bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4344: Consensus genomic subtypes of gastric adenocarcinoma and their therapeutic implication
Authors: Jeong Yun Seong , Eun Young-Gyu , Lee Sung Hwan , Kang Sang-Hee , Yim Sun Young , Kim Eui Hyun , Noh Joo Kyung , Sohn Bo Hwa , Lee Ju-Seog
Published: April 2023

Background: Gastric adenocarcinoma (GAC) is heterogeneous lethal disease in genomic and clinical level. Although the clinical relevance of genomic and molecular subtypes of GAC has been demonstrated, their translation to the clinic has been hindered by discrepancies in classification methods. We aim to examine a consensus of genomic subtypes and correlate them with clinical outcomes.

Method: We collected genomic data from 2527 GAC tumors and divided the data into discovery (n = 1427) and validation sets (n = 1100). By integrating 8 previously established genomic subtype algorithms, we identified 6 clinically and molecularly distinct genomic consensus subtypes (CGSs) in discovery set. For validation of clinical significance of new subtypes, we constructed GAC predictor of integrated consensus subtype with 120 genes (GPICS120) and applied it to validation data set. In systematic analysis of genomic and proteomic data, we estimated potential response rate of each subtype to standard and experimental treatments such as radiation therapy, target therapy, and immunotherapy and further validated their functional significance in cell line models.

Results: Among identified 6 subtypes. CGS1 is characterized by poorest prognosis, very high stem cell characteristics, and high IGF1 expression, but low genomic alterations. CGS2 showed canonical epithelial gene expression patterns. CGS3 and CGS4 are characterized by high copy number alterations and low immune activity. However, CGS3 and CGS4 are different in high HER2 activation (CGS3) and SALL4 and KRAS activation (CSG4). CGS5 has highest mutation burden and moderately high immune activity that is characteristics of MSI-high tumors. Most of CGS6 tumors are EBV-positive and shows extremely high methylation and high immune activity. Most interestingly, CSG1 is most responsive to immunotherapy while CGS3 is significantly associated with benefit of chemoradiation therapy due to high basal level ferroptosis. In addition, we also identified potential therapeutic targets for each subtype.

Conclusion: Consensus subtype is robust classification system and can be the basis for pre-clinical investigation of subtype-based targeted interventions and future clinical trials.

Citation Format: Yun Seong Jeong, Young-Gyu Eun, Sung Hwan Lee, Sang-Hee Kang, Sun Young Yim, Eui Hyun Kim, Joo Kyung Noh, Bo Hwa Sohn, Ju-Seog Lee. Consensus genomic subtypes of gastric adenocarcinoma and their therapeutic implication. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4344.
Abstract 4349: C-C chemokine receptor 2-positive monocytic myeloid-derived suppressor cells predicts chemotherapeutic responses of the metastatic lesions in breast cancer patients
Authors: Kim Ju Won , Sa Jason K. , Jung Seung Pil , Park Kyong Hwa , Park Serk In
Published: April 2023

Anti-tumoral T cell immunity is counterbalanced by several types of immunosuppressive cells such as Myeloid-Derived Suppressor Cells (MDSC). Circulating MDSC levels correlate with poor prognosis and metastatic progression of breast cancer patients. However, the value of MDSC in predicting chemotherapeutic treatment responses in the metastatic lesions is not clearly understood. We performed a prospective cohort clinical study to measure MDSC before and after chemotherapy in metastatic breast cancer patients (N=64). Multicolor flow cytometry was used to quantitate two major subtypes, i.e. monocytic (M-) and polymorphonuclear (PMN-) MDSC, and one additional subtype, C-C chemokine receptor 2 (CCR2)-positive M-MDSC in the fresh peripheral blood mononuclear cells. M-MDSC were defined as HLA-DRlow/−CD45+CD11b+CD15−CD14+ PBMC, and PMN-MDSC were defined as CD45+CD11b+CD33+CD15+ CD14− PBMC. PMN-MDSC and CCR2+ M-MDSC, but not M-MDSC, were significantly increased after chemotherapy. Subsequently, we divided the patients by two groups (therapy-sensitive vs. -resistant groups) by treatment responses. Treatment-resistant patients had significantly increased CCR2+ M-MDSC. Unsupervised clustering of patients based on MDSCs populations dichotomized patients PMN-MDSC high or M-MDSC high groups. M-MDSC high patients had increased bone and lymph node metastasis. In addition, CCR2+ M-MDSC levels correlated with the progression-free survival (P<0.05). Altogether, our results showed that CCR2+ M-MDSC levels potentially predict the therapeutic responses in the metastatic lesions of breast cancer patients.

Citation Format: Ju Won Kim, Jason K. Sa, Seung Pil Jung, Kyong Hwa Park, Serk In Park. C-C chemokine receptor 2-positive monocytic myeloid-derived suppressor cells predicts chemotherapeutic responses of the metastatic lesions in breast cancer patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4349.
Abstract 4359: Interventions to improve pathologic nodal staging of curatively resected lung cancer: A population-based implementation study
Authors: Ray Meredith A. , Fehnel Carrie , Akinbobola Olawale , Saulsberry Andrea , Dortch Kourtney , Matthews Anberitha , Anga Amal , Giampapa Christopher , Sales Elizabeth , Okun Sherry , Robbins Edward T. , Wolf Bradley , Levy Paul , Wiggins Horace L. , Ng Thomas , Sachdev Vishal , Valaulikar Ganpat , Patel Hetal D. , Faris Nicholas R. , Smeltzer Matthew , Osarogiagbon Raymond U.
Published: April 2023

Introduction. Despite its importance, pathologic nodal (pN) staging of lung cancer remains poor. We evaluated the quality and survival impact of two interventions to improve pN staging.

Methods. Using a non-randomized stepped-wedge design, we implemented use of a lymph node (LN) specimen collection kit to improve intraoperative LN collection (surgical intervention) and a novel gross dissection method for intrapulmonary LN retrieval (pathology intervention) in 12 hospitals in five contiguous Hospital Referral Regions in AR, MS and TN (2009-2021). With appropriate statistical methods we compared surgical quality and survival of patients among: neither (Group 1), pathology only (Group 2), surgical only (Group 3), and both (Group 4) interventions.

Results. Of 4,019 patients, 50%, 5%, 21% and 24%, were in Groups 1-4 respectively. Rates of non-examination of LNs and non-examination of mediastinal LNs: 11%, 9%, 0% and 0%; 29%, 35%, 2% and 2% respectively in Groups 1-4 (p<0.0001). Attainment of American College of Surgeons Operative Standard 5.8: 22%, 29%, 72%, 85%; International Association for the Study of Lung Cancer’s stringent definition of ‘complete resection’: 14%, 21%, 53%, 61% (p<0.0001). Compared to Group 1, adjusted hazard ratios, aHR (95% CI), were: Group 2, 0.93 (0.76-1.15); Group 3, 0.91 (0.78-1.03); Group 4, 0.75 (0.64-0.87). Compared to Group 2, Group 4 aHR- 0.72 (0.57-0.91); compared to Group 3, was 0.83 (0.69-0.99). These relationships remained after excluding wedge resections.

Discussion. Combining a LN collection kit and novel gross dissection method significantly improved pN evaluation and survival in a population-based cohort.

Citation Format: Meredith A. Ray, Carrie Fehnel, Olawale Akinbobola, Andrea Saulsberry, Kourtney Dortch, Anberitha Matthews, Amal Anga, Christopher Giampapa, Elizabeth Sales, Sherry Okun, Edward T. Robbins, Bradley Wolf, Paul Levy, Horace L. Wiggins, Thomas Ng, Vishal Sachdev, Ganpat Valaulikar, Hetal D. Patel, Nicholas R. Faris, Matthew Smeltzer, Raymond U. Osarogiagbon. Interventions to improve pathologic nodal staging of curatively resected lung cancer: A population-based implementation study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4359.
Abstract 4417: Activity of novel RNA therapeutics to overcome resistance to immune checkpoint blockade
Authors: O'Ketch Marvin , Kiefer Jeffrey A. , Rasale Dnyanesh , Weiner Warren S. , Castaño Lizette A. , Azorsa Nathalie A. , Lee David W. , Barnhart Kerry M. , Mousses Spyro , Azorsa David O.
Published: April 2023

T-cells in the tumor microenvironment can often become exhausted and dysfunctional due to chronic and prolonged exposure to antigen. The resulting condition of T cell exhaustion represents an important mechanism of clinical resistance to immune checkpoint blockade. The transcription factor NR4A1 is known to be upregulated in tumor-specific T-cells and is a mediator of T cell dysfunction including driving exhaustion during chronic antigen stimulation of T-cells. Pro-oncogenic activities of NR4A1 have been observed in various solid tumors, including colorectal and breast cancers, and co-expression of NR4A1 with known immune checkpoint markers such as PD-L1 and B7 family members has also been observed in various cancer cell lines. These findings have identified NR4A1 as an important target for overcoming resistance to cancer immunotherapy. We hypothesize that targeted silencing of the NR4A1 and other immunomodulatory genes can reverse T-cell exhaustion and expand the clinical benefit of immune checkpoint blockade in solid tumors resistant to immune therapy. To reverse T-cell exhaustion with a targeted approach, we developed T cell targeting SeekRsTM, which are chimeric RNA therapeutics containing dual-flanking aptamer binders connected by a double-stranded bridge containing siRNA silencers that can target multiple immunomodulatory genes. First-generation aptamer-siRNA chimeras designed with a CTLA-4 targeting aptamer containing a STAT3 siRNA demonstrated effectiveness in silencing its target. Modifications to the structure included the addition of chemically modified nucleotides to improve serum stability. Additionally, dimerization of the structure to form a SeekR dimer molecule greatly improved internalization and target silencing compared to the monomeric chimera. Newly developed SeekRs targeting T cells and designed to silence NR4A1 have effectively downregulated NR4A1 in model cell lines and activated T cells. These results demonstrate that dual targeting SeekRs can be used to specifically deliver siRNA to T cells for targeted silencing that can reverse exhaustion and potentially overcome resistance to cancer immunotherapy in the clinic.

Citation Format: Marvin O'Ketch, Jeffrey A. Kiefer, Dnyanesh Rasale, Warren S. Weiner, Lizette A. Castaño, Nathalie A. Azorsa, David W. Lee, Kerry M. Barnhart, Spyro Mousses, David O. Azorsa. Activity of novel RNA therapeutics to overcome resistance to immune checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4417.
Abstract 4469: Disruption of ATG9A-dependent basal autophagy sensitizes cancer cells to innate inflammatory signaling
Authors: Jayatunge Dasun N. , Tsang Tsz-Ming , McEwan Colten M. , Andersen Joshua L.
Published: April 2023

Pathogen-derived nucleic acids are recognized by pattern recognition receptors (PRRs), including cGAS and RIG-I, which trigger the innate immune response by activating IRF3-mediated interferon gene expression. In tumors, high levels of chromosomal instability can also lead to DNA fragments leaking into the cytosol to trigger PRR-mediated interferon response, resulting in pro-inflammatory cytokine secretion from the tumor. This could potentially expose tumor cells to immune attack. However, through mechanisms that are not completely understood, tumor cells dampen the interferon response to escape immune recognition. Previous work demonstrated that loss of ATG9A, but not ATG5, increased inflammatory signaling through the STING-IRF3 cascade, suggesting that perhaps an autophagy-independent function of ATG9A regulates inflammation. Here we found that loss of ATG9A or other upstream regulators of basal autophagy (e.g., ATG101), but not core LC3 lipidation machinery (ATG5 or ATG7), sensitizes cells to dsDNA-induced IRF3 activation and interferon gene expression. We also found that loss of ATG9A or ATG101, but not ATG5 or ATG7, increases the basal activity of the ubiquitin-sensing, IRF3-targeted kinase TBK1, which increases further upon dsDNA treatment. In these ATG9A and ATG101 deficient cells, TBK1 is clustered around large p62-positive condensates that previously been shown to contain large accumulations of LC3. Our preliminary data suggest a model in which ATG9A deletion causes the accumulation of LC3-positive membrane at p62 condensates, which may act as a platform for inflammatory signaling. Our current work focuses on identifying the upstream pathways and core mechanism that regulates IRF3 activation in ATG9A-deficient cells and exploiting this mechanism to improve anti-tumor immunity.

Citation Format: Dasun N. Jayatunge, Tsz-Ming Tsang, Colten M. McEwan, Joshua L. Andersen. Disruption of ATG9A-dependent basal autophagy sensitizes cancer cells to innate inflammatory signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4469.
Abstract 447: Genetic and pharmacological inhibition of TUT4 and TUT7 sensitizes AML cells to BCL-2 inhibition
Authors: Fyfe Corey , Fiorentino Gabriella , Steinbaugh Michael J. , Campos Joana , Lawson Hannah , Durko Jozef , O'Carroll Donal , Kranc Kamil , Harvey Darren , Ghosh Shomir , Campbell Robert M.
Published: April 2023

The terminal uridyltransferases TUT4/7 (ZCCHC11/6) are important mediators of RNA stability, RNA decay, histone mRNA regulation, viral RNA sensing, and miRNA biogenesis. Regulation of RNA processing pathways can be mediated by a number of RNA-binding proteins (RBPs), and dysfunctional RNA processing has implications as a therapeutic target in leukemia and lymphoma. We performed genetic and pharmacological perturbation of the activity of terminal uridyltransferases TUT4/7 (ZCCHC11/6) in MLL-rearranged acute myeloid leukemia (AML). A combination of catalytic inactivation and knockout by CRISPR/Cas9 of TUT4 and TUT7, respectively, were used to abrogate uridylation in THP-1 cells, with enzyme function measured by a cellular miRNA uridylation assay. Inhibition of uridylation in THP-1 cells reduced proliferation, induced changes in cell cycle progression, and enhanced sensitivity to the BCL-2 inhibitor, venetoclax, alone and in combination with azacitidine. BCL-2 expression was found to be reduced at the mRNA and protein levels, while increases in expression of BCL-2 regulating miRNAs miR139-5p and miR494-3p were observed. GSEA performed on uridylation deficient cells highlighted changes in cell cycle regulation, mitochondrial metabolism, and cell death pathways relative to unedited controls. These effects could be recapitulated with pharmacological inhibition using sub-inhibitory doses of a novel, selective inhibitor of TUT4/7, TS-002266, while the weakly active enantiomer TS-002455 did not show combination activity. Treatment of primary AML with combination of TS-002266 and venetoclax improved activity over venetoclax alone in a number of primary AML samples. Thus, we propose that TUT4/7 and BCL-2 inactivation may be a therapeutic strategy in leukemia.

Citation Format: Corey Fyfe, Gabriella Fiorentino, Michael J. Steinbaugh, Joana Campos, Hannah Lawson, Jozef Durko, Donal O'Carroll, Kamil Kranc, Darren Harvey, Shomir Ghosh, Robert M. Campbell. Genetic and pharmacological inhibition of TUT4 and TUT7 sensitizes AML cells to BCL-2 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 447.
Abstract 4583: An essential role for Cmtr2 in endothelial cell function and vascular development
Authors: Yermalovich Alena , Mohsenin Zarin , Giotti Bruno , Wheeler Douglas , Xu Kelly , Tsankov Alexander , Meyerson Matthew
Published: April 2023

Cancer genome sequencing studies have identified multiple novel tumor suppressor genes, including the FTSJD1 or CMTR2 gene that is subject to statistically significant levels of both nonsense and frameshift mutations in lung adenocarcinoma. However, these analyses are insufficient to determine the importance and modes of action of the gene in the disease. Follow-up functional studies are essential to elucidate these gene functions. The protein product of CMTR2 has been described as an mRNA cap methyltransferase, which O-methylates the ribose of the second guanine residue of the 5’ cap, likely serving to modify efficiency of transcript processing, translation and stability, but the details of how and why specific transcripts undergo methylation by the enzyme, as well as its role in oncogenesis, remain to be elucidated. In this study we report the first comprehensive analysis of the physiologic consequences of Cmtr2 deficiency in knockout (KO) mice and primary human cells. Constitutive deletion of Cmtr2 results in embryos that die in midgestation with defects in embryo size, placental malformation and yolk sac vascularization. Endothelial-specific deletion of Cmtr2 in mice also results in vascular defects and perinatal lethality, suggesting that vascular development is a key function of Cmtr2 in animals. In summary, our study reveals that Cmtr2 plays a critical role in vascular development, maturation and remodeling. Further studies of CMTR2 function may provide insight not only into lung cancer biology but also to its broader roles in ontogeny.

Citation Format: Alena Yermalovich, Zarin Mohsenin, Bruno Giotti, Douglas Wheeler, Kelly Xu, Alexander Tsankov, Matthew Meyerson. An essential role for Cmtr2 in endothelial cell function and vascular development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4583.
Abstract 4592: In vivo efficacy evaluation of anti-human VEGFR2 antibodies in humanized B-hVEGFR2 mice
Authors: Zhao Lei , Shang Chengzhang , Soto Rebecca , Liu Chonghui , Yu Zhaoxue
Published: April 2023

Angiogenesis, vascular development, vascular permeability, and embryonic hematopoiesis is partly regulated by VEGF binding to VEGFR2, which forms receptor dimers and activates downstream signaling pathways. In addition to developmental cues, VEGFR2 is also associated with disease progression for psoriasis, rheumatoid arthritis, diabetic retinopathy, and more significantly, tumor growth and drug resistance. Given this, inhibition of VEGFR2 signaling is actively being studied, and multiple VEGFR2 inhibitors have entered various phases of clinical studies and have shown promising results. To evaluate in vivo efficacy of anti-human VEGFR2 antibodies, we generated a human VEGFR2 knock-in (B-hVEGFR2) mouse model by replacing the murine Vegfr2 extracellular domain sequence with the corresponding human sequence. We validated human VEGFR2 gene and protein expression in B-hVEGFR2 mice by RT-PCR and flow cytometry, respectively. Furthermore, we observed an anti-tumor effect with anti-human VEGFR2 antibodies in homozygous B-hVEGFR2 mice subcutaneously implanted with MC38 colon cancer cells. Taken together, we established a novel preclinical B-hVEGFR2 mouse model for evaluation of VEGFR2-targeted immunotherapy.

Citation Format: Lei Zhao, Chengzhang Shang, Rebecca Soto, Chonghui Liu, Zhaoxue Yu. In vivo efficacy evaluation of anti-human VEGFR2 antibodies in humanized B-hVEGFR2 mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4592.
Abstract 4695: Expansion of the extracellular proximity labeling (ePL) technique to novel matrisome targets
Authors: Rich Joshua , Gurung Sadeechya , Kaur Sukhbir , Roberts David , Stetler-Stevenson William , Peeney David
Published: April 2023

The extracellular matrix (ECM) is a complex and dynamic meshwork of extracellular macromolecules that plays a key role in cancer progression and metastasis. The matrisome encompasses the genes encoding ECM and ECM-associated secretory proteins, and elucidating the protein-protein interactions (PPIs) of the matrisome is a promising area of study in the quest for a better understanding of cancer pathology and therapeutics. Proximity labeling, a powerful technique for investigating protein interactomes based on enzyme-fusion constructs and affinity chromatography, has considerable potential in detailing the protein-protein interactions of the matrisome. We have recently had success in developing a proximity labeling approach based on fusion constructs containing the biotinylating enzymes BioID2 and TurboID. We apply this approach to gain new insights into the interactome of the matrisome protein tissue inhibitor of metalloproteinase 2 (TIMP2), a protein found to have antitumorigenic potential. At present, we seek to expand this approach to other ECM protein targets, including tissue inhibitor of metalloproteinase 3 (TIMP3) and thrombospondin-1 (THBS-1). TIMP3 has been shown to possess anti-tumor properties, with loss of TIMP3 function associated with poor prognosis in multiple cancers. THBS-1 is a large (129 kDa) ECM protein that is relatively promiscuous with diverse functions in the tumor microenvironment. Expression of THBS-1 is frequently lost during oncogenesis, and such loss is associated with poor prognosis in some cancers. We describe how ePL can be expanded to these matrisome targets in different cellular systems (adherent vs. suspension) and show that ePL assays can be performed with stable and transient transgene expression. We show that ePL is a suitable method for studying PPIs in novel matrisome targets and expect to apply the technique to further targets in the near future.

Citation Format: Joshua Rich, Sadeechya Gurung, Sukhbir Kaur, David Roberts, William Stetler-Stevenson, David Peeney. Expansion of the extracellular proximity labeling (ePL) technique to novel matrisome targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4695.
Abstract 4701: Spatial resolution of tumor and immune cell lineages in the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC)
Authors: Arvidson Lisa , Prioli Reginaldo , Jensen Samuel , Smith Michael J. , White Katie O. , Heil-Chapdelaine Richard A. , Huang Chi-Chou , Phan Tuan H. , Sasaki Hideki , Angus-Hill Melinda L.
Published: April 2023

Understanding how the tumor microenvironment (TME) evolves during tumorigenesis and therapeutic response is crucial to developing personalized treatments with the goal of improving cancer therapy. With robust and comprehensive multiplexed imaging technologies, immune biomarker antibodies can be used to interrogate immune cell lineages and structures. When combined with specific oncology biomarker antibodies, this approach can capture the immune response within the TME in a variety of neoplasms. The Cell DIVETM Multiplex Imaging Solution allows probing and imaging of dozens of biomarkers on a whole single tissue section using an iterative staining and dye inactivation workflow. The broad portfolio of robust IHC-validated antibodies from Cell Signaling Technology (CST) enables the detection of key proteins in the TME, allowing immune cell detection and phenotyping in tissue. Here, we demonstrate multiplexed Cell DIVE imaging using a novel CST panel to probe pancreatic ductal adenocarcinoma (PDAC). These biomarker antibodies define the immune cell landscape in the hypoxic tumor. Development of the antibody panel required minimal optimization, enabled the identification of complex cell types and revealed their cell-to-cell interactions within the tumor microenvironment. The availability of cell type specific biomarkers, combined with the ability to interrogate using multiplexed tissue imaging, provides unprecedented and novel insights and spatial resolution of immune cell populations with many cell types in the TME.

Citation Format: Lisa Arvidson, Reginaldo Prioli, Samuel Jensen, Michael J. Smith, Katie O. White, Richard A. Heil-Chapdelaine, Chi-Chou Huang, Tuan H. Phan, Hideki Sasaki, Melinda L. Angus-Hill. Spatial resolution of tumor and immune cell lineages in the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4701.
Abstract 4776: NRF2 is overexpressed in esophageal adenocarcinoma and its targeting sensitizes tumor cells to cisplatin through induction of ferroptosis and apoptosis
Authors: Ballout Farah , Lu Heng , Chen Zheng , Hu Tianling , Chen Lei , El-Rifai Wael , Peng Dunfa
Published: April 2023

Background: Esophageal adenocarcinoma (EAC) is the predominant type of esophageal cancer in the United States, with the 5-year survival rate is below 20%. EAC develops through Barrett’s esophagus (BE)-dysplasia-carcinoma cascade. Gastroesophageal reflux disease (GERD), where acidic bile salts refluxate into the esophagus, is the main risk factor for the development of BE and its progression to EAC. The NFE2-related factor 2 (NRF2) is the master cellular antioxidant regulator, involving in many cancer hallmarks.

Methods and Results: Using western blotting and immunohistochemistry technologies, we detected high NRF2 protein levels in EAC cell lines and primary EAC tissues, as compared with normal esophagus and non-neoplastic Barrett’s esophagus samples. The knockdown of NRF2 using NRF2 specific siRNAs significantly increased oxidative stress in response to cellular stress stimuli by bile salts or cisplatin. This was associated with an increase in DNA damage and inhibition of EAC cell growth. Brusatol, a NRF2 inhibitor, significantly inhibited NRF2 transcriptional activity and downregulated NRF2 target genes. We discovered that in addition to inducing apoptosis, Brusatol alone or in combination with CDDP induced significant lipid peroxidation and ferroptosis as evidenced by reduced xCT and GPX4 expression, two known ferroptosis markers. Moreover, the combination of Brusatol and CDDP significantly inhibited EAC tumor xenografts growth in vivo. We confirmed the in vitro data showing ferroptosis as an important mechanism in the xenografted tumors treated with Brusatol or Brusatol and CDDP combination using IHC staining.

Conclusion: Our data support the role of NRF2 in protecting against stress-induced apoptosis and ferroptosis in EACs. Targeting NRF2 in combination with platinum therapy can be an effective strategy for eliminating cancer cells in EAC.

Citation Format: Farah Ballout, Heng Lu, Zheng Chen, Tianling Hu, Lei Chen, Wael El-Rifai, Dunfa Peng. NRF2 is overexpressed in esophageal adenocarcinoma and its targeting sensitizes tumor cells to cisplatin through induction of ferroptosis and apoptosis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4776.
Abstract 478: A novel dual-acting RAS/β-catenin inhibitor activates antitumor immunity to potentiate cancer immunotherapy
Authors: Gazi Md Yeashin , Okoko Ogacheko , Wang Xin , Brandle Caitlin , Ding Zhi-chun , Mitra Amit , Ramirez-Alcantara Veronica , Chen Xi , Keeton Adam , Maxuitenko Yulia , Piazza Gary , Zhou Gang
Published: April 2023

Previous research suggests that the antineoplastic activity of sulindac is mediated by a cyclooxygenase (COX) independent mechanism involving dual inhibition of RAS and β-catenin signaling. ADT030 is a novel non-COX inhibitory sulindac-derivative designed to target RAS and phosphodiesterase 10 (PDE10) to block MAPK/AKT signaling and activate cGMP/PKG signaling to suppress oncogenic β-catenin transcription, respectively. ADT-030 potently and selectively inhibits growth and induces apoptosis of a variety of cancer cell lines and has robust antitumor activity in multiple mouse tumor models by oral administration. However, the impact of ADT-030 on the tumor microenvironment (TME) has not been examined. Here we report the antitumor activity and immunomodulatory effect of ADT-030 in preclinical models. We show that oral administration of ADT030 significantly inhibited the growth of multiple types of murine tumors in immunocompetent mice. In a metastatic breast tumor model (4T1), ADT-030 not only delayed the growth of the primary tumor but also reduced tumor metastasis, leading to improved mouse survival. Immunological analysis revealed marked reduction of MDSCs in mice, and increased T cell tumor infiltration after ADT-030 treatment. The antitumor activity of ADT030 was diminished in immunodeficient mice or immunocompetent mice depleted of CD8+ T cells, suggesting the dependence of ADT-030 on host immunity. Moreover, the combination of ADT-030 and anti-PD-1 antibody therapy led to further improved tumor growth control and mouse survival. Bulk and single-cell RNAseq is being conducted to determine the impact of ADT-030 on the cell compositions and transcriptomic changes in the TME. Altogether, our study provides insight into the mechanism of action of ADT-030 and underscores the potential of ADT-030 as a monotherapy or in combination with immunotherapies for RAS and Wnt/β-catenin driven cancers.

Citation Format: Md Yeashin Gazi, Ogacheko Okoko, Xin Wang, Caitlin Brandle, Zhi-chun Ding, Amit Mitra, Veronica Ramirez-Alcantara, Xi Chen, Adam Keeton, Yulia Maxuitenko, Gary Piazza, Gang Zhou. A novel dual-acting RAS/β-catenin inhibitor activates antitumor immunity to potentiate cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 478.
Abstract 4812: Inhibition of Smurf2 E3 ubiquitin ligase by heclin and its analogues enhances HIF-1α expression and transcriptional activity in normoxia or hypoxia
Authors: Zhao Shuai , El-Deiry Wafik S.
Published: April 2023

Hypoxia-inducible factors (HIFs) act as transcription factors and play an essential role in cellular and systemic responses to low oxygen environments. HIF-1α expression is induced in acute hypoxia typically through failure of its ubiquitination and degradation mediated by Von Hippel-Lindau (VHL). Previously we discovered a non-canonical mechanism where the SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) promotes the ubiquitination of HIF-1α and reduces HIF-1α level in HCT116 colorectal cancer cells. Smurf2 is a HECT-type ubiquitin ligase known to interact with Smad proteins, leading to their ubiquitination and proteasomal degradation. Overexpression of Smurf2 decreased HIF-1α expression in HCT116 and SW480 colorectal cancer cells under hypoxia as well as in RCC4 VHL-deficient kidney renal clear cell carcinoma cells under normoxia. Treatment with MG132 at least partially rescued the expression of HIF-1α following Smurf2 overexpression, indicating involvement of proteasome-dependent degradation in Smurf2-mediated HIF-1α destabilization. Knockdown of SMURF2 increased HIF-1α expression under normoxia in HCT116 and RCC4 cells. To investigate the effect of Smurf2 inhibition, we tested a selective reversible inhibitor of HECT E3 ubiquitin ligases, heclin, along with its analogues, PYR-41, C646 and 4E1RCat. Treatment with heclin increased HIF-1α expression in HCT116 under hypoxia as well as in SW480 and RCC4 cells under normoxia in a dose-dependent manner. PYR-41 elevated the level of HIF-1α level in HCT116 cells under hypoxia, in RCC4 cells under normoxia and in SW480 cells under both normoxia and hypoxia. To examine the effect on HIF transcriptional activity, we performed luciferase reporter assay using plasmids containing the hypoxia response element (HRE) from the PGK1 promoter and the VEGF promoter, respectively. PYR-41 induced transcriptional activity on PGK1-HRE in both HCT116 and SW480 cells. 4E1RCat robustly activated transcription on both VEGF-HRE and PGK1-HRE in HCT116 and SW480 cells. In summary, Smurf2 targets HIF-1α for ubiquitination and degradation independently of oxygen concentration and inhibition of Smurf2 to stimulate HIF activity under normoxia or hypoxia may be beneficial in pathological circumstances featuring anemia and hypoxemia.

Citation Format: Shuai Zhao, Wafik S. El-Deiry. Inhibition of Smurf2 E3 ubiquitin ligase by heclin and its analogues enhances HIF-1α expression and transcriptional activity in normoxia or hypoxia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4812.
Abstract 4899: Inhibition of murine tumor growth by decitabine is correlated with enhanced expression of the mouse mammary tumor virus
Authors: Zhang Jieyu , Higgins Savannah , Smith Riley , Reginald Tania , Qi Andrew , Li Jiayi , King Anna , Liu Yingguang
Published: April 2023

The purpose of this study is to clarify the mechanisms of the antineoplastic effect of decitabine (DAC), a DNA methyltransferase inhibitor. We previously reported that DAC inhibited the proliferation of the 4T1 mammary cancer cell line in vitro while inhibiting MMTV env and pol expression in the same cells. Here we present findings in vivo using two murine tumor models. We inoculated 4T1 cells into BALB/c mice from which the cell line was derived. In addition, we inoculated the murine colon carcinoma cell line, MC38, into C57BL/6 mice, the mouse strain of its origin. We treated the mice with DAC when tumors became palpable. DAC inhibited tumor growth in both tumor models and inhibited metastasis of 4T1 cells into the lungs. Quantitative PCR detected elevated expression of MMTV genes in the tumor tissues of DAC-treated mice in both models. The levels of MMTV env RNA in tumor tissues were negatively correlated with tumor mass in both models, so were the levels of the MMTV Env protein in 4T1 tumors. DAC also significantly mitigated tumor-induced splenomegaly. Hepatotoxicity of DAC was observed in C57BL/6 mice. Our data suggest the possibility that decitabine inhibits tumor growth in vivo and in vitro via different mechanisms. The findings also support the current understanding of decitabine upregulating endogenous retroviruses (ERVs) which subsequently activate an interferon response. The negative correlation between tumor mass and MMTV Env protein expression suggests a possible role of ERV proteins serving as tumor-associated antigens and decitabine enhancing adaptive immunity against cancer by stimulating ERV expression on tumor cells. Further studies of the interaction between decitabine and MMTV using immunodeficient mice may help us understand the possible involvement of adaptive immunity in the pharmacodynamics of decitabine as well as the role of ERVs in tumor development.

Citation Format: Jieyu Zhang, Savannah Higgins, Riley Smith, Tania Reginald, Andrew Qi, Jiayi Li, Anna King, Yingguang Liu. Inhibition of murine tumor growth by decitabine is correlated with enhanced expression of the mouse mammary tumor virus. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4899.
Abstract 4918: Natural compounds ursolic acid and digoxin exhibit anti-cancer effects through nuclear receptor RORγ-dependent and -independent mechanisms
Authors: Zou Hongye , Yang Yatian , Cai Demin , Chen Hong-Wu
Published: April 2023

Natural compounds such as ursolic acid (UA) and digoxin isolated from fruits and other plants display potent anti-cancer effects in preclinical studies. UA and digoxin have also been at several clinical trials for treatment of different cancers including prostate cancer, pancreatic cancer and breast cancer. However, they displayed limited benefit to patients. Currently, a poor understanding of their direct targets and mechanisms of action (MOA) in tumor cells severely hinders their further development. We previously identified nuclear receptor RORγ as a novel therapeutic target in castration-resistant prostate cancer (CRPC) and triple-negative breast cancer (TNBC). Previous studies implicated UA and digoxin as potential RORγ antagonists in modulating the functions of immune cells such as Th17 cells. Here we showed that UA displays a strong activity in inhibition of RORγ-dependent transactivation function, while digoxin exhibits no effect at clinically relevant concentrations. In prostate cancer cells, UA down-regulates RORγ-stimulated AR expression and AR signaling, whereas digoxin up-regulates AR signaling pathway. UA also showed potent anti-growth effects in AR-positive prostate cancer cells compared to AR-negative prostate cancer cells. In TNBC cells, UA but not digoxin alters RORγ-regulated cell proliferation and apoptosis gene programs. Together, our study shows for the first-time that UA, but not digoxin, acts as a natural antagonist of RORγ in prostate cancer and TNBC cells. Our finding that RORγ is a direct target of UA in specific type of cancer will help select patients with tumors that likely respond to UA treatment.

Citation Format: Hongye Zou, Yatian Yang, Demin Cai, Hong-Wu Chen. Natural compounds ursolic acid and digoxin exhibit anti-cancer effects through nuclear receptor RORγ-dependent and -independent mechanisms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4918.
Abstract 4983: Anaplastic thyroid cancer cells upregulate mitochondrial one-carbon metabolism to meet purine demand, eliciting a targetable vulnerability
Authors: Sugarman Adam J. , Huynh Luong Do , Cristofano Antonio Di
Published: April 2023

Anaplastic thyroid cancer (ATC) is one of the most aggressive and lethal tumor types, characterized by loss of differentiation, epithelial-to-mesenchymal transition, extremely high proliferation rate, and generalized resistance to therapy. To identify novel relevant, targetable molecular alterations, we have analyzed gene expression profiles from a genetically engineered ATC mouse model and from two human patient datasets, and have found consistent and coordinated upregulation of several genes encoding enzymes involved in the one-carbon metabolic pathway, which uses serine and folates to generate nucleotides, glutathione and glycine. We have used a combination of cell line and in vivo approaches to assess the role of this pathway in the progression and maintenance of ATC and the effect of its inhibition on tumor behavior. Genetic and pharmacological inhibition of both MTHFD2 and SHMT2, key enzymes of the mitochondrial arm of the one-carbon pathway, rendered mouse and human ATC cells auxotroph for glycine and led to significant inhibition of tumor cell proliferation and colony forming ability, which was primarily caused by depletion of the purine pool leading to arrest in S phase. Notably, these growth-suppressive effects were significantly amplified when cells were grown in the presence of physiological types and levels of folates. Genetic blockage of the one-carbon pathway dramatically impaired tumor growth in vivo, both in immunocompetent allograft models and in immunocompromised xenograft models of ATC. Together, these data establish the upregulation of the one-carbon metabolic pathway as a novel and targetable vulnerability of ATC cells, which can be exploited for therapeutic purposes.

Citation Format: Adam J. Sugarman, Luong Do Huynh, Antonio Di Cristofano. Anaplastic thyroid cancer cells upregulate mitochondrial one-carbon metabolism to meet purine demand, eliciting a targetable vulnerability. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4983.
Abstract 4995: Targeting PLK1 effectively suppresses growth of small cell lung cancer
Authors: Schmitz John C. , Zhang Guojing , Ivanov Andrey A. , Pannucci Abbe , Ridinger Maya , Owonikoko Taofeek K.
Published: April 2023

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor characterized by rapid disease progression and poor patient survival. Key transcription factors implicated as drivers of unique biological phenotypes of SCLC include ASCL1, NEUROD1, YAP1, and POU2F3. Strategies to exploit these phenotypes for innovative precision medicine approaches will be impactful. Using an unbiased agnostic preclinical drug screen to uncover therapeutic opportunities in SCLC, we identified a strong signal with PLK1 inhibitors (PLK1i) with low nanomolar IC50. We extended the in vitro findings by testing the efficacy of PLK1i in vivo using traditional xenograft of SCLC H526 cell line and patient derived xenografts (PDX). Volasertib achieved significant tumor growth inhibition relative to control in H526 xenografts. Also, onvansertib significantly inhibited growth of platinum-resistant and platinum-sensitive PDXs. The combination of PLK1i with standard chemotherapeutic agents identified promising synergy of the combination of onvansertib and paclitaxel. We further interrogated for predictive biomarkers of PLK1i sensitivity using gene expression profile comparing highly sensitive to less sensitive cell lines. High expression of C-MYC but not PLK1, TP53 or RB was associated with resistance to PLK1i. Conversely, while TP53 expression level did not correlate, TP53 gene mutation status (inactivating disruptive mutations) correlated with cell sensitivity to PLK1i. We queried the publicly available CCLE and the Cancer Therapeutics Response Portal to evaluate whether any of the SCLC subtypes have therapeutic vulnerability to PLK1i. In general, high expression of YAP1 in SCLC cell lines correlated with greater sensitivity to PLK1i. A YAP1 positive cell line, SW1271, with strong TP53 expression was particularly resistant to PLK1i. CRISPR knockout of YAP1 in this cell line enhanced SW1271 sensitivity to PLK1i suggesting that YAP1 expression as a marker of vulnerability to PLK1i could be context dependent especially when co-occurring with TP53 mutations. The mechanism of this interaction will be discussed.

Citation Format: John C. Schmitz, Guojing Zhang, Andrey A. Ivanov, Abbe Pannucci, Maya Ridinger, Taofeek K. Owonikoko. Targeting PLK1 effectively suppresses growth of small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4995.
Abstract 5006: Discovery of novel heterobifunctional degraders of mutant EGFR proteins for NSCLCs harboring various EGFR mutations
Authors: Ki Dong Hyuk , Joo Min Sung , Nam Joonwoo , Choi Hunmi , Seo Kyoungwan , Kim Eun-Jung , Kim Jiyeon , Kim Chulwon , Jin Jimmy Taiguang , Han Wooseok
Published: April 2023

Abnormal expression and activation of Epidermal Growth Factor Receptor (EGFR) contribute to malignancy development, especially in non-small cell lung cancers (NSCLCs). Although the first- to third-generation EGFR small molecule inhibitors have made significant progress in the treatment of EGFR mutant NSCLC patients, acquired mutations have been developed rapidly, resulting in drug resistance. Targeted protein degradation (TPD) technologies including PROteolysis Targeting Chimeras (PROTACs) have recently emerged as a promising alternative modality to address the confronting issues of small molecule inhibitors such as drug resistance. Recently, we have developed a series of heterobifunctional degraders of EGFR mutant proteins, which showed strong degradation ability against a variety of mutant EGFRs including triple mutants with C797S. DC50 (half-maximal degradation concentrations) values of the degraders were obtained using HiBiT assay in NCI-H1975 (EGFR L858R/T790M), HCC827 (EGFR Del19), and H1299 (EGFR WT) cells. Our lead compounds demonstrated excellent EGFR degradation potency with high selectivity against EGFR wild type. Moreover, C-09045, C-09066, and C-13951 strongly inhibited the cell growth of Ba/F3 cells harboring one of the EGFR mutations including L858R/C797S, Del19/C797S, L858R/T790M/C797S, Del19/T790M/C797S, and Exon20 NPH insertion. By contrast, these compounds showed much weaker potency in normal lung fibroblast (HFL-1) and Ba/F3 cells harboring EGFR wild-type. In PK analysis, C-09066 showed good oral bioavailability in mice (F%=41.4) with desirable pharmacokinetic parameters and robust tumor growth inhibition efficacy in the NCI-H1975 EGFR L858R/T790M/C797S xenograft and the Ba/F3 Del19/C797S allograft mouse models. In summary, we have identified selective, potent, and orally bioavailable degraders of various EGFR mutant proteins with broad spectrum anti-tumor efficacy both in vitro and in vivo.

Citation Format: Dong Hyuk Ki, Min Sung Joo, Joonwoo Nam, Hunmi Choi, Kyoungwan Seo, Eun-Jung Kim, Jiyeon Kim, Chulwon Kim, Jimmy Taiguang Jin, Wooseok Han. Discovery of novel heterobifunctional degraders of mutant EGFR proteins for NSCLCs harboring various EGFR mutations. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5006.