bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2334: Inhibition of DHFR modulates anti-tumor immunity
Authors: Boese Austin , Young Isabelle , Kang Jihoon , Malin Courteney , Hwang Jung Seok , Kang Sumin
Published: June 2022

Lung cancer is the second most common type of cancer in both men and women and has the highest mortality rate. Non-small cell lung cancer (NSCLC) makes up most cases. Immune checkpoint inhibitors have significantly improved the clinical management of some NSCLC cases, yet many lung cancer patients still fail to respond to conventional immunotherapy. Therefore, more research is needed to characterize cellular pathways contribute to immune checkpoint inhibitor (ICI) resistance in NSCLC patients. Dysregulated cellular metabolism is a common feature of many cancer types, and metabolites are now known to modulate several functions inside cells such as signaling pathways and epigenetic control of gene expression. Therefore, targeting tumor metabolism may be able to combat NSCLC immunotherapy resistance. To identify potential metabolic pathways involved in the anti-tumor immune response, we employed a metabolic inhibitor-based drug screen in human NSCLC cell lines and tested for expression changes in a panel of known immune regulator genes. Notably, pharmacologic inhibition of dihydrofolate reductase (DHFR) downregulated the mRNA expression of anti-phagocytic surface protein cluster of differentiation 24 (CD24) in lung cancer cells. In line with this, shRNA knockdown and overexpression of DHFR, decreased and increased CD24 RNA and protein levels, respectively. These results show that the metabolic enzyme DHFR is involved in CD24 gene expression. Cancer cells heavily rely on DHFR for enhanced nucleotide biosynthesis to facilitate rapid tumor growth, and DHFR inhibitors are already used in the clinic for cancer therapy. This study highlights a novel interaction between reprogrammed cancer metabolism and the anti-tumor immune response, with possible clinical implications for DHFR inhibitors such as methotrexate in combination with conventional cancer immunotherapy agents.

Citation Format: Austin Boese, Isabelle Young, Jihoon Kang, Courteney Malin, Jung Seok Hwang, Sumin Kang. Inhibition of DHFR modulates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2334.
Abstract 2340: Evaluation of metabolic and hormonal disorders in Yemeni women with breast cancer attending national oncology center in Mukalla, Yemen
Authors: Dahman Lotfi Saeed Bin , Sanoon Zaki Farag , Kaleem Waleed Khalid , Humam Mariam Ahmed , Bamhraz Shaima Adeeb , Bamatraf Noor Adeeb , Humran Khadija Mohmmed , Albada Fatima Lutfi , Obad Yaser Saeed
Published: June 2022

Background: Metabolic syndrome has been linked to many adverse health consequences, including breast cancer. This study aimed to evaluate the metabolic and hormonal disorders and assess the effect on estrogens in Yemeni women with breast cancer.

Patients and Methods: A total of 69 Yemeni women with breast cancer were recruited into the cross-sectional study. This study was conducted at the National Oncology Center in Mukalla, Yemen, from15th March to 30th July 2021. Anthropometric data and blood pressure were taken from each participant. Fasting blood glucose FBG, HbA1c, lipid profile, C-reactive protein (CRP), were measured using the chemical autoanalyzer (Mindray Bs230 autoanalyzer, Mindray Diagnostics). Insulin, estradiol, and testosterone were measured using Mindray CL-960i, Mindray Diagnostics ). Data were analyzed by using SPSS.

Results: The mean age of the participants was 48.17±11.0 years. The majority were normal weight(40.6%), and some were obese (30.4%) and overweight (29.0%). The majority had breast cancer stage2 (53.6%), and some were stage 4 (24.6%) and stage 3 (21.7%). Obese breast cancer women had significantly higher weight (P<0.001), BMI (P<0.001), waist (P<0.001), and hips (P<0.001) than the normal weight group. Furthermore, obese breast cancer women had significantly higher HbA1c(P=0.016), TC (P<0.001), and LDL-C (P=0.032) than the normal weight group. Insulin was positively associated with FBG (P=0.001) and HOMA-IR (P<0.001). Estradiol was positively associated with total cholesterol (P=0.004), triglycerides (P=0.007), and CRP (P=0.026). testosterone was negatively associated with CRP (P=0.011) and estradiol (P=0.019) in the combined groups studied.

Conclusion: Metabolic syndrome is more prevalent in breast cancer patients and is more likely to develop breast cancer.

Citation Format: Lotfi Saeed Bin Dahman, Zaki Farag Sanoon, Waleed Khalid Kaleem, Mariam Ahmed Humam, Shaima Adeeb Bamhraz, Noor Adeeb Bamatraf, Khadija Mohmmed Humran, Fatima Lutfi Albada, Yaser Saeed Obad. Evaluation of metabolic and hormonal disorders in Yemeni women with breast cancer attending national oncology center in Mukalla, Yemen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2340.
Abstract 2349: SAP30, a novel drug response specific transcription factor in high-risk neuroblastoma
Authors: Prathipati Philip , Pathania Anup S. , Chaturvedi Nagendra K. , Gupta Subash C. , Byrareddy Siddappa N. , Coulter Don W. , Challagundla Kishore B.
Published: June 2022

Neuroblastoma is the most devastating extracranial solid malignancy in children. Despite an intense treatment regimen, the prognosis for high-risk neuroblastoma patients remains poor, with less than 40% survival. So far, MYCN status/amplification is considered the most prognostic factor but corresponds to only 25% of neuroblastoma patients. Therefore, it is essential to identify a better prognosis and prediction marker of therapy response in neuroblastoma patients. The identification of master regulators with good prognostic and drug response abilities are not successful due to the complexity and lack of data-driven bioinformatic workflows. We applied robust bioinformatic data mining tools such as Weighted Gene Co-expression Network Analysis, cisTarget, and Single-Cell rEgulatory Network Inference and Clustering on three neuroblastoma patient datasets (n=1252). We found Sin3A Associated Protein 30 (Sap30), a driver transcription factor positively associated with high-risk, progression, stage 4, and poor survival in high-risk neuroblastoma patient cohorts. Tumors of high-risk neuroblastoma patients and relapse-specific patient-derived xenografts showed higher Sap30 levels. The Genomics of Drug Sensitivity in Cancer, The Cancer Cell Line Encyclopedia, and CRISPR-Cas9 screens indicated that Sap30 essentiality is associated with Cisplatin resistance and further showed higher levels in Cisplatin resistant patient-derived xenograft tumor cell lines. The silencing of Sap30 in cells reduced cell growth, induced cell death, mitochondrial membrane potential loss in vitro, and reduced tumorigenicity in vivo. Altogether, these results indicate that Sap30 is a novel prognostic and Cisplatin resistant marker and thus a potential drug target in high-risk neuroblastoma patients.

Citation Format: Philip Prathipati, Anup S. Pathania, Nagendra K. Chaturvedi, Subash C. Gupta, Siddappa N. Byrareddy, Don W. Coulter, Kishore B. Challagundla. SAP30, a novel drug response specific transcription factor in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2349.
Abstract 2405: Expression of the low affinity neurotrophin receptor (p75NTR) correlates with perineural invasion in adenoid cystic carcinoma of the lacrimal gland
Authors: Moeyersoms Acadia H. , Gallo Ryan A. , Weiss Madison , Maeng Michelle M. , Tse David T. , Pelaez Daniel
Published: June 2022

Lacrimal gland adenoid cystic carcinoma (LGACC) is a rare but highly lethal indolent cancer originating in the cells of secretory glands, primarily of the head and neck. With the 10-year survival rate of 20%, the only life extending technique is to remove the eye and surrounding socket contents followed by extensive chemoradiation. Despite radical treatment, LGACC has a poorer prognosis than head and neck adenoid cystic carcinoma in part due to its propensity for perineural invasion (PNI) and intracranial spread. Analysis of LGACC tumors has revealed signatures enriched for pathways known to be involved in normal LG development, suggesting LGACC tumor cells re-engage or hijack developmental programs during its progression. This project sought to evaluate if developmental pathways involved in embryologic LG innervation were similarly recapitulated by LGACC tumors, and whether any of those markers could be correlated to perineural invasion phenotypes. We performed microarray analysis of embryonic and adult murine lacrimal glands and generated a differential expression signature associated with normal LG developmental pathways. Among other pathways, Notch and FGF signaling are significantly upregulated during LG organogenesis and our group and others have shown that activating mutations in these pathways are correlated with poor prognosis in LGACC. Interestingly, among the highest differentially expressed genes in the embryonic LG was the low-affinity nerve growth factor receptor (p75NTR). We thus subsequently evaluated the expression of p75NTR in LGACC tumors and its correlation to PNI. Our immunofluorescent imaging of histologically confirmed tumors with PNI demonstrated that p75NTR is highly correlated to cancer cells that have invaded the nerve. This correlation of p75NTR expression in nerve infiltrating LGACC cells can inform our investigation into the mechanisms for LGACC PNI and elucidate possible therapeutic targets to limit this aggressive manifestation in LGACC.

Citation Format: Acadia H. Moeyersoms, Ryan A. Gallo, Madison Weiss, Michelle M. Maeng, David T. Tse, Daniel Pelaez. Expression of the low affinity neurotrophin receptor (p75NTR) correlates with perineural invasion in adenoid cystic carcinoma of the lacrimal gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2405.
Abstract 2415: Loss of Krüppel-like factor 4 facilitates disruption of epithelial barrier function in gastric cancer and promotes metastasis
Authors: Goswami Sandeep , Sarkar Chandrani , Frankel Wendy L. , Basu Sujit , Chakroborty Debanjan
Published: June 2022

Stomach or gastric cancer (GC) is one of the most common malignancies observed in men and women worldwide. It is also a leading cause of cancer-related deaths. The disease is potentially curable when diagnosed early with survival records of more than 90%. This number, however, sharply declines to less than 20% when diagnosed at advanced stages. Therefore identification of functional molecular markers that can predict the progression of the disease is of utmost importance as early diagnosis enhances the chances of successful therapeutic intervention. Krüppel-like Factors (KLFs) are a family of evolutionarily conserved transcriptional regulators with zinc finger DNA binding domains. KLFs have been shown to play important roles both in normal development as well as in disease progression. In cancers, KLFs have been reported to regulate diverse cellular processes like cell growth, proliferation and differentiation. Of the 17 KLFs identified to date, KLF4 and KLF 5 show significantly altered expression in GC tissues compared to normal gastric tissues (TCGA sample analysis of gene expression pattern for KLFs). WhileKLF5 typically pro-proliferative and acts as a tumor promoter, KLF4 has been both oncogenic and tumor-suppressive. KLF4 downregulation is associated with a poor prognosis in GC. However, the specific role of KLF4 in GC progression has not been fully elucidated. Using human GC tissues, orthotropic gastric tumors developed in athymic nude mice, and in vitro functional assays, we hereby report that loss of KLF4 in GC disrupts tight junction stability of epithelial cells. Loss of KLF4 expression correlates with altered expression of tight junction proteins such as claudins and ZO1 in GC. Altered expression of these proteins results in disruption of epithelial barrier integrity leading to cell detachment and metastasis. Therefore, our work indicates that loss of KLF4 observed in GC may promote cell metastasis and can serve as a prognostic marker for GC metastasis. The knowledge generated from this study not only imparts insight into the pathogenesis of GC metastasis but also will be helpful in the development of newer treatment strategies in the future.

Citation Format: Sandeep Goswami, Chandrani Sarkar, Wendy L. Frankel, Sujit Basu, Debanjan Chakroborty. Loss of Krüppel-like factor 4 facilitates disruption of epithelial barrier function in gastric cancer and promotes metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2415.
Abstract 2438: Determining the role of non-mutated C-Raf kinase in metastatic disease
Authors: Ta Lisa H. , Carr-Ascher Janai R. , Deng Weixian , Tsai Brandon L. , Tran Wendy , Gun Donny , Cheng Donghui , Sha Jihui , Hwang Yeonjoo , Phillips John W. , Obusan Matthew B. , Bangayan Nathanael J. , Noguchi Miyako , Mao Zhiyuan , Chen Chia-Chun , Wang Liang , Varuzhanyan Grigor , Gordon John D. , Wohlschlegel James W. , Witte Owen N.
Published: June 2022

Many aggressive epithelial cancers do not carry targetable driver mutations (Mendiratta et al. 2021, NCI-MATCH Clinical trial NCT02465060). Commonly altered signaling pathways highlight the propensity for tumors to depend on such pathways for growth and survival where genomic alterations may not be required for pathway activation. The Raf family kinases are crucial mediators of the Ras/Raf/MEK/ERK (MAPK) cascade, which has been shown to be upregulated in a subset of metastatic cancers and are mutated in many epithelial cancers. A recent study suggests non-mutated C-Raf can drive metastasis to the lungs, bone, and various other tissues (Faltermeir et al. 2016). Though the mechanism of C-Raf driven metastasis is likely through MAPK activation, C-Raf has been demonstrated to possess MAPK independent roles that may also contribute to this phenotype. In this study, we explored various C-Raf functions as drivers of metastasis in an intracardiac mouse model system. Using a series of Raf knock-out cell lines and mutants targeting functional residues in C-Raf, we demonstrated that C-Raf dimerization is necessary to drive metastasis. We further showed that the metastasis-promoting activity of C-Raf is dependent on co-expression of its family member, B-Raf, for an accelerated metastatic phenotype. However, overexpression of a kinase-dead C-Raf mutant was still able to produce metastases albeit with lower efficiency, suggesting C-Raf possesses non-kinase dependent functions that also contributed to metastasis. Together, these results point to the importance of Raf non-canonical roles in oncogenic processes that may be unappreciated in metastatic disease.

Citation Format: Lisa H. Ta, Janai R. Carr-Ascher, Weixian Deng, Brandon L. Tsai, Wendy Tran, Donny Gun, Donghui Cheng, Jihui Sha, Yeonjoo Hwang, John W. Phillips, Matthew B. Obusan, Nathanael J. Bangayan, Miyako Noguchi, Zhiyuan Mao, Chia-Chun Chen, Liang Wang, Grigor Varuzhanyan, John D. Gordon, James W. Wohlschlegel, Owen N. Witte. Determining the role of non-mutated C-Raf kinase in metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2438.
Abstract 2440: Investigating the role of Eya3 in the regulation of NF-kB signaling and tumor progression in TNBC
Authors: Hughes Connor J. , Mallo Rebecca , Fields Kaiah , Zhou Hengbo , Gustafson Annika , Kong Deguang , Slansky Jill , Zhao Rui , Ford Heide L.
Published: June 2022

Triple Negative Breast Cancer (TNBC), characterized by low/absent expression of the estrogen receptor and the HER2 growth factor receptor, has high rates of metastasis and an overall poor prognosis largely due to a lack of targeted therapeutic options. The Eyes absent (Eya) family of proteins are transcriptional cofactors with both intrinsic tyrosine phosphatase and associated serine/threonine phosphatase activities. In addition to developmental functions, Eyas have been shown to play a role in promoting many hallmarks of cancer. Published work from our laboratory demonstrated that Eya3 is capable of upregulating expression of PD-L1 in TNBC tumors thereby suppressing anti-tumor CD8+ T-cell responses, and preliminary data suggests that Eya3 may also regulate the expression of various cytokines in this context. In tumor cells, innate immune signaling pathways, such as NF-kB, have been shown to promote tumor progression, immune evasion, and metastasis in many distinct tumor types. We hypothesize that, in addition to regulating CD8+ T-cell responses, Eya3 regulates innate immune signaling in TNBC cells to facilitate enhanced primary tumor growth and metastasis. Preliminary findings suggest that Eya3 is a key mediator of tumor progression and metastasis in TNBC and a strong regulator of NF-kB signaling in this context. Further exploration is warranted to determine the mechanism through which Eya3 regulates these phenotypes, whether Eya3-mediated NF-kB activation is a major contributor to the pro-metastatic phenotype of Eya3, and whether Eya3 could serve as a therapeutic target for preventing or treating disseminated TNBC.

Citation Format: Connor J. Hughes, Rebecca Mallo, Kaiah Fields, Hengbo Zhou, Annika Gustafson, Deguang Kong, Jill Slansky, Rui Zhao, Heide L. Ford. Investigating the role of Eya3 in the regulation of NF-kB signaling and tumor progression in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2440.
Abstract 2456: Mass spectrometry imaging reveals distinct differences in glycogen accumulation in lung tumors from Appalachian patients
Authors: Buoncristiani Michael D. , Conroy Lindsey R. , Clarke Harrison , Young Lyndsay E. , Sun Ramon C.
Published: June 2022

Lung cancer is the leading cause of cancer related death in the United States, with patients from rural Appalachia having drastically higher incidence and mortality rates, driven by largely unknown mechanisms. Identifying molecular features that contribute to this health disparity is a critical step in lung cancer research that could lead to predictive biomarkers and personalized therapy for the Appalachia population. Recently, aberrant glycogen accumulation in lung tumors has been reported to promote lung cancer progression. However, the full clinical implications of dysregulated glycogen metabolism and its role in lung cancer health disparities has yet to be explored. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a new and innovative technique that combines mass spectrometry with high resolution imaging, enabling the detection of hundreds of analytes with spatial distribution. We recently developed an advanced method to analyze glycogen content in formalin-fixed paraffin-embedded tissues using MALDI-MSI. With this technique, we analyzed glycogen chain length and glycogen phosphate content from tissue microarrays containing over 100 lung cancer patient samples banked at the University of Kentucky. Our patient cohort included adenocarcinoma and squamous cell carcinoma patients from Appalachian and non-Appalachian counties, with diverse cytopathological features. We found significant glycogen accumulation and higher glycogen phosphate content in lung tumor tissue compared to match normal lung across all patients. Further, we found glycogen accumulation is markedly high in adenocarcinoma patients compared to other lung cancer subtypes. Interestingly, Appalachian adenocarcinoma patients exhibit increased glycogen accumulation and higher glycogen phosphate content than patients from non-Appalachian counties. These data suggest differential glycogen accumulation is a unique feature of lung tumors from Appalachian adenocarcinoma patients. Our study highlights the potential clinical applications of MALDI-MSI for digital pathology, biomarker applications, and reveals molecular features that contribute to the health disparity in Appalachian lung cancer patients that warrant further molecular analysis.

Citation Format: Michael D. Buoncristiani, Lindsey R. Conroy, Harrison Clarke, Lyndsay E. Young, Ramon C. Sun. Mass spectrometry imaging reveals distinct differences in glycogen accumulation in lung tumors from Appalachian patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2456.
Abstract 247: Targeting Notch4 with a novel neutralizing antibody inhibits tumor growth and alters immune cell populations within the tumor microenvironment
Authors: Eng Jason , Naiche L.A. , Swaminathan Bhairavi , Kato Yu , Thakkar Krishna , Vadakath Rahul , Herts James , Emmadi Rajyasree , Matsui Junji , Kitajewski Jan
Published: June 2022

Endothelial Notch signaling is critical for tumor angiogenesis and growth. Inhibition of Notch1 or its ligand, Dll4, can induce tumor vessel hypersprouting, which interferes with perfusion and reduces tumor development. Unfortunately, blocking Notch1 in endothelial cells of normal tissue can lead to tissue hypoxia and vascular neoplasms. Notch4, another member of the Notch family, is upregulated and increased in the endothelium of certain tumor types compared to normal vasculature. Reducing Notch4, unlike Notch1, causes reduction in developmental and tumor angiogenesis. Using a newly developed anti-Notch4 antibody, E7011, we assessed the effects on Notch4 inhibition on developmental retinal angiogenesis. E7011 treatment did not alter capillary density within the vascular plexus or endothelial tip cell number, but did decrease radial outgrowth and vein diameter, supporting the hypothesis that Notch4 functions as a pro-angiogenic signal. These findings suggest that the targeting of Notch4 could interfere with tumor angiogenesis and offer a novel therapeutic avenue. To establish the role of Notch4 within the tumor microenvironment, we analyzed its expression in both human and murine tumor samples. Immunofluorescent staining revealed a range of Notch4 expression in both tumor cells and endothelium in human triple negative breast cancers. Several murine syngeneic tumors examined expressed Notch4 primarily in the endothelium with only rare tumor expression. We treated orthotopically implanted Py8119 murine breast carcinomas and B16F10 melanomas with E7011, which significantly reduced tumor size compared to IgG treated controls, indicating that suppression of Notch4 is anti-tumorigenic. To determine mechanistically how E7011 delays tumor growth, we performed single cell RNA sequencing on E7011 and control treated Py8119 tumors. Surprisingly, we found that while Notch4 transcripts were present only in endothelial cells, E7011 administration dramatically altered macrophage and monocyte populations within the tumor. Taken together, these findings indicate that inhibition of tumor endothelial Notch4 by E7011 can regulate the presence of immune cells within the tumor microenvironment and improve tumor control.

Citation Format: Jason Eng, L.A. Naiche, Bhairavi Swaminathan, Yu Kato, Krishna Thakkar, Rahul Vadakath, James Herts, Rajyasree Emmadi, Junji Matsui, Jan Kitajewski. Targeting Notch4 with a novel neutralizing antibody inhibits tumor growth and alters immune cell populations within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 247.
Abstract 2487: Distinct venous brain vessels provide structures for T lymphocyte recruitment to brain tumors in mouse models of intracranial melanoma
Authors: Messmer Julia M. , Piechutta Manuel , Venkataramani Varun , Mayer Chanté , Berghoff Anna S. , Hinze Daniel , Hölzel Michael , Helfrich Iris , Schadendorf Dirk , Wick Wolfgang , Karreman Matthia A. , Winkler Frank
Published: June 2022

Immune checkpoint therapy has demonstrated potential for the treatment of brain metastases, but the mechanisms of lymphocyte recruitment to brain tumors are poorly understood. To monitor T cell infiltration and brain tumor growth in vivo in real time, we performed intravital microscopy of adoptively transferred and endogenous T cells in mouse models of intracranial melanoma. By spatio-temporal analysis of T cell infiltration dynamics in brain tumors we could identify a distinct type of venous blood vessels in the tumor vicinity, “peritumoral venous vessels” (PVVs). Intraluminal T cell attachment and T cell extravasation was increased at PVVs compared to intratumoral vessels. Moreover, PVVs were characterized by high perivascular T cell density, high T cell motility parameters and high expression of ICAM-1. PVVs were specifically exploited for the recruitment of T cells to melanoma brain tumors, whereas alternative T cell routes via brain structures such as brain capillaries, intratumoral blood vessels and parasagittal regions were not observed. Immune checkpoint inhibition increased T cell motility in the brain, facilitating the advance from PVV to tumor, which resulted in intracranial tumor growth inhibition. Our findings describe a distinct mechanism by which the immune system targets melanoma brain tumors, and, potentially, other intracranial pathologies.

Citation Format: Julia M. Messmer, Manuel Piechutta, Varun Venkataramani, Chanté Mayer, Anna S. Berghoff, Daniel Hinze, Michael Hölzel, Iris Helfrich, Dirk Schadendorf, Wolfgang Wick, Matthia A. Karreman, Frank Winkler. Distinct venous brain vessels provide structures for T lymphocyte recruitment to brain tumors in mouse models of intracranial melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2487.
Abstract 2488: PET imaging of innate immunity activation In Vivo with redox-tuned 4-[18F]fluoro-1-naphthol
Authors: Gammon Seth T. , Pisaneschi Federica , Paolillo Vincenzo , Qureshy Sarah , Piwnica-Worms David
Published: June 2022

High redox potential reactive oxygen and nitrogen species (ROS/RNS), such as O2 free radicals, superoxide, and hypochlorous acid, generated by activities of the NADPH oxidase-2 (NOX2)/myeloperoxidase (MPO) axis and related enzymes, are key effector molecules of innate immunity in physiological and diseased inflammatory states. Other lower energy species (H2O2, NO) provide adjuvant signaling functions. NOX2- and MPO-derived high energy radicals are known to oxidize naphthol species, wherein the naphthol products bind to proximate proteins and activated myeloid cells. Herein, we present 4-[18F]fluoro-1-naphthol ([18F]4FN), a novel redox-tuned radiopharmaceutical that selectively detects by positron emission tomography (PET) high energy radicals produced by activated innate immunity. The products of human MPO plus H2O2, but not H2O2 alone, rapidly and completely oxidized [18F]4FN. All-trans-retinoic acid-differentiated HL-60 "neutrophil-like" human cells activated with phorbol-12-myristate-13-acetate (PMA) retained [18F]4FN 5-fold over unstimulated cells. 4-ABAH, an MPO-specific inhibitor, or DPI, a broad oxidase inhibitor, blocked cellular retention by >95%. [18F]4FN PET/CT imaging readily discriminated foci of inflammation in vivo in three distinct murine models of acute inflammation: endotoxin-induced whole-body toxic shock, PMA-induced mild contact dermatitis of the ear, and lipopolysaccharide (LPS)-induced ankle arthritis. Mechanistically, in mice in vivo, 4-ABAH reduced inflammation-induced [18F]4FN retention, and Cybb-/- (Nox2-/-) gene-deletion strongly and significantly abrogated PMA-induced [18F]4FN retention. Thus, [18F]4FN shows promise as a robust redox-tuned reporter for imaging activation states of innate immunity by PET/CT, is ready for translation. [18F]4FN PET imaging may find application in a variety of inflammatory states associated with cancer therapy, immunotherapy-related adverse events, as well as other diseases, including arthritis, hepatitis, atherosclerosis, COVID-19, as well as up-staging and monitoring multi-organ inflammation.

Citation Format: Seth T. Gammon, Federica Pisaneschi, Vincenzo Paolillo, Sarah Qureshy, David Piwnica-Worms. PET imaging of innate immunity activation In Vivo with redox-tuned 4-[18F]fluoro-1-naphthol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2488.
Abstract 2495: Clinical and demographic predictors of survival for fibrolamellar hepatocellular carcinoma patients - A patient-community registry-based study
Authors: Berkovitz Amichai , Migler Rachael D. , Qureshi Adam , Vaughan Roger , Marcotte Erin L. , Simon Sanford M.
Published: June 2022

Fibrolamellar Carcinoma (FLC), a rare liver cancer, has yet no specific tools to evaluate its prognosis. This study aims, in partnership with the patient community, to search for associations between various demographic and clinical factors and overall survival of patients, to assess prognostic factors. We performed a retrospective analysis on a community-based patient registry (The Fibrolamellar Registry http://fibroregistry.org) to examine relations between survival and different variables. The variables examined were demographic (gender), clinical (age at diagnosis, tumor size, number of tumors inside the liver, presence of positive lymph nodes or distant metastasis) as well as treatment modalities (surgery or systemic therapy). Kaplan-Meier survival plots were created for the overall research population and for stratifying by unadjusted variables. Cox proportional hazards model was used for multivariable analysis of hazard ratio (HR) for death at any time among groups, adjusting for variables found significant in the univariate analysis. Among 150 patients, (89 females and 61 males), median overall survival time from diagnosis to death or loss of follow-up was 8 years and 2 months. Features with independent positive effect on survival were female gender, as well as being treated with surgery alone. In contrast, having 10 or more tumors inside the liver had a negative effect on survival. Positive lymph nodes at diagnosis, and distant metastasis at diagnosis, were both associated with poorer prognosis, though did not remain independent when adjusting for covariables. We could not find an effect of age at diagnosis or tumor size at its largest measurement, on survival. In conclusion, our study suggests gender is a significant prognostic factor for FLC patients, with females having a better outcome than males. Surgical removal of the tumor, without additional systemic therapy is a positive prognostic factor as well. Having a large number of tumors of 10 and above, despite all being inside the liver, is a negative prognostic factor.

Citation Format: Amichai Berkovitz, Rachael D. Migler, Adam Qureshi, Roger Vaughan, Erin L. Marcotte, Sanford M. Simon. Clinical and demographic predictors of survival for fibrolamellar hepatocellular carcinoma patients - A patient-community registry-based study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2495.
Abstract 2499: Identifying DNA methylation signatures of retinoblastoma via aqueous humor, a novel ocular liquid biopsy
Authors: Xu Liya , Li Hongtao , Weisenberger Dan , Liang Gangning , Berry Jesse
Published: June 2022

Introduction: Retinoblastoma (Rb) is the most common childhood intraocular cancer. Tissue biopsy of Rb can cause tumor spread, so it is contraindicated. We demonstrated that aqueous humor (AH), an ocular fluid, is a high yield liquid biopsy enabling in vivo detection of tumor-derived cell free DNA (cfDNA) thus overcoming the contraindication to biopsy. Prognostic genomic cfDNA biomarkers in Rb AH enables objective real time disease monitoring.

In ~13% of Rb, tumor progression is driven by epigenetic deregulation of tumor-promoting pathways without detectable genomic alterations. However, epigenetic studies have been done only on tumor from surgically removed eyes. The frequency and effect of epigenomic regulation in eyes that have been saved with therapy is not clear due to no access to in vivo tumor. Therefore, epigenetic analysis of AH cfDNA is highly desired to understand the broader spectrum of Rb tumorigenesis and prognosis.

Materials and Methods: 16 AH samples and 4 Rb tumors from 12 patients with 14 Rb eyes were included in the study. We conducted global DNA methylation profiling of tumor tissues from surgically removed Rb eyes, AH samples collected from different clinical stages with different treatment outcomes using the Illumina Infinium EPIC DNA methylation BeadArray platform. Publicly available DNA methylation data of normal retina, Rb eyes, and Rb patients were obtained from Gene Expression Omnibus (GEO, GSE57362) for cell type DNA methylation comparisons.

Results: Our preliminary studies revealed a high degree of concordance in genome-wide differential DNA methylation patterns between paired AH and tumor samples. Integrating our data with large public datasets, we identified reliable RB DNA methylation signatures in cfDNA that have potential diagnostic and prognostic values. We also identified hypermethylation at RB1 promoter, which suggested RB1 is druggable to DNA methylation inhibitors. By integrating DNA methylation data with gene expression data, we identified over 300 differentially expressed genes potentially regulated by DNA methylation change. Pathway analysis indicated several tumor suppressor pathways (e.g., RB1 and P53 pathways) were suppressed and some oncogenic pathways were activated (e.g., E2F pathways).

Conclusions: Our study sets the stage for exploiting epigenetic markers in AH and identifying potential therapeutic targets to improve the clinical management of patients with RB.

Citation Format: Liya Xu, Hongtao Li, Dan Weisenberger, Gangning Liang, Jesse Berry. Identifying DNA methylation signatures of retinoblastoma via aqueous humor, a novel ocular liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2499.
Abstract 2504: Next generation sequencing identifies novel potential biomarkers for DICER1 syndrome
Authors: Wu Mona K. , Lopez Raphael D. , Warren Mikako , Shah Rachana , Neviani Paolo , Amatruda James F.
Published: June 2022

MicroRNAs (miRNAs) are small RNA molecules that play a critical role in regulating gene expression. The DICER1 enzyme is a key component of the microRNA biogenesis pathway. Reflecting this central role, patients who inherit or acquire mutations in the DICER1 gene have an increased risk of developing a spectrum of benign and malignant tumors —thereby earning the recognition as a pediatric cancer predisposition syndrome known as DICER1 syndrome (OMIM 601200). DICER1 syndrome is definitively diagnosed by DNA sequencing demonstrating presence of a loss of function mutation on one copy of DICER1 plus a “hotspot” mutation on the other copy of DICER1. As tumor and germline DNA sequencing can pose logistical and/or cost difficulties, a complementary diagnostic method based on marker expression is valuable for both diagnostics and surveillance. Biomarkers that are candidate therapeutic targets are also appealing as these may offer a more tailored approach for therapy. As there are no approved biomarkers for identification of DICER1-associated tumors, we performed RNAseq and miRNAseq in an engineered murine mesenchymal cell model that incorporates a specific DICER1 hotspot mutation to identify candidates. We validated candidate biomarkers at the RNA (Eya2, Vipr1, miR-139-3p, and miR-30d-3p) and protein (Eya2, and Vipr1) levels in vitro, to provide proof of principle data for their eventual clinical utility. The miRNA candidate biomarkers levels were evaluated in both total and small extracellular vesicle-enriched fractions. These novel data pave the way for additional tools for diagnosis and surveillance in patients with DICER1 syndrome.

Citation Format: Mona K. Wu, Raphael D. Lopez, Mikako Warren, Rachana Shah, Paolo Neviani, James F. Amatruda. Next generation sequencing identifies novel potential biomarkers for DICER1 syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2504.
Abstract 2505: Predicting prognosis in PFA ependymoma and group 3/4 medulloblastoma methylomes using interpretable epigenetic models
Authors: Ng Indy , Fricke Alexander , Pai Shraddha
Published: June 2022

Introduction: Our goal is to develop interpretable, prognostic models for Group 3/4 medulloblastoma (G3/4 MB) and PFA ependymomas (PFA-EP) using brain tumor DNA methylomes. Our models use prior knowledge of genome regulation and may identify cellular processes that help develop molecular therapies. We previously developed a classifier algorithm, netDx, which uses similarity networks to integrate heterogeneous patient data and predict outcome1. netDx demonstrates excellent performance and allows measures to be grouped into biologically meaningful features (e.g. grouping genes into pathway features).

Ependymoma Methods: We predicted survival in PFA-EP using 569 methylomes2 (Illumina 450K). Using netDx, we evaluated a model that grouped CpG-level methylation into sets reflecting 25 cell types from the developing human cerebellum, 3 chromatin states, EZH2 binding sites, and brain super enhancers (80:20 train/test split, feature selection >=8/10; 10 splits). This model was compared to a baseline lacking prior knowledge.

Preliminary Results: Organizing methylomes by prior knowledge significantly improves prognostic prediction (Table 1, p < 5x10-3, one-sided WMW). Features that predict prognosis are consistent with known dysregulation in PFA-EP2 (Table 1).

G3/4 MB Methods: We predicted binarized survival using 285 methylomes3 (Illumina 450K), using identical methods as above.

Preliminary results: Tumor methylomes carry predictive signal for survival prediction (Table 1), consistent with previous findings. We are currently evaluating the effect of including prior knowledge.

Interim Conclusion: Prior knowledge can improve survival prediction in PFA-EP and identifies features reflecting tumor biology. We are interested in extending interpretable modeling to other tumours.1. Pai et al. (2019) Mol Sys Biol. 15. 2. Pajtler et al. (2018) Acta Neuropathol. 136. 3. Northcott et al. (2017) Nature 547.

Table 1. Average model performance and top features. Dataset Predictor design AUPR (mean+/- SD, 10 train/test splits) Predictive features PFA-EP; 252 good/317 poor survivors2 DNAm, no prior knowledge 64.2 +/- 2.3 n/a * Covariates: CXOrf67 mutation status, sex DNAm, prior knowledge* 67.5 +/- 3.5(p < 5x10-3; one-sided WMW test compared to baseline model) CXOrf67mut, methylation in: {H3K9me3 sites; marker genes for ependymal cells of choroid plexus; marker genes for multiple interneuron classes} G3/4 MB: 146 good/139 poor survivors3 DNAm, no prior knowledge 0.61 +/- 6.3 n/a ** Covariates: age, sex DNAm, prior knowledge** In progress

Citation Format: Indy Ng, Alexander Fricke, Shraddha Pai. Predicting prognosis in PFA ependymoma and group 3/4 medulloblastoma methylomes using interpretable epigenetic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2505.
Abstract 2513: MDSC-derived TNF is a novel regulator of T-cell dysfunction in pancreatic cancer
Authors: Bianchi Anna , De Castro Silva Iago , Deshpande Nilesh U. , Mehra Siddharth , Singh Samara , Dosch Austin R. , Garrido Vanessa T. , Rafie Christine I. , Nagathihalli Nagaraj , Merchant Nipun , Datta Jashodeep
Published: June 2022

Introduction: Abundance of myeloid-derived suppressor cells (MDSC) and a dysfunctional T-cell compartment are defining hallmarks of therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC). Using congenic in vivo murine models to phenocopy extremes of T-cell enrichment or exclusion, we sought to interrogate central MDSC-mediated mechanisms that govern immune tolerance in PDAC.

Methods: Orthotopically implanted T-cell-excluded (Tcelllo) vs T-cell-enriched (T-cellhi) congenic KPC tumors, and intratumoral Ly6G+F4/80- MDSCs from both clones, were subjected to RNA sequencing. Ex vivo co-cultures evaluated the effects of intratumoral MDSC on splenic T-cells. Orthotopically injected KPC-T-celllo mice were treated with etanercept vs. vehicle, and immunophenotyping via flow cytometry was performed.

Results: RNA-seq of KPC T-celllo vs. T-cellhi tumors revealed enrichment of myeloid immunoregulatory pathways, and downregulation of leukocyte activation/cytotoxicity pathways. Flow cytometry revealed a dramatic increase in MDSCs infiltrating KPC-Tcelllo tumors (P<0.001). To decipher MDSC-intrinsic mechanisms associated with T-cell exclusion, RNA-seq of MDSCs infiltrating T-cellhi clones revealed relative downregulation of MAPK signaling, and cytokine profiling of MDSCs conditioned with MAPK inhibitor trametinib revealed marked reduction in TNF secretion. Confocal microscopy confirmed striking decrease in TNF in MDSCs isolated from KPC- T-cellhi vs. Tcelllo tumors. Ex vivo MDSC-T-cell co-cultures significantly attenuated T-cell proliferation and activation (via IFN-γ release) while favoring T-cell apoptosis, which could be rescued by pre-conditioning MDSCs with either etanercept (TNFR2 decoy receptor) or MAPK pathway inhibitors. Orthotopically injected KPC-T-celllo tumor-bearing mice treated with etanercept demonstrated a remodeled TME vs. vehicle-treated mice, with attenuation in MDSC trafficking, enrichment in CD4+/CD8+ T-cell infiltration, and reduction in T-cell exhaustion.

Conclusion: MDSC-derived TNF regulates T-cell dysfunction in PDAC via a MAPK-dependent mechanism. Compartment-specific inhibition of TNF may be a provocative strategy to overcome immune tolerance in PDAC.

Citation Format: Anna Bianchi, Iago De Castro Silva, Nilesh U. Deshpande, Siddharth Mehra, Samara Singh, Austin R. Dosch, Vanessa T. Garrido, Christine I. Rafie, Nagaraj Nagathihalli, Nipun Merchant, Jashodeep Datta. MDSC-derived TNF is a novel regulator of T-cell dysfunction in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2513.
Abstract 2514: Pancreatic cancer subtype-specific secreted factors determine the immunosuppressive tumor microenvironment
Authors: Bärthel Stefanie , Falcomatà Chiara , Widholz Sebastian A. , Toska Albulena , Boniolo Fabio , Schmitt Constantin , Gölling Vanessa , Swietlik Jonathan , Mir Jonas , Jesinghaus Moritz , Meissner Felix , Rad Roland , Schmidt-Supprian Marc , Saur Dieter
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is a complex disease displaying genetic heterogeneity along with a tumor microenvironment (TME) unique in its composition. Whereas the PDAC TME is known to be highly immunosuppressive, it remains elusive how distinct genetic alterations affect TME cell type recruitment and how this, in turn, influences progression and treatment outcome. Therefore, we systematically investigated the TME composition and tumor-immune crosstalk in molecular PDAC subtypes. We delineated the TME landscape of a comprehensive tumor cohort derived from Kras-driven PDAC mouse models by integrating single cell RNA-sequencing (scRNA-seq) with histopathological analysis, tissue RNA-seq followed by deconvolution and flow cytometry immunophenotyping. Our results show that distinct molecular PDAC subtypes recruit divergent subpopulations of infiltrating immune cells, most notably evidenced by major differences in frequency and phenotype of macrophages and neutrophils. Functional secretome analysis of well-characterized Kras-driven mouse and primary human PDAC cell lines revealed subtype-specific factors which differ between the two main transcriptional subtypes of PDAC (classical and mesenchymal). Accordingly, crosstalk between tumor and immune cells inferred by interaction probability analysis through ligand-receptor pairs from scRNA-seq data nominates the CSF signaling network as a key player in mesenchymal PDAC, whereas CXCL signal hubs mediate higher infiltration of immunosuppressive neutrophils in classical tumors. Our findings contribute to the understanding of PDAC subtype-specific biology and aid the identification of potential immunotherapeutic vulnerabilities thereof.

Citation Format: Stefanie Bärthel, Chiara Falcomatà, Sebastian A. Widholz, Albulena Toska, Fabio Boniolo, Constantin Schmitt, Vanessa Gölling, Jonathan Swietlik, Jonas Mir, Moritz Jesinghaus, Felix Meissner, Roland Rad, Marc Schmidt-Supprian, Dieter Saur. Pancreatic cancer subtype-specific secreted factors determine the immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2514.