or data at the interpersonal, organizational, community, and policy levels, is rarely sought after but may be used to improve risk prediction by providing information about a patient and the many groups to which they belong. Colorectal cancer screening promotion can be expensive and not all patients need it. Objective: This study sought to develop a risk prediction model using individual data available in the EHR, and then compared a multilevel risk prediction model using publicly available multilevel data. Study design and analysis: Retrospective data was used to develop a risk model, using logistic regression, first in data available in the EHR, and then adding multilevel data. Setting or dataset: This retrospective data only study was conducted at Kaiser Permanente Northwest. Population studied: Patients were eligible who were 50-75 years old, had been members at KPNW for at least a year before their birthday in 2018, and if they were due for screening. Results: The individual level model was sufficient and included 14 characteristics, and had a bootstrap corrected C-statistic of 0.722. The multilevel model added 9 variables, and the bootstrap corrected C-statistic was 0.724. Conclusions: Models can be used to identify patients unlikely to screen, although the performance of the model after adding multilevel data did not improve. Screening models could help in population health management of CRC screening.

Citation Format: Amanda F. Petrik. The development of a multilevel risk prediction model to identify risk of failure to complete CRC screening [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A001.
Abstract A015: Targeting necroptosis and RIP3 in colorectal cancer to overcome chemotherapeutic resistance
Authors: Ermine Kaylee A. , Chen Dongshi , Wang Peng , Yu Jian , Zhang Lin
Published: December 2022

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States. When metastatic, chemotherapeutic drug 5-fluorouracil (5-FU) is typically used for treatment, but resistance often occurs. Many studies have shown that 5-FU induces apoptosis in cancer cells, but when apoptosis is inhibited, cell death can still occur, suggesting that other forms of cell death are occurring. Necroptosis, or regulated necrosis, is an alternative form of cell death, controlled by RIP1, RIP3, and MLKL, and its role in anticancer therapy remains unclear. TCGA RNAseq data show that RIP3 is significantly downregulated in primary CRC tumors, with RIP3 downregulation correlating with worse clinical outcomes. Analysis of numerous established CRC cell lines revealed that RIP3 expression was downregulated in ~50% of the cell lines, with low RIP3 expression being associated with decreased 5-FU sensitivity. These data led us to hypothesize that RIP3 expression and necroptosis may play a critical role in response to anticancer therapies, such as 5-FU, and that restoring defective necroptosis may enhance therapeutic sensitivity. Our preliminary studies have shown that RIP3-expressing (RIP3+) CRC cell lines are able to undergo both apoptosis and necroptosis in response to 5-FU, which could explain the enhanced sensitivity. As expected, knock-out of RIP3 in RIP3+ cells resulted in decreased 5-FU sensitivity, as well as abrogated necroptosis induction. Additionally, transient restoration of RIP3 in RIP3-silenced (RIP3-) CRC cells appeared to enhance 5-FU sensitivity, and so, we generated a stable RIP3-expressing cell line through retroviral infection. Although the stable RIP3-expressing cell line did not appear to be more sensitive to 5-FU alone, it did show enhanced sensitivity under conditions where apoptosis is inhibited. Together, our results suggest that RIP3 plays an important role in 5-FU-induced cell death in CRC, and that restoring RIP3 when the apoptosis pathway is implicated could provide a way to enhance 5-FU sensitivity and overcome resistance. The results from this project provide novel insight into the role of RIP3 in mediating response to anticancer therapy in CRC, which could be used for development of improved therapies and personalized medicine based on RIP3 status in the clinic.

Citation Format: Kaylee A. Ermine, Dongshi Chen, Peng Wang, Jian Yu, Lin Zhang. Targeting necroptosis and RIP3 in colorectal cancer to overcome chemotherapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A015.
Abstract B001: Inverse relationship between tissue fusobacterium nucleatum amount and CD274 (PD-L1) expression of colorectal carcinoma
Authors: Ugai Tomotaka , Shimizu Takashi , Kawamura Hidetaka , Takashima Yasutoshi , Väyrynen Juha P. , Kahaki Seyed Mostafa Mousavi , Okadome Kazuo , Masugi Yohei , da Silva Annacarolina , Zhang Xuehong , Chan Andrew T. , Wang Molin , Meyerhardt Jeffrey A. , Nowak Jonathan A. , Song Mingyang , Giannakis Marios , Ogino Shuji
Published: December 2022

Background: The CD274 (programmed cell death ligand 1, PD-L1)/PDCD1 (programmed cell death 1, PD-1) immune checkpoint axis is known to regulate the antitumor immune response. Evidence suggests that Fusobacterium nucleatum (F. nucleatum) promotes colorectal carcinogenesis through its suppressive effect on antitumor immunity. We hypothesized that tumor CD274 overexpression and intratumor abundance of F. nucleatum might tend to be mutually exclusive immune evasion mechanisms in colorectal cancer. Methods: We assessed tumor CD274 expression by immunohistochemistry and F. nucleatum DNA within tumor tissue by quantitative polymerase chain reaction in 812 cases among 4,465 incident colorectal cancer cases that had occurred in the Nurses’ Health Study and the Health Professionals Follow-up Study. To adjust for potential confounders and selection bias due to tissue data availability, inverse probability weighting was integrated into multivariable ordinal logistic regression analyses to examine the association of tumor CD274 expression with the amount of intratumor F. nucleatum. Results: Tumor CD274 expression was negative in 93 (11%), low in 231 (28%), intermediate in 210 (26%), and high in 278 (34%) of 812 cases. F. nucleatum DNA was detected in tumor tissue in 109 (13%) cases of 812 cases. Tumor CD274 expression level was inversely associated with the amount of F. nucleatum in colorectal cancer tissue. For one category increase in three ordinal F. nucleatum categories (negative, low, and high), multivariable-adjusted odds ratios (with 95% confidence intervals) of the low, middle, and high CD274 expression categories (vs. negative) were 0.78 (0.41-1.51), 0.64 (0.32-1.28), and 0.50 (0.25–0.99), respectively (Ptrend=0.032). Conclusions: We found that CD274 expression was inversely associated with the amount of F. nucleatum in colorectal cancer tissue. Our findings suggest that a colorectal tumor tends to have either of the immune evasion mechanisms, i.e., PDCD1 (PD-1) immune checkpoint activation and intratumor abundance of F. nucleatum.

Citation Format: Tomotaka Ugai, Takashi Shimizu, Hidetaka Kawamura, Yasutoshi Takashima, Juha P. Väyrynen, Seyed Mostafa Mousavi Kahaki, Kazuo Okadome, Yohei Masugi, Annacarolina da Silva, Xuehong Zhang, Andrew T. Chan, Molin Wang, Jeffrey A. Meyerhardt, Jonathan A. Nowak, Mingyang Song, Marios Giannakis, Shuji Ogino. Inverse relationship between tissue fusobacterium nucleatum amount and CD274 (PD-L1) expression of colorectal carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B001.
Abstract B007: LIN28B promotes collective cell invasion and colorectal cancer metastasis via a novel CLDN1 and NOTCH3 axis
Authors: Shin Alice E. , Sugiura Kensuke , Masuike Yasunori , Suzuki Kensuke , Lengner Christopher J. , Rustgi Anil K.
Published: December 2022

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second most common cause of cancer death worldwide. The 5-year survival rate is 90% in patients with localized tumors, but the survival rate drastically decreases to 14% in patients with distant metastasis. Therefore, it is imperative to elucidate the molecular mechanisms underlying CRC metastasis. The RNA-binding protein LIN28B is overexpressed in over 30% of patients with CRC and is associated with poor prognosis. Indeed, our previous work revealed that LIN28B promotes liver metastasis in a subcutaneous xenograft model of CRC as well as a portal vein injection model. In the present study, we investigated the mechanism by which LIN28B promotes colorectal tumor progression and metastasis. To assess biological changes induced by LIN28B, we established CRC cells (DLD-1 and LoVo) with high expression of LIN28B (LIN28Bhigh). LIN28B overexpression upregulated Claudin-1 (CLDN1), a protein that functions as a major constituent of the tight junction complexes. RNA immunoprecipitation revealed that LIN28B directly binds to and stabilizes CLDN1 at a post-transcriptional level. Knockdown of CLDN1 expression in LIN28Bhigh cells suppresses cell aggregation, wound healing rate, and collective invasion. Using a mouse model of metastatic CRC, we reveal that knockdown of CLDN1 inhibits liver metastasis of LIN28Bhigh CRC cells. RNA-sequencing of metastatic liver tumors from LIN28Bhigh cells showed that NOTCH3 works downstream of the LIN28B-CLDN1 axis to induce cell aggregation, collective invasion, and metastatic liver tumor formation. Inhibition of Notch signaling by DAPT, an inhibitor of the γ-secretase complex, significantly reduced metastatic liver tumors in vivo. Taken together, our results indicate that LIN28B promotes cell aggregation, invasion, and liver metastasis in CRC through post-transcriptional induction of CLDN1 and upregulation of NOTCH3. Development of new therapies that target LIN28B-CLDN1-NOTCH3 axis may provide an effective strategy for stage 4 CRC. This work was supported by 9R01CA277795-22.

Citation Format: Alice E. Shin, Kensuke Sugiura, Yasunori Masuike, Kensuke Suzuki, Christopher J. Lengner, Anil K. Rustgi. LIN28B promotes collective cell invasion and colorectal cancer metastasis via a novel CLDN1 and NOTCH3 axis [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B007.
Abstract B014: Genotype-tailored ERK/MAPK pathway and HDAC inhibition rewires the apoptotic rheostat to trigger colorectal cancer cell death
Authors: Jenkins Laura J. , Luk Ian Y. , Fairlie Walter D. , Lee Erinna F. , Palmieri Michelle , Schoffer Kael L. , Tan Tao , Ng Irvin , Vukelic Natalia , Tran Sharon , Tse Janson , Nightingale Rebecca , Alam Zakia , Chionh Fiona , Iatropoulos George , Ernst Matthias , Afshar-Sterle Shoukat , Desai Jayesh , Gibbs Peter , Sieber Oliver M. , Dhillon Amardeep S. , Tebbutt Niall C. , Mariadason John M.
Published: December 2022

The EGFR/RAS/MEK/ERK signaling pathway (ERK/MAPK) is hyper-activated in most colorectal cancers (CRCs). A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in CRC cells. Nevertheless, these drugs do induce expression of pro-apoptotic factors, suggesting they may prime CRC cells to undergo apoptosis. As histone deacetylase inhibitors (HDACi) induce expression of multiple pro-apoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger CRC cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in CRC cell lines and patient-derived tumor organoids in vitro,and attenuated Apc-initiated adenoma formation in vivo. Mechanistically, combined MAPK/HDAC inhibition enhanced expression of the BH3-only pro-apoptotic proteins BIM and BMF, and their knockdown significantly attenuated MAPK/HDAC inhibitor-induced apoptosis. Importantly, we demonstrate that the paradigm of combined MAPK/HDAC inhibitor treatment to induce apoptosis can be tailored to specific MAPK genotypes in CRCs, by combining a HDAC inhibitor with either an EGFR, KRASG12C or BRAFV600 inhibitor in KRAS/BRAFWT ; KRASG12C , BRAFV600E CRC cell lines respectively. These findings identify a series of ERK/MAPK genotype tailored treatment strategies that can readily undergo clinical testing for the treatment of colorectal cancer.

Citation Format: Laura J. Jenkins, Ian Y. Luk, Walter D. Fairlie, Erinna F. Lee, Michelle Palmieri, Kael L. Schoffer, Tao Tan, Irvin Ng, Natalia Vukelic, Sharon Tran, Janson Tse, Rebecca Nightingale, Zakia Alam, Fiona Chionh, George Iatropoulos, Matthias Ernst, Shoukat Afshar-Sterle, Jayesh Desai, Peter Gibbs, Oliver M. Sieber, Amardeep S. Dhillon, Niall C. Tebbutt, John M. Mariadason. Genotype-tailored ERK/MAPK pathway and HDAC inhibition rewires the apoptotic rheostat to trigger colorectal cancer cell death [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B014.
Abstract B016: Inhibition of KRASG12C in colon cancer illustrates a link between beta-catenin, WNK, and the GID complex
Authors: Nayak Kasturi , Hwang Yeonjoo , Wang LeeAnn , Swaney Danielle L. , Krogan Nevan J. , Gordan John D.
Published: December 2022

KRAS is mutated in 35%-45% of colorectal cancers (CRC), and KRAS mutational status determines the prognosis and therapeutic options available to patients with advanced CRC. Direct KRASG12C inhibitors have proven to be highly effective for patients with non-small cell lung cancers, but unfortunately are relatively ineffective in the treatment of CRC. Thus, we have investigated tissue-specific mechanisms of resistance to direct KRAS inhibition. We used multiplex inhibitor bead kinome profiling (MIBs) and global phosphoproteomic analysis to determine the signaling response to the KRASG12C inhibitor ARS-1620 in four human colon cancer cell lines. Analyzing the kinome revealed a profound reprogramming beyond the Ras/MAPK pathway. We used network propagation to integrate analysis across these lines and define essential signaling nodes modified by direct KRAS inhibition, including two distinct signaling nodes containing RAS/MAPK and WNT-regulating kinases. Two additional smaller nodes contained WNK kinases and their effectors, followed by Hippo and a number of cell cycle-related kinases. The WNK kinases have been identified to modulate beta-catenin, a major driver of CRC biology, via the GID E3 ubiquitin ligase complex. Thus, to understand the signaling that links KRAS and beta-catenin, we used small molecule inhibitors of these kinases along with ARS-1620, we tested how they affected the transcription of beta-catenin targets. Additionally, we assessed the correlation between WNK/GID complex, and beta-catenin transcriptional output using western blot and quantitative real time PCR and observe that inhibition of KRASG12C also modulates beta-catenin transcriptional output. Our results identify new families of possible kinase targets in CRCs expressing KRAS mutations and shed light on the relationship of KRAS, beta-catenin, and WNK/GID in CRC maintenance. By dissecting these signaling relationships, we hope to identify potential drug combinations to overcome primary resistance KRASG12C inhibition in CRC.

Citation Format: Kasturi Nayak, Yeonjoo Hwang, LeeAnn Wang, Danielle L. Swaney, Nevan J. Krogan, John D. Gordan. Inhibition of KRASG12C in colon cancer illustrates a link between beta-catenin, WNK, and the GID complex [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B016.
Abstract B022: The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer
Authors: Invrea Federica , Carugo Alessandro , Petti Consalvo , Draetta Giulio , Medico Enzo
Published: December 2022

Colorectal cancer (CRC) is a heterogeneous disease with a wide spectrum of clinical outcomes, from indolent resectable disease to aggressive-metastatic cases. Primary and acquired resistance limits the efficacy of available treatments, and the identification of effective drug combinations is needed to further improve patients' outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induces tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a drop-out loss-of-function synthetic lethality screening with a shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at various levels could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. Our results suggested possible therapeutic opportunities in specific CRC clinical settings. We further focused on HER2-amplified CRC and found marked sensitivity to pevonedistat in a CRC cell line carrying HER2 amplification. In these cells, long-term in vitro treatment with a combination of lapatinib and trastuzumab induces the emergence of colonies of long-term persisters (i.e., cells surviving to several weeks of HER2/EGFR blockade). These persisters display extremely slow growth and senescence features, however they fully recover upon removal of the blockade, mimicking disease relapse in patients after treatment suspension. Interestingly, persister colonies maintained sensitivity to pevonedistat, displaying a marked decrease when pevonedistat was added after six weeks of HER2/EGFR blockade, independently of the continuation of the blockade. These results unveil the possibility of employing pevonedistat in HER2-amplified CRC patients subsequently to tumor stabilization or regression by HER-2/EGFR blockade.

Citation Format: Federica Invrea, Alessandro Carugo, Consalvo Petti, Giulio Draetta, Enzo Medico. The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B022.
Abstract B030: Dipeptidase-1 is a highly upregulated protein in CRC exosomes: Potential clinical relevance
Authors: Bechard Matthew E. , Glass Sarah E. , Coffey Robert J.
Published: December 2022

Bert Vogelstein and co-workers identified DPEP1 in 2001 as one of the top six upregulated genes encoding a membrane or secreted protein in colorectal adenomas and cancers (CRCs). DPEP1 is a GPI-linked, zinc-dependent dipeptidase whose enzymatic activity includes processing extracellular glutathione and converting LTD4 to LTE4. Recently, it was reported to act nonenzymatically as a receptor for neutrophils on liver and lung endothelial cells and to influence monocyte migration. DPEP1 has been reported to have roles in cancer proliferation, invasion and metastasis. We recently showed that DPEP1 is the most abundant protein in small extracellular vesicles (sEVs) released from CRC cell lines by mass spectrometry and is present in a subset of EGFR+/CD81+ exosomes along with other clinically relevant biomarkers by fluorescence-activated vesicle sorting (FAVS) (Q Zhang et al., Nature Cell Biology 23:1240-1254, 2021). We also found that DPEP1/CEACAM5 double-positive exosomes are increased in the plasma from three CRC patients compared to normal controls. Immunohistochemical staining for DPEP1 in clinically well-annotated adenoma and CRC tissue microarrays has revealed staining in 27% of adenomas and 71% of CRCs, whereas no staining was observed in normal colonic tissue. Moreover, diffuse cytoplasmic staining in CRCs was associated with a worse progression-free and overall survival. We have now generated adenoma organoids, some of which express endogenous DPEP1 that localizes to the apical plasma membrane in 3D organoids and when cultured as polarized monolayers on Transwell filters. We plan to examine the bi-directional effects of cellular DPEP1 and DPEP1-containing EVs and freshly isolated neutrophils and/or their EVs in a co-culture system that is part of our recently funded Translational and Biological Science in Early Lesions (TBEL) NCI U54 grant.

Citation Format: Matthew E. Bechard, Sarah E. Glass, Robert J. Coffey. Dipeptidase-1 is a highly upregulated protein in CRC exosomes: Potential clinical relevance [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B030.
Abstract IA003: ctDNA Guiding adjuvant treatment in early stage colon cancer: Are we moving the needle?
Authors: Tie Jeanne
Published: December 2022

My presentation will summarize the accumulating evidence on the ability of circulating tumor DNA to detect minimal residual disease and track adjuvant treatment efficacy in colorectal cancer and the potential for ctDNA to reshape the adjuvant treatment paradigm. I will also discuss the rationale and results of the DYNAMIC trial (ctDNA-guiding adjuvant therapy study in stage II colon cancer) and other ongoing ctDNA-based trials.

Citation Format: Jeanne Tie. ctDNA Guiding adjuvant treatment in early stage colon cancer: Are we moving the needle? [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr IA003.
Abstract IA004: Moving the needle: It is time for universal germline and somatic genetic testing in all colorectal cancer patients
Authors: Goldberg Richard M.
Published: December 2022

The rapid evolution and commercialization of new techniques for analyzing DNA have extended our knowledge of inherited genetic abnormalities that can predispose a person to develop colorectal and other cancers. Concomitantly, the genetic fingerprinting of individual tumors has fueled the development of targeted therapies and informed their appropriate deployment for the management of individual patients. Recently, Cercek and colleagues at Memorial Sloan Kettering Cancer Center reported that all 14 patients with MSI-H rectal cancers treated in a clinical trial in the neoadjuvant setting with the PD-L1 inhibitor dostarlimab had complete remissions of their disease without chemotherapy, radiation, or surgery and those individuals are being followed to determine if, as hoped and expected, those remissions are durable (PMID:35660797). These insights have led and will continue to lead us to develop specific strategies to manage and minimize risk for affected individuals and their family members with inherited cancer susceptibility syndromes. They are also enabling us to decipher the practical value of the heterogeneity of colorectal cancers based upon the identification of inherited or acquired genetic abnormalities that drive the development and progression of an individual patient’s cancer. Now it is time to deploy germline and somatic genetic testing as a standard of care in the clinic, universally and equitably, for individualizing the assessment of inherited colorectal cancer susceptibility and for individualizing care in patients with colorectal cancer. Implementing this strategy will require resource allocation to ensure that health care teams and the patients that they care for both understand and derive the maximum possible benefit from implementing this new approach.

Citation Format: Richard M. Goldberg. Moving the needle: It is time for universal germline and somatic genetic testing in all colorectal cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr IA004.