bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3030: Integrated in vitro and in silico screening of breast cancer reveals serine metabolism as a precision oncology target
Authors: Slayton Mark Daniel , Achreja Abhinav , Jeon Jin Heon , Bao Liwei , Liu Alisa , Collard Mason , Nagrath Deepak , Merajver Sofia
Published: June 2022

Cellular metabolism sits at the forefront of growth and has been identified as an essential element for the progression of cancer. Large-scale chromosomal alterations and mutations incurred by cancerous cells can predispose cancer cells to dysfunctional metabolic programming independent of microenvironmental pressure. Cancer cells are adaptable to this reprogramming and find alternative measures to sustain elevated metabolic demands. However, this process reduces the number of functional metabolic pathways. Dysregulated metabolic pathways within cancerous cells create an opportunity for therapeutic intervention by targeting genetic chokepoints within these pathways. We analyzed metabolic landscape of breast cancers by combining genomic and transcriptomic data from cancer patient datasets, to identify metabolic pathways prone to dysregulation due to genomic influence. Our platform identified disruption in the serine biosynthesis, which some cancerous cells rely upon to sustain elevated growth in a subset of breast carcinoma. We utilized gain- and loss-of-function techniques in multiple breast cancer cell lines to recapitulate metabolic disruption observed in tumors. These in vitro screens are complemented by metabolic flux analysis and stable-isotope tracer analysis that reveal the vulnerable metabolic pathways in breast cancers with dysregulated serine metabolism. Our data demonstrate that specific metabolic targets can be identified through this integrated screening and validated empirically to confirm their significance as avenues for potential therapeutics.

Citation Format: Mark Daniel Slayton, Abhinav Achreja, Jin Heon Jeon, Liwei Bao, Alisa Liu, Mason Collard, Deepak Nagrath, Sofia Merajver. Integrated in vitro and in silico screening of breast cancer reveals serine metabolism as a precision oncology target [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 3030.
Abstract 3055: Field cancerization and microbiome in lung cancer
Authors: Alhammad Rayan I. , Vuong Ngoc B. , Zhou Weidong , Mueller Claudius , Johann Donald J , Katta Amith R. , Hoek Monique V. , Alsaab Fahad M. , Luchini Alessandra , Liotta Lance
Published: June 2022

Lung Cancer carcinogenesis is the outcome of a field of premalignant changes that occur in the bronchial tree leading to the overt emergence of the malignant lung cancer. We sought to study the proteome of the microbiome associated with the premalignant bronchial mucosa epithelia. The overall goal is the identify functional relationships between the bronchial mucosa microbiome and the risk of developing lung cancer. We evaluated the proteome of the microbiome using mass spectrometry and specialized microbiome authentication software. This method was applied to a case-control study of plasma samples donors of physician health study who donated plasma at different times prior to the onset of Non-Small Cell Lung Cancer. We also examined bronchial mucosa tissue samples near to, and distant from, the tumor harvested from surgical lobectomy specimens. Highly significant protein biomarker differences were found in the plasma and in the tissue samples that were blindly associated with cancer. These included specific proteins from the mucosal microbiome that may have functional role in the carcinogenic process. These key diagnostic risk markers were shared between the premalignant bronchial mucosa tissue and the plasma, supporting the concept that risk markers of bronchial mucosa lung cancer carcinogenesis can be detected in the peripheral circulation.

Citation Format: Rayan I. Alhammad, Ngoc B. Vuong, Weidong Zhou, Claudius Mueller, Donald J Johann Jr, Amith R. Katta, Monique V. Hoek, Fahad M. Alsaab, Alessandra Luchini, Lance Liotta. Field cancerization and microbiome in lung 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 3055.
Abstract 3066: Functional modeling and characterization of high frequency mutations associated with gastric cancer in US racial and ethnic minority populations
Authors: Arana Alexa Morales , Halmai Nicole B. , Zhang Hongyong , Lott Paul , Estrada-Florez Ana , Toal Ted W. ,   University of California Minority Patient-DerivedXenograft (PDX) Development and Trial (UCaMP) Cent, Carvajal-Carmona Luis G.
Published: June 2022

Gastric cancer (GC) is the 5th most common neoplasm and the 2nd most deadly cancer worldwide, with an estimated 783,000 deaths in 2018. Furthermore, Hispanic/Latino Americans continue to be underrepresented in GC genomic studies despite being twice as likely to be diagnosed with and die from GC compared to non-Hispanic whites (NHW). In order to address this pervasive health disparity, our lab has led efforts to characterize tumor biopsies from racial/ethnic minority cancer patients to inform more effective cancer treatment and advance precision medicine among these high-risk populations. We performed whole-exome sequencing of 31 GC patient tumors with more than 60% of the samples coming from minority patients. We identified higher somatic mutation frequencies in the ARID1A, BRCA2, and APC genes compared with the TCGA GC cohort, which is made up of mostly NHW patients. To better understand the role these genes play in cancer tumorigenesis among racial/ethnic minority GC patients, our lab has established a CRISPR/Cas9 genome-editing platform in minority patient-derived organoids (mPDOs). In this study, we utilized our platform to generate mPDO models of APC-TP53 double knockouts (dKO), ARID1A-TP53 dKO, and TP53-ARID1A-BRCA2 triple knockouts in organoid lines derived from normal gastric tissue. We developed medium to select for functional mutants following organoid cell electroporation that yielded near-complete selection for the desired gene knockouts, allowing us to generate isogenic mPDO lines modeling each gene KO combination. Our organoid models will provide a critical method to elucidate therapeutic sensitivity and resistance mechanisms in minority groups towards the ultimate goal of addressing GC health disparities in US racial/ethnic minorities.

Citation Format: Alexa Morales Arana, Nicole B. Halmai, Hongyong Zhang, Paul Lott, Ana Estrada-Florez, Ted W. Toal, University of California Minority Patient-DerivedXenograft (PDX) Development and Trial (UCaMP) Cent, Luis G. Carvajal-Carmona. Functional modeling and characterization of high frequency mutations associated with gastric cancer in US racial and ethnic minority populations [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 3066.
Abstract 3068: Identify targetable molecular drivers of chemotherapy resistance in gastroesophageal adenocarcinoma
Authors: Kong Mingyang , Bailey Swneke , Sangwan Veena , Ferri Lorenzo
Published: June 2022

Introduction: Gastroesophageal adenocarcinoma (GEA) is one of the most lethal malignancies worldwide. Even with the standard-of-care chemotherapy, 40% of the patients are innately resistant and half of the initial responders develop acquired resistance. The molecular mechanisms underlying the development of resistance in GEA remain largely unknown.

Methods: 30 GEA patients who received chemotherapy were included in this cohort. Both pre-and post-treatment primary tissues were collected to generate patient-derived organoids (PDOs). 29 PDOs were treated with chemotherapeutic agents and the cell viability was measured. We also evaluated the correlation between ex-vivo drug responses of PDOs and in-patient clinical responses. Data obtained from whole-exome (WES) and RNA sequencing of the primary tumor and their PDOs were analyzed to identify targetable molecules associated with chemo-resistance.

Results: After treatment, the cell viability of the PDOs obtained from chemo-resistant patients was significantly higher than that of chemo-sensitive patients. The variant allele frequency of the PDOs recapitulated their parental tumors. WES of one patient tumor revealed an increased copy number of EGFR, and their PDOs showed high sensitivity to an EGFR inhibitor in the subsequent drug screens. Furthermore, 40% of chemo-sensitive patients had mutations in one or more genes belonging to the adhesion G protein-coupled receptors (GPCRs) family. The adenylate cyclase-activating G protein-coupled receptor signaling pathway was found to be significantly activated only in chemo-sensitive patients. Single-cell RNA sequencing of pre-and post-treatment tissue revealed a broadly shifted transcriptome in epithelial cells. Patients with upregulated cell stemness genes including CTNNB1, MYC, ETS2, and KLF4 had disease recurrence 6 months post-surgery, while patients with unaltered or downregulated stemness genes remain disease-free 18 months until today.

Conclusion: Our PDOs not only reflected the genetic profile of primary tumors but also recapitulated patient responses to chemotherapy. Genomic analysis revealed that mutations in a few GPCR genes might have a role in increased sensitivity to chemotherapy. In addition, the transcriptomic analysis showed stemness genes were associated with disease recurrence. Our GEA PDO platform allows the identification and the subsequent verification of resistant-conferring targets, providing insight for novel therapeutic combinations.

Funding: This project is supported by the Cancer Research Society (CCS), and the Department of Defence (DoD), USA.

Citation Format: Mingyang Kong, Swneke Bailey, Veena Sangwan, Lorenzo Ferri. Identify targetable molecular drivers of chemotherapy resistance in gastroesophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3068.
Abstract 3095: Breaking the crosstalk of the cellular tumorigenic network in NSCLC by a highly effective drug combination
Authors: Gürgen Dennis , Conrad Theresia , Becker Michael , Sebens Susanne , Röcken Christoph , Hoffmann Jens , Langhammer Stefan
Published: June 2022

Background: Non-small cell lung cancer (NSCLC) is commonly diagnosed at advanced stages with limited treatment options particularly in tumors without targetable driver mutations.

Methods: Considering previously published criteria for selecting drug combinations for overcoming drug resistances1, we have established a low dose combination therapy with cabozantinib, afatinib, etoricoxib and plerixafor. Utilizing NSCLC patient-derived xenograft (PDX) models we aimed to use this drug regimen to disrupt paracrine substitutional signaling within the tumorigenic network in order to circumvent pre-existing and adaptive drug resistances. In contrast to daily dosing for respective monotherapies the low dose treatment was paused every five days for two days.

Result: All PDX tumors treated, including highly therapy-resistant adeno- and squamous cell carcinomas without identifiable driver mutations, were completely suppressed by this drug regimen leading to an Objective Response Rate of 81% and a Clinical Benefit Rate of 100%. After treatment discontinuation growth suppression remained until the end of the follow-up period with only slight increase in tumor growth. The application and the safety profile of this low dose therapy regimen was well manageable and without pronounced side effects in this pre-clinical setting.

Discussion: For the first time we show that simultaneous inhibition of pathways forming the cellular tumorigenic network in NSCLC tumors is highly effective and overcomes drug resistances for monotherapies. All of the drugs used in this combination regimen are approved for different indications and therefore readily available. Conclusion: Our results emphatically encourage a setup for a clinical study in advanced stage NSCLC patients without targetable driver mutations combining the drugs cabozantinib, afatinib, plerixafor and etoricoxib in a low dose treatment regimen as novel therapy strategy.

Indication of Source: 1. Langhammer S and Scheerer J. Oncotarget 8, 43555-43570 (2017).

Citation Format: Dennis Gürgen, Theresia Conrad, Michael Becker, Susanne Sebens, Christoph Röcken, Jens Hoffmann, Stefan Langhammer. Breaking the crosstalk of the cellular tumorigenic network in NSCLC by a highly effective drug combination [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 3095.
Abstract 310: Harness small peptide to improve anti-cancer effect of PD-1 checkpoint inhibitor in melanoma-bearing obese mice
Authors: Han Seungmin , Decker Kelsey E. , Won Young-Wook
Published: June 2022

Obesity-associated chronic inflammation and adipokine increase the cancer risk through promotion of tumor growth and malfunction of immune cells. Leptin, an adipokine abundantly found in obesity, is positively correlated with PD-1 expression and up-regulation of PD-1 on immune cells leads to immune cell exhaustion. PD-1 blockade (checkpoint inhibitor) has shown promising anti-tumor efficacy in patients with obesity through direct effects on the up-regulated PD-1. However, there are still need for improving the response rate and the efficacy of PD-1 blockage. In our previous study, we have verified that prohibitin binding peptide (PBP) inhibits glucose metabolism of cancer cells, resulting in alterations in pro-tumor tumor microenvironment (TME) to pro-immune TME. The pro-immune TME reinvigorates anti-cancer activity of immune cells and results in better suppression of tumor growth. Based these findings, we hypothesized that PBP would improve the anti-cancer effect of PD-1 checkpoint inhibitor on cancer with obesity. Melanoma was embedded in lean or obese mice, then the mice were assigned to one of following groups: i) PBS, ii) PBP, iii) anti-PD-1 antibody (aPD-1) and iv) combination of PBP and aPD-1 (PBP+aPD-1). The PBP+aPD-1 group showed improved anti-cancer efficacy in tumor-bearing obese mice (not in lean mice) compared to single aPD-1 injection. Immune cells in PBP+aPD-1 injected obese mice showed the more activated characteristics in terms of its cytokine secretion and phenotype. In addition, the serum leptin level was decreased upon PBP treatment, indicating that the PBP-mediated decrease in the serum leptin level attributed the rejuvenation of immune cells through the balanced PD-1 expression on immune cells. PBP improves the anti-cancer efficacy of aPD-1 on tumor with obesity through recovering anti-cancer activity of immune cells that is driven, at least in part, by a decrease in the glucose metabolism of cancer cells and the circulating leptin levels. Consequently, combination of PBP and aPD-1 would have the great potential to treat cancer patients with obesity.

Citation Format: Seungmin Han, Kelsey E. Decker, Young-Wook Won. Harness small peptide to improve anti-cancer effect of PD-1 checkpoint inhibitor in melanoma-bearing obese mice [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 310.
Abstract 3102: MiniPDX and omics analysis for clinical indication and biomarker identification
Authors: Wang Shuai , Di Yu , Li Rongyu , Liu Yang , Gu Xiaorong , Hu Kaimeng , Wen Danyi
Published: June 2022

MiniPDX as an in vivo version of the organoid assay represents a novel platform of fast turnaround (7 days) in vivo drug sensitivity test, using either fresh patient tumor samples or tissues from established PDX models. We systematically evaluated and compared the response rates of MiniPDX assays and PDX assays pair-wise in 26 PDX models involving 3 types of cancers to 12 clinical relevant regimens, and found a high correlation between drug responses between the two assays, with sensitivity and specificity of 80% and 93%, respectively. LIDE has completed over 2,400 MiniPDX assays for precision medicine in clinical, including over 50 indications. Using MiniPDX for ranking the drug/treatment approved in clinical practice facilitates prioritization of the best strategy for benefiting patient in precision medicine, while screening a series of small/large molecules within the similar scaffold enables selection of the best candidate for further drug R&D. Importantly, performing MiniPDX Mouse Trial using fresh tumor samples generated from clinic is beneficial for determination of potential clinical indication that would be fit for the investigating new drug. RNA or DNA can be extracted and enriched from only thousands of cells left from MiniPDX preparation by K cell technology, while RNAseq can be further analyzed via functional genomic imaging (FGI). The Omics data would be useful for determination of potential biomarkers to distinguish responders vs non-responders in population with certain indication(s) and further application in patient stratification to confirm inclusive/exclusive criteria in clinical trials.

Citation Format: Shuai Wang, Yu Di, Rongyu Li, Yang Liu, Xiaorong Gu, Kaimeng Hu, Danyi Wen. MiniPDX and omics analysis for clinical indication and biomarker identification [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 3102.
Abstract 3108: Preclinical studies of chemotherapies and targeted therapies to address gastric cancer disparities
Authors: Zhang Hongyong , Estrada-Florez Ana Patricia , Lott Paul , Toal Ted William , Morales-Arana Alexa , Poceros-Coba Alma ,   UCaMP Consortium, Ma Ai-Hong , Halmai Nicole Britney , Carvajal-Carmona Luis
Published: June 2022

Gastric cancer (GC) is one of the most common malignant tumors of the digestive system, and its prognosis remains poor because most tumors were detected in late stages and the 5-year survival is less than 5%. The Cancer Genome Atlas (TCGA) study found that 70% of all GCs have mutations that can be targeted with existing drugs, yet only four molecularly-guided therapies have been approved. GCs have strong disparities in incidence, mortality and survival that disproportionately affect different populations of color. No large-scale cancer genomic research has been carried out in U.S. minority populations. GC molecular data from U.S. minority residents is crucial to improve outcomes, as genomic phenotypes have been associated with differential prognosis and response to treatments. To our knowledge, minority patient-derived models have not been used to test GC therapies. In this study, several organoids lines from minority patients were established and used as an in vitro platform for screening effective therapies. After genomic data analyses, six minority patient-derived organoids with or without cell cycle regulator or PI3K/AKT/mTOR pathway gene alterations were tested for responses to first-line therapies, targeted drugs and combination treatments. Among the 24 PDX models established, the efficacy study of PDX model PDX015 which has amplifications of CDK and AKT genes showed the targeting therapy (a CDKi) can effectively prolong overall survival of mice bearing xenograft treated with the CDKi (60 days), compared with those mice in control group (22 days). In addition, an AKT inhibitor was found significantly inhibited tumor growth in CDKi-resistant mice treated with the CDKi/AKTi combination. In conclusion, investigating the efficacy of targeted therapies in these U.S. minorities will empower precision medicine applications in GC patients and improve GC treatment outcomes. Minority-patient derived tumor models have great potential in preclinical studies of chemotherapies and targeted therapies to address gastric cancer disparities.

Citation Format: Hongyong Zhang, Ana Patricia Estrada-Florez, Paul Lott, Ted William Toal, Alexa Morales-Arana, Alma Poceros-Coba, UCaMP Consortium, Ai-Hong Ma, Nicole Britney Halmai, Luis Carvajal-Carmona. Preclinical studies of chemotherapies and targeted therapies to address gastric cancer disparities [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 3108.
Abstract 3110: Identification of the phosphorylation network in PDX and corresponding organoid (PDXO) models upon targeted therapy treatment using deep phosphoproteomic analysis[MB1]
Authors: Xu Xiaoxi , Tognetti Marco , Feng Yuehan , Shang Limei , Chen Leilei , Wang Jessie , Bruderer Roland , Bourré Ludovic , Li Henry
Published: June 2022

In precision medicine, genomic, transcriptomic, and proteomic data has contributed to the identification of novel driver genes and the molecular-level characterization of cancers. This data has led to a better understanding of drug modulation and resistance mechanisms. Patient-derived tumor models, including patient-derived xenograft (PDX) and organoid counterparts (PDXO), have been increasingly viewed as predictive preclinical cancer models. These models closely recapitulate tumor complexity, enabling the study of tumor identity for personalized medicine. Leveraging our large PDX collection models which are genomically and phenotypically annotated and validated, we have established and characterized a series of PDXO models to be used as scalable and high throughput compatible drug screening platforms. Here we conducted methodical analysis of drug response in paired PDX and PDXO models. In addition, trough deep whole- and phospho-proteomic analysis, we have analyzed and compared the effect of targeted therapies on protein expression and phosphorylation in both PDX and PDXO tumor models. Indeed, one of the most common post-translational modifications that is involved in cell regulation and intracellular signal transduction is reversible protein phosphorylation catalyzed by protein kinases. For this purpose, we have tested a KRAS inhibitor (AMG510), BCR-ABL TKI (Ponatinib) and EGFR TKI (Afatinib) respectively in lung NSCLC model carrying KRAS G12C mutation, colorectal model with RET fusion and Lung NSCLC model with EGFR exon 19 deletion. According to our historical data, a specific relationship between area under the curve value of organoid drug dose response and in vivo tumor growth has been observed, irrespective of the drug treatment. Furthermore, comparison between PDX and PDXO models deploying quantitative proteomic data enabled deep characterization of both global expression and signaling cascades modulated through small molecule inhibitors. Thus, we demonstrate the predictivity of organoid cultures to not only model in vivo drug responses but also to serve as a powerful platform to investigate target identification, mechanism of action and resistance mechanism via deep proteomics analysis.

Citation Format: Xiaoxi Xu, Marco Tognetti, Yuehan Feng, Limei Shang, Leilei Chen, Jessie Wang, Roland Bruderer, Ludovic Bourré, Henry Li. Identification of the phosphorylation network in PDX and corresponding organoid (PDXO) models upon targeted therapy treatment using deep phosphoproteomic analysis[MB1] [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 3110.
Abstract 3116: Development of bioluminescent PDX models to study metastasis and evaluate therapeutic interventions
Authors: Nakashima Jonathan , Sperry Jantzen , Pedros Christophe , Carapia Bianca , Yan Deborah , Rivera Giovanni , Federman Noah , Singh Arun , Eilber Fritz C. , Datnow Brian
Published: June 2022

Metastatic disease continues to be a significant cause of mortality among cancer patients. Despite advancements in patient-derived xenografts (PDXs), translational models of metastasis are lacking. To overcome this challenge, we have developed luciferase tagged PDX cultures and utilized optical imaging to create robust translational in vivo models of metastasis. Here, we show that these models can be implanted orthotopically into common metastatic sites, or injected intravenously or intracardiacally to study spontaneous metastasis. Furthermore, we apply these models in humanized NOG-EXL mice to demonstrate immunophenotypic differences among tumors in different anatomical sites.

Citation Format: Jonathan Nakashima, Jantzen Sperry, Christophe Pedros, Bianca Carapia, Deborah Yan, Giovanni Rivera, Noah Federman, Arun Singh, Fritz C. Eilber, Brian Datnow. Development of bioluminescent PDX models to study metastasis and evaluate therapeutic interventions [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 3116.
Abstract 3129: TWEAK cytokine supports ovarian cancer tumor-initiating cell (TIC) phenotypes important for relapse
Authors: Stevenson Denay N. , Holmberg Ryne M. , Gilbert Samuel F. , Robinson Mikella , House Carrie D.
Published: June 2022

Ovarian cancer is the deadliest gynecological cancer with over 13,000 deaths estimated in the United States this year. While there is an initial high response rate to cytotoxic chemotherapy, over 80% of advanced cases relapse within 24 months, presenting an urgent need to better understand the molecular mechanisms permitting relapse. Current research suggests that cancer recurrence can be attributed to a small subpopulation of cancer stem-like tumor-initiating cells (TICs) that possess the ability to both resist chemotherapy and initiate tumorigenesis. Our lab previously demonstrated an essential role for alternative NF-kB signaling in maintaining ovarian TICs, yet it is not fully understood how this pathway becomes activated in ovarian cancer. To begin to address this, we used an NF-kB reporter system to screen soluble factors known to be in the ovarian tumor microenvironment (TME) to identify the strongest inducers of NF-kB activation in ovarian cancer cells. We found that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a strong inducer of alternative NF-kB signaling. In vitro experiments demonstrate enhanced expression of stemness genes including SOX2, OCT4 and NANOG and increased spheroid formation ability after stimulation with TWEAK. Moreover, flow cytometry analysis of several TIC markers shows a significant enrichment of TICs when chemotherapy was combined with TWEAK, relative to chemotherapy alone. Stimulation of CD117+ cells led to higher expression of stemness genes relative to stimulation of CD117- cells, suggesting TWEAK activity may be specific to TICs and enhances their stem-like phenotype. In support of this, we found that CD117+ cells have higher expression of the TWEAK receptor FN14. We investigated the role of TWEAK in aiding relapse in vivo using an intraperitoneal xenograft mouse model and found that administration of a small molecule inhibitor of TWEAK-FN14 signaling as a maintenance treatment following chemotherapy significantly prolonged survival. These findings highlight a potential mechanism of TIC enrichment in response to chemotherapy that contributes to relapse.

Citation Format: Denay N. Stevenson, Ryne M. Holmberg, Samuel F. Gilbert, Mikella Robinson, Carrie D. House. TWEAK cytokine supports ovarian cancer tumor-initiating cell (TIC) phenotypes important for relapse [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 3129.
Abstract 314: Amplifying STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy
Authors: Zhou Xingwu , Sun Xiaoqi , Gong Wang , Lei Yu Leo , Moon James J.
Published: June 2022

Background: Innate immune pathway of stimulator of interferon genes (STING) orchestrates antitumor immune responses and can elicit potent antitumor efficacy. However, clinical development of cyclic-dinucleotide (CDN) STING agonists is mostly limited to intratumoral administration because of the undesirable pharmacological properties of CDN. This limits their applications in treating metastasis, hematologic malignancies, and hard-to-reach solid tumor. Here, we aim to develop a pharmacological formulation that facilitate systemic delivery of CDN STING agonist to broaden its applications for cancer treatment.

Methods: Nutritional metal ions physiologically mediate various immune processes, which could potentially be utilized for modulating the STING pathway. By screening different nutritional metal ions, we discovered manganese ion (Mn2+) and cobalt ion (Co2+) synergize with CDN STING agonists and significantly enhance type-I interferon (IFN-I) responses. As Mn2+ has been used in FDA approved drugs, we further developed the formulation to enable systemic codelivery of Mn2+ and CDN.

Results: Mn2+ could significantly potentiate the STING activation by up to 77-fold. In addition, Mn2+ interact with CDNs and self-assemble into nanocrystals that could be stabilized as uniform nanoparticles (~120 nm) through lipid layer coating (CDN-Mn Particle, CMP). After either intratumoral or intravenous administrations, CMP effectively elevate the serum levels of inflammatory cytokines (IFN-β, TNF-α, CXCL-9, and CXCL-10), mitigates the suppressive functions of myeloid cells, relieve the immunosuppressive tumor microenvironment (TME), and induce antigen-specific CD8+ T cells expansion. Minute dose of CMP given intravenously exert remarkable antitumor efficacy in CT26, B16F10, and immune checkpoint blocker (ICB)-resistance tobacco-associated NOOC1 murine tumor models, leading to efficient tumor regression. Furthermore, the systemic biodistribution of CMP could be modulated by changing the composition of the lipid layer coating, allowing for organ-specific delivery of CMP to target tumor malignancies in certain organs.

Conclusions: CMP provides a novel and versatile platform for local and systemic cancer treatment. More importantly, it highlights the potential of harnessing metal ions in treating various immune-mediated diseases.

Citation Format: Xingwu Zhou, Xiaoqi Sun, Wang Gong, Yu Leo Lei, James J. Moon. Amplifying STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy [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 314.
Abstract 3146: EGFL6 induces immunosuppressive functions of tumor-associated myeloid cells and mediates resistance to anti-PDL1 therapy
Authors: Sinno Sarah , Bai Shoumei , Coronnello Claudia , Vlad Anda , Buckanovich Ronald J. , Cascio Sandra
Published: June 2022

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is Epidermal growth factor-like 6 (EGFL6). EGFL6 is a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. Similarly, EGFL6 promotes endothelial cell migration and proliferation. Indicating a potential role for EGFL6 as a myeloid cell regulatory factor, we found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of Syk/ERK signaling, (i) acts as a chemotactic factor for human myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling and flow cytometry analysis of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Consistent with Egfl6 driving an immune suppressive TME, EGFL6 expression in an otherwise immune ‘hot’/anti-PD-L1 responsive tumor model completely inhibited response to anti-PD-L-1 therapy. We are currently evaluating the impact of Egfl6 neutralizing antibody on the efficacy of ICI therapy and anti-tumor immunity. Combined our data show that EGFL6 acts as a chemotactic factor to both recruit myeloid cells to the ovarian TME and subsequently promotes their differentiation to an immunosuppressive phenotype. This suggests EGFL6 is a potential novel therapeutic target to ovarian tumor mediated immunosuppression and enhance response to immune therapy in ovarian cancer patients.

Citation Format: Sarah Sinno, Shoumei Bai, Claudia Coronnello, Anda Vlad, Ronald J. Buckanovich, Sandra Cascio. EGFL6 induces immunosuppressive functions of tumor-associated myeloid cells and mediates resistance to anti-PDL1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3146.
Abstract 3158: Defining the mechanisms that lead to mutational heterogeneity in the normal respiratory epithelium
Authors: Gowers Kate H. , Clarke Sarah E. , Syaraf Ayu Hutami , Yoshida Kenichi , Przybilla Moritz J. , Selway Hugh , Pennycuick Adam , Campbell Peter J. , Janes Sam M.
Published: June 2022

It is well established that tobacco smoking causes lung cancer and this is thought to be the result of a progressive accumulation of genomic mutations induced by tobacco smoke exposure; however, until recently a detailed understanding of the effects of smoke exposure on the genomes of cells in the normal bronchial epithelium was lacking.

To address this, we defined the genomic architecture of normal airway basal cells in children and non-smoking, smoking and ex-smoking adults. We discovered two populations of basal cells in individuals with a smoking history: those with high mutational burden, driver gene mutations and tobacco-associated mutational signatures, and those with low mutational burden, similar to basal cells from never smokers and with an absence of mutational signatures associated with tobacco exposure. These cells expand preferentially in ex-smokers, accounting for 20-40% of basal cells compared with 2-5% in current smokers. We hypothesise that these cells are cancer-protective and their expansion reduces the risk of lung cancer development after smoking cessation.

Understanding the functional differences between high- and low-mutant airway basal cells and the mechanisms by which some cells resist the accumulation of smoking-induced mutations will be crucial to understanding the earliest stages of lung carcinogenesis.

To address this, we performed RNAseq on a subset of whole-genome sequenced airway basal cells. Preliminary data show that expression of markers of stemness is significantly different between high- and low-mutant basal cells. In addition, initial analysis shows that pathways such as carcinogen metabolism and MHC class I antigen presentation are higher in high-mutant basal cells. Further analysis in more patients is ongoing. To complement these data, we expanded whole-genome sequenced basal cell clones in culture and performed a range of assays to assess their progenitor and differentiation capacity. We assessed proliferation, colony-forming efficiency, longevity and differentiation and found no apparent differences between low- and high-mutant basal cells.

Initial analysis suggests that carcinogen metabolism and MHC class I antigen presentation may be key pathways in establishing heterogeneity in mutational burden and clonal dynamics in the normal airway epithelium. Additional assays and analysis are ongoing and will be the focus of future research.

Citation Format: Kate H. Gowers, Sarah E. Clarke, Ayu Hutami Syaraf, Kenichi Yoshida, Moritz J. Przybilla, Hugh Selway, Adam Pennycuick, Peter J. Campbell, Sam M. Janes. Defining the mechanisms that lead to mutational heterogeneity in the normal respiratory epithelium [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 3158.
Abstract 3162: Macrophage-derived C/EBPb regulates adult mammary gland homeostasis
Authors: Rojo Michelle D. , Burke Caitlin M. , Dow Alexa M. , Schwertfeger Kathryn L. , Machado Heather L.
Published: June 2022

Throughout mammary gland development, there are distinct stages that are “hot spots” of breast cancer risk. During these critical windows, tissue resident macrophages function to maintain the homeostatic balance within the stromal microenvironment and the ductal epithelium, and alterations in these interactions disrupt tissue homeostasis and impact breast cancer risk. However, the signaling and mechanisms by which macrophages regulate mammary gland homeostasis are poorly understood. Our recent studies (and others) have identified transcriptional profiles of distinct tissue resident macrophage subpopulations in the mammary gland. We found that Cebpb, a transcription factor important for myeloid cell differentiation and mammary gland development, is highly expressed in Cx3cr1+ ductal macrophages and localizes to macrophages in close contact with the epithelial ducts. Genetic ablation of C/EBPβ in CSF1R-expressing macrophages results in deficient alveolar budding during diestrus, while branching morphogenesis was normal. Interestingly, this phenotype is also seen in macrophage null models and suggests that C/EBPβ in macrophages plays a crucial role in signaling to the mammary epithelium. Analysis of serum hormone levels show that circulating estrogen and progesterone levels are not changed in CebpbΔMφ mice, however, the number of progesterone receptor (PR)-expressing cells are significantly increased in the ductal epithelium, suggesting altered local progesterone signaling. Further examination of the mammary epithelium shows alterations in a number of Wnt family members, including Wnt1, Wnt2b, Wnt3a and Axin2, indicative of decreased canonical Wnt signaling. Importantly, canonical Wnt signaling has been shown to be critical for regulating mammary stem cell expansion during diestrous budding. Analysis of the C/EBPβ-null macrophages show decreased expression of TGFβ, which is known to signal to basal epithelial stem cells to induce Wnt expression. Finally, other signals, such as Notch 2, which have important roles in the mammary gland development are altered. Ongoing studies are aimed at delineating how macrophage-derived C/EBPβ facilitates mammary stem cell function during the estrous cycle in a progesterone-independent manner. The results of these studies will have critical implications for understanding how the signaling between macrophages and the ductal epithelium regulate tissue homeostasis and the inherent dysregulations that lead to tumorigenesis.

Citation Format: Michelle D. Rojo, Caitlin M. Burke, Alexa M. Dow, Kathryn L. Schwertfeger, Heather L. Machado. Macrophage-derived C/EBPb regulates adult mammary gland homeostasis [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 3162.
Abstract 317: Isolation of highly selective antibodies against claudin 18.2 for the treatment of solid tumors
Authors: Screnci Brad , Barnes Trevor , Shema Kristen , Rimkunas Rebecca , Kannan Shruthi , Phillips Tim , Navia Carmen , Azuelos Charles , Charpentier Tom , Houtmann Jennifer , Miller Lisa , Stafford Lewis J. , Doranz Benjamin J. , Rucker Joseph B. , Chambers Ross
Published: June 2022

Monoclonal antibodies (MAbs) are a well-established treatment approach in oncology and other diseases. Nevertheless, many multipass membrane proteins are largely inaccessible as antibody targets due to their poor expression, membrane-dependent structure, small extracellular regions, and high sequence conservation between humans and rodents. Integral Molecular’s MPS Antibody Discovery platform specifically addresses each of these challenges. A key enabling feature of MPS is the use of chickens as an evolutionarily divergent host species for immunization, allowing a more robust immune response for targets that are highly conserved in mammals. We will present on antibodies isolated against challenging membrane protein targets in oncology including Claudin 18.2 (CLDN18.2).CLDN18.2 is a transmembrane adhesion protein undetectable in most adult healthy tissues but highly expressed in gastric, pancreatic, esophageal, and lung cancers. Antibody discovery efforts against this validated target are challenging due to the abundant expression of the splice isoform CLDN18.1 differing by only 8 amino acids in the extracellular domain. As part of the MPS platform, we used virus-like particles (Lipoparticles) to immunize chickens with a high concentration of native CLDN18.2 protein and obtain high-titer immune responses. This enabled us to generate and isolate a large and diverse collection of MAbs (48 unique clones) and select candidates for optimization. We present a panel of three highly specific, humanized CLDN18.2 MAbs with picomolar affinities that are superior to the clinical-stage benchmark. We will show in-depth profiling data for the MAb panel that were used for lead selection and de-risking clinical development. These data include biosensor binding kinetics, amino-acid resolution epitope mapping, and specificity testing against the Membrane Proteome Array (MPA) consisting of 6,000 membrane proteins. This panel of preclinical antibodies are being developed for therapeutic use in various formats, including bispecifics, antibody-drug conjugates, and CAR-T applications.

Citation Format: Brad Screnci, Trevor Barnes, Kristen Shema, Rebecca Rimkunas, Shruthi Kannan, Tim Phillips, Carmen Navia, Charles Azuelos, Tom Charpentier, Jennifer Houtmann, Lisa Miller, Lewis J. Stafford, Benjamin J. Doranz, Joseph B. Rucker, Ross Chambers. Isolation of highly selective antibodies against claudin 18.2 for the treatment of solid tumors [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 317.
Abstract 3188: Modulating UGDH expression in ovarian cancer tumor-initiating cells alters the tumor microenvironment
Authors: Harrington Brittney S. , Kamdar Rahul , Wong Nathan , House Carrie D. , Annunziata Christina M.
Published: June 2022

Epithelial ovarian cancer (EOC) is a global health burden, with the poorest five-year survival rate of the gynecological malignancies due to diagnosis at advanced stage and high recurrence rate. Recurrence in EOC is driven by the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that are supported by a complex extracellular matrix (ECM) and immunosuppressive microenvironment. To target TICs to prevent recurrence, we identified genes critical for TIC viability from a whole genome siRNA screen. A top hit was the cancer-associated, ECM synthesis enzyme UDP-glucose dehydrogenase (UGDH). Immunohistochemistry was used to delineate UGDH expression in histological and molecular subtypes of EOC. Clustering analysis was performed to characterize EOC cell lines to aligned to the molecular subtypes observed in tumors. UGDH expression was modulated in the cell lines using inducible shRNA, and the effects on TICs and on the cells in the supporting microenvironment were examined. High UGDH expression was observed in the majority of high-grade serous ovarian cancers and variable expression was observed in clear cell, mucinous and endometrioid histotypes. A distinctive survival prognostic for UGDH expression was revealed when serous cancers were stratified by molecular subtype, where high UGDH was associated with a poor prognosis in the mesenchymal subtype. Using OV90 as a representative cell line for the mesenchymal subtype, we examined the effect of UGDH knockdown on the tumor cells alone, and on mesothelial support cells in co-culture with the tumor cells. Knock down of UGDH in the OV90 cells reduced the viability, sphere-formation and colony formation capacity of TICs and reduced extracellular hyaluronan production. Knocking down UGDH in the tumors affected the mesothelial cells in co-culture by significantly reducing the expression of ECM signaling and remodeling proteins Versican, Vitronectin, Laminin and matrix metalloprotease 1. These data show that modulation of UGDH expression in tumors influences cells in the microenvironment and reveal a distinct role for UGDH in the mesenchymal molecular subtype of EOC. UGDH is a strong prospective therapeutic target in TICs, for the treatment of metastatic and recurrent EOC, particularly in patients with the mesenchymal molecular subtype.

Citation Format: Brittney S. Harrington, Rahul Kamdar, Nathan Wong, Carrie D. House, Christina M. Annunziata. Modulating UGDH expression in ovarian cancer tumor-initiating cells alters 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 3188.
Abstract 3194: Cancer-associated fibroblast heterogeneity in neuroblastoma
Authors: Peterson Nicolas B. , Thomas Paul G.
Published: June 2022

Extracellular matrix (ECM) remodeling is a critical process within the solid tumor microenvironment. Cancer associated fibroblasts (CAFs) are the primary producers of matrix proteins and remodeling enzymes that cross-link and degrade matrix proteins. CAFs are enigmatic in that they can promote or impede tumor growth, suggesting heterogeneity that is not completely understood. How CAF heterogeneity evolves over tumor progression and how this evolution impacts ECM remodeling within solid tumors remain an outstanding question across cancer models. Recent studies have focused on CAF function in models of adult cancer, but little attention has been directed toward understanding CAF heterogeneity and ECM remodeling in childhood cancers, which are etiologically different diseases. Neuroblastoma (NB) is a childhood cancer of neuroendocrine cells that accounts for 15% of pediatric cancer deaths. Like other solid tumors, the NB tumor microenvironment includes malignant cells, CAFs, and immune cells. Our laboratory studies NB using a penetrant, spontaneous model of NB (TH-MYCN, AlkF1178L/+) in immunocompetent mice, allowing in vivo investigation of the tumor immune microenvironment from tumorigenesis to progressive disease. Using this model, we previously demonstrated the pro-tumorigenic role of macrophages and CD4+ T cells by showing that their depletion significantly reduced the formation of tumors and extended survival. In this study, 10X Chromium gene expression analysis of stromal cells from nascent and mature tumors identified two distinct subsets of CAFs characterized by alpha smooth muscle actin (Acta2) and Wilms tumor 1 (Wt1) expression. Interestingly, only Wt1 CAFs expressed chemokines and cytokines involved in recruitment of myeloid and T cells. Wt1 CAFs were enriched in the early tumor microenvironment and expressed genes encoding ADAMTS enzymes involved in degradation of the proteoglycan versican. Our preliminary findings indicate that Wt1 CAFs may be a crucial early recruiter of pro-tumorigenic immune cells in our model of neuroblastoma.

Citation Format: Nicolas B. Peterson, Paul G. Thomas. Cancer-associated fibroblast heterogeneity in 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 3194.