bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3035: Proliferation induced by IL4R is regulated via Glut1 activity in metastatic breast cancer
Authors: Hargrove-Wiley Ebony , Valent Daniel , Williams Demond , Bindeman Wendy , Fingleton Barbara
Published: June 2022

Metabolic reprogramming is a characteristic of all cancers including breast cancer; however, the signaling pathways involved are not fully understood. Altered metabolism, like a shift to aerobic glycolysis, supports cancer cell growth and metastatic behavior. Similarly, immune signals from the tumor microenvironment can promote these same phenotypes and therefore, immune signals likely influence the metabolic state of cancer cells. More specifically, the cytokine interleukin-4 (IL4) promotes increased tumor cell survival, proliferation, and invasion upon stimulation of the type II IL4 receptor alpha (IL4Rα) which is aberrantly expressed on epithelial cancer cells. Previously, we have shown that metastatic mouse mammary cancer cells (4T1) rely on IL4 signaling for colonization of metastatic sites in the lung and liver. Additionally, these cells uptake more glucose when treated with IL4 in vitro. Here, we investigated the significance of glucose consumption on pro-tumorigenic phenotypes induced by IL4/IL4Rα. In 4T1 cells, IL4/IL4Rα signaling increased protein expression and global glycosylation of a primary glucose transporter, Glut1. In human metastatic breast cancer cells (MDA-MB-231), CRISPR-mediated knockout (KO) of Glut1 significantly reduced basal and IL4-induced glucose consumption when assayed by 2-NBDG uptake, a fluorescently labeled glucose analog. Glut1 KO reversed the proliferative effects of IL4/IL4Rα activation on human cancer cells in vitro. Glut1 depletion attenuated tumor cell dependence on glucose consumption as shown by decreased expression of Hexokinase-2, a rate-limiting enzyme in glycolysis. In addition, KO cells have elevated levels of the glutamine transporter, ASCT2, that are further enhanced by IL4 treatment which suggest a greater reliance on glutamine albeit, insufficient for cell expansion supported by IL4/IL4Rα stimulation. Our data indicate that IL4-induced glucose consumption supports the increased metabolic activity necessary for cancer cell proliferation. Targeting the IL4/IL4Rα signaling axis could be a therapeutic strategy to hinder tumor-associated metabolism and reduce tumor burden at primary and metastatic sites.

Citation Format: Ebony Hargrove-Wiley, Daniel Valent, Demond Williams, Wendy Bindeman, Barbara Fingleton. Proliferation induced by IL4R is regulated via Glut1 activity in metastatic breast 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 3035.
Abstract 3044: C. albicans infection mediates upper gastrointestinal tract malignancy independently of Il17a in an APECED-like mouse model
Authors: Zhu Feng , Phan Quynh T. , Ferre Elise M. , Willette-Brown Jami , Salcedo Rosalba , Shen Tsai-Wei , Fan Yu , Zhao Yongmei , Mehta Monika , Meerzaman Daoud , Tran Bao , Trinchieri Giorgio , Lionakis Michail S. , Filler Scott G. , Hu Yinling
Published: June 2022

Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) develop chronic mucocutaneous candidiasis during childhood and about 10% of senior patients develop upper GI tract squamous cell carcinoma (SCC), suggesting that C. albicans infection may be relevant to the cause of SCC development in APECED patients. In this study, we investigated the role of C. albicans infection in upper GI tract SCC development using an APECED-like IKKα kinase-dead (KA/KA) mouse model. We found that KA/KA mice developed upper GI tract SCC after oral infection of WT C. albicans weekly for three months. Interestingly, while upper GI tract SCC development in C. albicans - infected KA/KA mice is IL-17a-independent, EGFR pathway plays an important role in C. albicans infection-mediated tumorigenesis in KA/KA mice, as evidenced by significantly lower SCC incidence in the upper GI tract of KA/KA mice infected with Als3Δ/Δ C. albicans. Moreover, upper GI tract SCCs from WT C. albicans-infected KA/KA mice exhibited robust p-EGFR and Ki67 signals. However, both p-EGFR and Ki67 signals were only mildly increased in upper GI tract of Als3Δ/Δ C. albicans-infected KA/KA mice compared to WT control mice. Meanwhile, EGFR inhibition dramatically reduced C. albicans colonization in upper GI tract and the weight of stomach in KA/KA mice. Furthermore, we found that aberrant infiltrations of neutrophils and macrophages with significant inflammatory signatures were present in the upper GI tract SCCs in KA/KA mice. Interestingly, KA/KA neutrophils exhibited a defect in C. albicans killing capacity due to functional defects in ROS generation, degranulation, NETs formation, and apoptosis. We reasoned that KA/KA neutrophils’ inability in C. albicans killing capacity may further enhance the tumor-promoting microenvironment. Finally, whole genome sequencing data suggested that C. albicans infection may lead to genome instability. Currently we are investigating the role of neutrophil infiltration in C. albicans infection-mediated upper GI tract tumorigenesis.

Citation Format: Feng Zhu, Quynh T. Phan, Elise M. Ferre, Jami Willette-Brown, Rosalba Salcedo, Tsai-Wei Shen, Yu Fan, Yongmei Zhao, Monika Mehta, Daoud Meerzaman, Bao Tran, Giorgio Trinchieri, Michail S. Lionakis, Scott G. Filler, Yinling Hu. C. albicans infection mediates upper gastrointestinal tract malignancy independently of Il17a in an APECED-like mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3044.
Abstract 3082: Leveraging 3D organoids to identify molecular changes underlying HNSC initiation and development
Authors: Agee Robert , Flashner Samuel , Shimonosono Masataka , Klein-Szanto Andres , Nakagawa Hiroshi
Published: June 2022

Head and neck squamous cell carcinoma (HNSC) is a devastating disease accounting for 570,000 newly diagnosed cases and resulting in 344,000 deaths annually. HNSC arises from its histological precursor lesion, squamous dysplasia (HNSD); however, most patients are diagnosed with late-stage HNSC with no prior identification of pre-malignant disease. Earlier diagnosis and intervention could ameliorate the outsized burden of HNSC; therefore, there is an urgent unmet need to characterize the molecular changes underlying progression through HNSD to HNSC. To identify novel therapeutic targets or biomarkers that predict progression to HNSC, we have generated a three dimensional (3D) organoid library derived from dysplastic oral tissue harvested from mice treated with 4-Nitriquinoline N-oxide (4-NQO), a potent mimetic of tobacco smoke. Through RNA sequencing analysis of these organoids, we have identified two distinct gene expression states that accompany either early or late dysplasia. By performing gene set enrichment analysis of these two distinct gene signatures, we have identified c-Jun N-terminal Kinase (JNK) signaling as a potential biomarker that predicts or progression from HNSD to HNSC. Together, we have identified the salient transcriptional changes that accompany progression through the histological precursor lesion of HNSC. This study positions JNK signaling as a potential biomarker or therapeutic target for early intervention during the treatment of a deadly disease.

Citation Format: Robert Agee, Samuel Flashner, Masataka Shimonosono, Andres Klein-Szanto, Hiroshi Nakagawa. Leveraging 3D organoids to identify molecular changes underlying HNSC initiation and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3082.
Abstract 3107: Identifying therapeutic vulnerabilities in HR-positive, HER2-negative advanced breast cancer patient-derived xenograft models refractory to CDK4/6 inhibition
Authors: Kettner Nicole M. , Navarro-Yepes Juliana , Bui Tuyen N. , Rao Xiayu , Wang Jing , Meric-Bernstam Funda , Hunt Kelly K. , Tripathy Debu , Keyomarsi Khandan
Published: June 2022

Background: Clinically, the addition of CDK4/6 inhibitors (e.g. palbociclib) to endocrine therapy (ET) significantly delays progression of advanced hormone receptor-positive (HR+), HER2-negative (HER2-) breast cancer patients. However, most patients develop resistance and progress following long-term treatment. Thus, an ongoing clinical challenge has been the identification of biomarkers that predict response to CDK4/6 inhibitors. There is also an unmet need to identify actionable targets for patients who have progressed. Currently, there are no clinically useful biomarkers to predict response and/or resistance to anti-CDK4/6 therapy. Hence, the goal of our study was to identify the therapeutic vulnerabilities of CDK4/6 inhibitor resistant patient derived xenograft (PDX) models and identify key markers that longitudinally correlate with development of resistance and inform new treatment directions.

Methods: We obtained 4 different PDX models, two from patients who progressed after 2-4 months on treatment with palbociclib plus ET (i.e. intrinsic resistance) and the other two who developed resistance over time (i.e. acquired resistance) with disease progression between 12-18 months while on palbociclib plus ET. To elucidate mechanisms of resistance, we performed genome-wide expression analysis via RNA-sequencing.

Results: The molecular assessment of the PDX models revealed different transcriptomic signatures for intrinsic resistance compared to acquired resistance. We identified 3,386 upregulated and 2,829 downregulated significant differentially expressed genes (DEGs) in the acquired compared to intrinsic resistant PDX models. Further, gene set enrichment analysis (GSEA) revealed enrichment of distinct driver pathways in the acquired-compared to the intrinsic resistant models.

Conclusions: Collectively, these results have identified potential unique targets for intrinsic and acquired resistance to guide treatment strategies. Thus, our ongoing studies are geared towards identifying and targeting distinct therapeutic vulnerabilities of intrinsic and acquired resistance to palbociclib.

Citation Format: Nicole M. Kettner, Juliana Navarro-Yepes, Tuyen N. Bui, Xiayu Rao, Jing Wang, Funda Meric-Bernstam, Kelly K. Hunt, Debu Tripathy, Khandan Keyomarsi. Identifying therapeutic vulnerabilities in HR-positive, HER2-negative advanced breast cancer patient-derived xenograft models refractory to CDK4/6 inhibition [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 3107.
Abstract 3119: Circulating cancer stem cells as a tool for generating patient-derived xenografts on chick chorioallantoic membrane (CAM) in cancer patients
Authors: Pizon Monika , Schott Dorothea , Pachmann Ulrich , Schobert Rainer , Pachmann Katharina
Published: June 2022

Background: Circulating cancer stem cells (cCSCs) are a small aggressive subset of circulating tumor cells with cancer stem cells features: resistance to diverse cancer treatments and the capacity for generating new metastases. Patient-derived xenografts (PDX) are an increasingly accepted tool in oncology, providing biologically meaningful models of many cancer types, and potential platforms for the development of precision oncology approaches. Commonly, mouse models are used for the xenotransplant assessment of potential new therapeutic targets in cancers. However, animal models do not necessarily represent the real world scenario and are costly and time consuming. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay.

Methods: In this study, primary cultures from circulating cancer stem cells were established using sphere-forming assays. Subsequently, tumorspheres were transplanted onto the CAM membrane of fertilized chicken eggs to form secondary microtumors.

Results: We have developed an innovative reliable in vitro platform for cultivation of CSCs from peripheral blood of cancer patients. The number of tumorspheres increased significantly with tumor progression. Patients with metastatic disease had statistically more tumorspheres as compared to patients without metastasis (30 vs 10/100µl blood, p<0.05). Patients with multiple metastases had more tumorspheres compared to patients with single metastases (60 vs 30/100µl blood, p<0.05). The number of tumorspheres was positively correlated with Ki-67, Her2 status and grade score in primary breast tumors. Tumorspheres showed self-renewal, growth potential, invasion and differentiation in vivo. Tumorspheres could be successfully grafted onto the CAM and grafting positively correlated with aggressiveness and proliferation capacity of the primary tumor. These tumors growing on the CAM pathologically closely resembled the primary tumor.

Conclusion: The number of tumorspheres cultured from peripheral blood of cancer patients and the success rate of establishing PDX directly reflect the aggressiveness and proliferation capacity of the primary tumor. Our results support the CAM model using cCSC as a valuable alternative for xenotransplant models. It allows establishing new PDXs and providing a fast, low-cost, easy to use preclinical platform for cancer biology research, new drug development, treatment individualization, and biomarker discovery.

Citation Format: Monika Pizon, Dorothea Schott, Ulrich Pachmann, Rainer Schobert, Katharina Pachmann. Circulating cancer stem cells as a tool for generating patient-derived xenografts on chick chorioallantoic membrane (CAM) in cancer 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 3119.
Abstract 3155: "Super NK cells" - natural killer cells derived from engineered hiPSC with enhanced NK receptor expression demonstrate better anti-tumor effects for solid tumors
Authors: Wang Yiyun , He Xiangjun , Wang Linqin , Shao Mi , Yue Yanan , Gao Yangbin , Church George , Huang He , Yang Luhan
Published: June 2022

CAR-T cells expressing chimeric antigen receptors (CAR) have shown clinical success for treating hematologic malignancies, but their efficacy in solid tumors has been limited, largely due to tumor heterogeneity and immunosuppressive tumor microenvironment (TME). Natural Killer (NK) cells derived from human induced pluripotent stem cells (hiPSC) hold great potential to become the next-generation cell therapy products because of their established safety profile and their unique biological activities, especially their potential effectiveness for targeting solid tumor. In contrast to T cells, NK cells exert anti-tumor activity without requiring antigen recognition through MHC complex; they are instead activated by calculated signaling through ligation of multiple activating and inhibitory receptors. As various solid tumors highly express activation ligands for NK cells, we reason that enhanced expression of activation receptors on NK cells could improve their killing activity towards solid tumors. Here, we report that “super NK cells” with more than 10 folds higher expression of NK cell receptors such as NKG2D and NCRs derived from engineered hiPSC show improved cytotoxicity against solid tumors compared with non-engineered ones. The “super NK cells” can achieve more than ten rounds of serial killing against both K562 and THP-1 cell lines in vitro. More importantly, the “super NK cells” show improved cytotoxicity to multiple solid tumors in both in vitro system and in vivo xenograft mouse models. In summary, we have engineered hiPSCs which can be differentiated into "super NK cells" as a potential cell therapy product for improved efficacy against solid tumors.

Citation Format: Yiyun Wang, Xiangjun He, Linqin Wang, Mi Shao, Yanan Yue, Yangbin Gao, George Church, He Huang, Luhan Yang. "Super NK cells" - natural killer cells derived from engineered hiPSC with enhanced NK receptor expression demonstrate better anti-tumor effects for 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 3155.
Abstract 3171: ARL2 is required for homologous recombination repair and colon cancer stem cell maintenance
Authors: Choi Seok Gyeong , Lee Hani , Ha Sojung , Yoon Sukjoon , Kim Woo-Young
Published: June 2022

ARL2 is a small GTP binding protein mediates many cytosolic functions including localization of Ras, dynamics of microtubule and fission of mitochondria but its nuclear function is not well elucidated yet. Here we present the novel role of ARL2 in human cancer nucleus and cancer stem cells (CSC). The cancer tissue gene expression data in The Human Protein Atlas and The Cancer Genome Atlas showed possible pro-cancer and nuclear role of ARL2 in colon cancers. The single cell RNA seq analysis suggests its function in colon stem cells. The activation mutant of K-Ras harboring colon cancer cells expressed less ARL2 than the wild type cells but increased ARL2 expression in CSC spheres. The deprivation of ARL2 in colon cancer cell lines using RNAi preferentially eliminated the CSC spheres while it resulted in minimal impact on bulk cultured cell (BCC) numbers. Suppression of ARL2 halted the BCC at M phase as expected but did not change cell cycle in CSC spheres. However, it led to accumulation of a DNA double strand break marker and apoptosis only in CSC spheres which suggest that ARL2’s requirement for double strand DNA break repair in CSC. Out of 6 somatic RAD51 family genes which all mediate the critical early events of homologous recombination repair (HRR), 5 showed significant and positive association with ARL2 in gene expression in human colon cancer tissue suggesting functional link between ARL2 and HRR. Indeed, ARL2 was required for HRR when double strand DNA breaks were induced. These data collectively showed that ARL2 may contribute to the HRR in the nucleus and the ARL2 mediated HRR is more critical for colon CSC survival.

Citation Format: Seok Gyeong Choi, Hani Lee, Sojung Ha, Sukjoon Yoon, Woo-Young Kim. ARL2 is required for homologous recombination repair and colon cancer stem cell maintenance [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 3171.
Abstract 3179: Tumor educated lung epithelial cells promote osteosarcoma metastasis and chemotherapy resistance by inducing integrin dependent fibronectin fibrillogenesis
Authors: Reinecke James Brandon , Gross Amy , Cam Maren , Roberts Ryan
Published: June 2022

Metastasis is the leading cause of cancer related deaths for patients with solid tumors. While tumor genomic evolution is one possible mechanism whereby cancer cells acquire chemotherapy resistance during the metastatic process, increasing evidence demonstrates that non-cancerous cells within the secondary organ have crucial roles in mediating metastasis and therapy resistance. Therefore, disrupting tumor-stromal cell interactions is an exploitable therapeutic strategy to improve outcomes. Unfortunately, mechanistic insight into tumor-stromal interactions is limited for most cancers. Osteosarcoma, an aggressive bone tumor that primarily afflicts children and young adults, displays an overwhelming propensity for metastasis to the lungs, with overall survival plummeting to less than 20% at 5 years upon lung metastasis. Why osteosarcoma displays tropism for the lung and how it educates the lung to promote tumor growth in a vastly different microenvironment from the primary tumor remains poorly understood. In this study, we utilized single-cell RNA sequencing to define the cellular and molecular landscape of osteosarcoma metastases in immunocompetent mouse models. We unexpectedly found that alveolar epithelial cells are the most prominent non-immune stromal component within lung metastases. Not only are they present in significant numbers in lung metastases compared to unaffected lung, our molecular analysis demonstrated that tumor associated epithelial cells acquire a pro-fibrosis ‘wound-like’ phenotype, which was also observed in human metastasis samples. Furthermore, utilizing human and mouse culture systems, we found that osteosarcoma cells directly reprogramed epithelial cells through secretion of growth factors and cytokines such as FGF7 and IL6. These profibrotic epithelial cells in turn induced osteosarcoma cells to generate a complex, beta-1integrin (ITGB1)- dependent fibronectin matrix. The epithelial induced fibronectin matrix promoted osteosarcoma invasion and chemotherapy resistance. Accordingly, inhibition of ITGB1 sensitized osteosarcoma cells to chemotherapy. Collectively our results demonstrate that osteosarcoma cells educate the lung microenvironment into a ‘pro-wound’, pro-fibrosis state that is conducive to osteosarcoma lung colonization. Disruption of the lung metastatic niche is a promising therapeutic target for sensitizing osteosarcoma cells to chemotherapy.

Citation Format: James Brandon Reinecke, Amy Gross, Maren Cam, Ryan Roberts. Tumor educated lung epithelial cells promote osteosarcoma metastasis and chemotherapy resistance by inducing integrin dependent fibronectin fibrillogenesis [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 3179.
Abstract 3183: Targeting a myofibroblastic prostate cancer-associated fibroblast subtype through pharmacological inhibition of NADPH oxidase 4
Authors: Brunner Elena , Neumann Lucy , Damisch Elisabeth , Puhr Martin , Schäfer Georg , Szyndralewiez Cédric , Klocker Helmut , Sampson Natalie
Published: June 2022

Recent developments in single cell sequencing have revealed significant heterogeneity in the stromal tumor microenvironment. It is now widely accepted that there are different cancer-associated fibroblast (CAF) subtypes with different functions and effects on tumor progression. Since CAFs are generally considered an emerging therapeutic target, it is crucial to identify those subtypes that are tumor-promoting and to characterize their driver pathways. In this study we describe a population of CAFs in prostate cancer (PCa) that express elevated levels of NADPH oxidase 4 (Nox4) and localize adjacent to tumor foci. We previously showed that Nox4 is essential for TGFβ-mediated differentiation to a myofibroblastic CAF phenotype and that its increased expression is associated with biochemical relapse and reduced survival. Thus, this study aims to investigate whether pharmacological Nox4 inhibition can be used as an adjuvant therapeutic approach in PCa and which molecular pathways are regulated by Nox4 in the tumor microenvironment.

Experiments using GKT831, a small molecule Nox1/Nox4 inhibitor with promising effects in clinical trials for fibrotic diseases, so far support its use as a therapeutic target. Most importantly, Nox4 inhibition reduced CAF marker expression in primary prostate CAFs and attenuated the expression of PSA, the key clinical biomarker of PCa progression, in ex vivo cultured human PCa tissue. The translational application of Nox4 inhibition is currently under investigation in vivo using CAF-PCa cell xenografts. To elucidate the molecular mechanisms regulated by Nox4 we performed integrative bioinformatics and functional assays, which revealed that Nox4 regulates CAF adhesion and migration. In addition, we found that Nox4 regulated genes were associated with a YAP signature and the transcription factor TEAD1, a major cofactor of YAP. Ongoing experiments suggest that during adhesion Nox4 signals via oxidation of SHP2, a redox-sensitive phosphatase that has previously been implicated in regulating YAP transcriptional activity.

Previous studies have shown that adhesion and mechanotransduction pathways are commonly altered in myofibroblastic CAFs and that they support the formation of a tumor-promoting microenvironment. Our data suggest that Nox4 acts as a central regulator of these key oncogenic pathways and thus represents a promising therapeutic target.

Citation Format: Elena Brunner, Lucy Neumann, Elisabeth Damisch, Martin Puhr, Georg Schäfer, Cédric Szyndralewiez, Helmut Klocker, Natalie Sampson. Targeting a myofibroblastic prostate cancer-associated fibroblast subtype through pharmacological inhibition of NADPH oxidase 4 [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 3183.
Abstract 3203: Cutting edge biomarkers strategy to provide early insights into activity of EVT-801, a novel selective VEGFR-3 inhibitor that targets tumor angiogenesis during the FIH clinical trial
Authors: Paillasse Michael R. , Garner James , Fitzgerald Michael , Davenne Lise , Ancey Pierre-Benoit , Poussereau-Pomie celine , Esquerre Michael , Badet Gaelle , Tuyaret Joel , Mandron Marie , Rochaix Philippe , Ayyoub Maha , Scarlata Clara Maria , Ménétrier-Caux Christine , CAUX Chrsitophe , Cassier Philippe , Delord Jean-Pierre , Roca Carlos Gomez , Fons Pierre
Published: June 2022

Introduction: EVT801 is a highly selective small molecule inhibitor of VEGFR3 and acts by inhibiting lymphangiogenesis and tumor angiogenesis in and around the tumor. It has shown compelling activity in a wide range of cancer models and is well-tolerated in preclinical toxicology studies. A phase I study is underway and will focus primarily on understanding the safety, tolerability, and pharmacokinetics of EVT801 across a range of doses.

Methods: The first stage of the study is designed to determine the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) for EVT801, with a second stage aiming to confirm RP2D in selected cancer patient populations. Clinical samples from these patients will be used to explore preliminary signals of clinical efficacy and investigate the biological activity of the drug using several biomarkers. Efficacy biomarkers will include imaging approaches (DCE-MRI and CEUS) to characterize tumor vascularization, as well as analysis of protein and mRNA quantification in on-treatment biopsies vs pre-treatment biopsies. Moreover, the relationship between activity of EVT801 and expression of key markers at protein and mRNA level will be investigated to potentially establish biomarkers for patient stratification and selection. Lastly, target engagement and pharmacodynamics effects will be investigated by immuno-monitoring as well as assessment of a defined set of proteins as markers of inflammation and angiogenesis as identified in preclinical in vivo models. We expect that these analyses will help to better understand the effects of the drug in human subjects and may also help to select the most responsive patients and provide early signs of clinical efficacy.

Citation Format: Michael R. Paillasse, James Garner, Michael Fitzgerald, Lise Davenne, Pierre-Benoit Ancey, celine Poussereau-Pomie, Michael Esquerre, Gaelle Badet, Joel Tuyaret, Marie Mandron, Philippe Rochaix, Maha Ayyoub, Clara Maria Scarlata, Christine Ménétrier-Caux, Chrsitophe CAUX, Philippe Cassier, Jean-Pierre Delord, Carlos Gomez Roca, Pierre Fons. Cutting edge biomarkers strategy to provide early insights into activity of EVT-801, a novel selective VEGFR-3 inhibitor that targets tumor angiogenesis during the FIH clinical trial [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 3203.
Abstract 3245: Genes associated with Src-Met driven resistance of head & neck cancers to EGFR-PI3K cotargeting
Authors: Adeluola Adeoluwa A. , Amin A.R.M. Ruhul
Published: June 2022

Background: In recent years, oncology research has led to the discovery of targeted therapies aimed at specific genetic abnormalities unique to cancer cells. In particular, the overexpression of epidermal growth factor receptor (EGFR) and constitutive activation of the phosphoinositide-3-kinase/protein kinase B (PI3K/AKT) pathway are common aberrations in head and neck cancer. However, intrinsic and acquired resistance are continuously posing challenges to success. Previously, we reported that the combination of EGFR inhibitor erlotinib with PI3K inhibitor BKM120 had synergistic effects that induced efficient apoptosis in a panel of head and neck cancer cell lines, but some cells were still resistant. The purpose of the current study is to understand the mechanism of resistance to EGFR and PI3-K co-targeting.

Hypothesis: Src-Met signaling confers apoptosis resistance to EGFR and PI3K cotargeting by regulating downstream genes expresiion.

Methods: Apoptosis was measured by annexin V-PE staining. Western blotting and qPCR were used for measuring the expression of proteins and mRNA, respectively. RNASeq was conducted from RNA samples treated with (Erlotinib + BKM120) ± dasatinib (Src inhibitor)/crizotinib(Met inhibitor).

Results: Biochemical studies revealed that JHU022 cells, which were resistant to the combination treatment, had an overactivation of c-Met receptor tyrosine kinase that was regulated by Src kinases. Pharmacological or genetic inactivation of both Met or Src kinase sensitized this cell line to apoptosis induced by EGFR and PI3K co-targeting. RNA-Seq analysis identified that a total of 291 genes were modulated (2-folds) by both crizotinib and dasatinib. Pathway enrichment suggested that many of these genes were associated with cell cycle, DNA replication, and cellular response to DNA damage. CPA4 was identified as the most downregulated gene and GDF15 as one of the top upregulated genes.

Conclusions: SRC-Met signaling confers resistant to EGFR and PI3K cotargeting by modulating genes associated with cell cycle, DNA replication, and cellular response to DNA damage.

Citation Format: Adeoluwa A. Adeluola, A.R.M. Ruhul Amin. Genes associated with Src-Met driven resistance of head & neck cancers to EGFR-PI3K cotargeting [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 3245.
Abstract 3253: Probing the effects of retinoblastoma binding protein 6 (RBBP6) knockdown on the sensitivity of cisplatin in cervical cancer cells
Authors: Moela Pontsho , Mehta Harshini , Ambele Melvin
Published: June 2022

RBBP6 is considered a potential cancer biomarker due to its association with cell proliferation and the fact that it is overexpressed at cervical cancer sites where there is marked apoptosis and elevated p53. More information is emerging regarding the role of RBBP6 in cancer treatment, specifically its potential to sensitize cancer cells to radiation and certain chemotherapeutic agents via BCL-2 gene regulation. Cisplatin is an FDA-approved chemotherapeutic agent that still presents with acquired resistance in certain cervical cancer cases through p53 repression and BCL-2 upregulation, which is why there is ongoing research in trying to understand the molecular mechanisms involved in the response of cervical cancer cells to this drug. The present study therefore aims to investigate the possible relationship between cisplatin and RBBP6 expression in cervical cancer cells. RBBP6 was silenced in Hela, CaSki and Vero cells using the RNAi technology, followed by measurement of wild-type p53 and BCL-2 at mRNA level using qPCR. Cells co-treated with cisplatin and siRBBP6 were therefore analyzed for apoptosis induction and real time growth monitoring using flow cytometry and the xCELLigence system, respectively. Cancer cells in the co-treatment group showed a reduction in apoptosis compared to cisplatin-only group and real time growth monitoring revealed a reduced growth rate in RBBP6-knockdown cells treated with cisplatin. Although wild-type p53 remained unchanged in the co-treatment group of cancer cells, BCL-2 was completely repressed, suggesting that RBBP6 is necessary for the responsiveness of cervical cancer cells to via BCL-2 downregulation. Findings from this study suggest that RBBP6 expression promotes sensitivity of HeLa cells to cisplatin through BCL-2 downregulation. Knockdown of RBBP6 limits apoptosis induction and delays cell growth inhibition in response to cisplatin. The knowledge obtained here has the potential to help improve cisplatin efficacy through personalized administration based on the expression profile of RBBP6 among individual patients.

Citation Format: Pontsho Moela, Harshini Mehta, Melvin Ambele. Probing the effects of retinoblastoma binding protein 6 (RBBP6) knockdown on the sensitivity of cisplatin in cervical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3253.
Abstract 326: Engineered toxin bodies (ETBs) targeting Trop2
Authors: Cornelison Garrett L. , Bartos Adam , Brieschke Brigitte , Momb Jessica , Pedraza Ileana , Kapeel Elizabeth M. , Martin Rebecca , Pletka Channing , Gonzalez Adrian , Dekker Joseph D. , Zhao Jay , Majercak John , Robinson Garrett L.
Published: June 2022

Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forced internalization, undergoing retrograde translocation to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes resulting in the inhibition of protein synthesis and induction of apoptosis through ribotoxic stress mechanisms.

Additionally, Molecular Templates has developed the Antigen Seeding Technology (AST) platform to generate ETBs with the unique ability to deliver foreign protein antigen to targeted populations of tumor cells. This MOA allows for the intracellular processing of antigen and subsequent surface MHC-I presentation required for activation of a re-directed T lymphocyte response and the capacity to restore a functional immune clearance program against the tumor.

Three ETBs are in clinical development (MT-5111 targeting HER2, MT-0169 targeting CD38, and AST enabled MT-6402 targeting PD-L1). The novel mechanisms of action have potential benefit in different indications including in the relapsed setting, when disease has progressed after chemotherapies and other targeted therapies, and additionally may be able to combine with standard of care.

ETBs are being developed that target other cell surface receptors expressed on solid tumors including tumor-associated calcium signal transducer 2 (Trop2). Trop2 is a clinically validated target of antibody drug conjugate (ADC) therapy in metastatic triple-negative breast cancer (mTNBC) and other cancers such as metastatic urothelial carcinoma (mUC).

In vitro, tumor cells expressing Trop2 are effectively, specifically, and directly killed with picomolar activity by targeted ETBs. AST enabled Trop2 targeted ETBs are capable of delivering viral antigens for multiple HLA types and inducing cytokine secretion and T-cell mediated killing in a co-culture assay of Trop2 target cells with antigen matched HLA type and antigen specific T-cells. These pre-clinical in vitro data suggest AST enabled Trop2 targeted ETBs have the potential to deplete Trop2 positive malignancies through multiple unique mechanisms of action.

Citation Format: Garrett L. Cornelison, Adam Bartos, Brigitte Brieschke, Jessica Momb, Ileana Pedraza, Elizabeth M. Kapeel, Rebecca Martin, Channing Pletka, Adrian Gonzalez, Joseph D. Dekker, Jay Zhao, John Majercak, Garrett L. Robinson. Engineered toxin bodies (ETBs) targeting Trop2 [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 326.
Abstract 3269: Dual targeting of FLT3 and LSD1 disrupts the MYC super-enhancer complex in acute myeloid leukemia
Authors: Yashar William M. , Smith Brittany M. , VanCampen Jake , Kong Garth L. , Macaraeg Jommel , Coleman Daniel J. , Druker Brian J. , Maxson Julia E. , Braun Theodore P.
Published: June 2022

Clinical responses to kinase inhibitor therapy in acute myeloid leukemia (AML) are limited by development of resistance. A major contributor of resistance is epigenetic adaptation to kinase inhibitor therapy. We present evidence that inhibition of the epigenetic regulator lysine-specific demethylase 1 (LSD1) augments the response to inhibitors of FMS-like tyrosine kinase 3 (FLT3) in FLT3-mutant AML. We demonstrate that combined FLT3 and LSD1 inhibition results in synergistic cell death of FLT3-mutant AML cell lines and primary patient samples. High-resolution epigenetic sequencing revealed that the combination therapy synergistically suppresses pro-proliferative MYC-bound promoters and activates pro-differentiative PU.1-bound enhancers. Regulon enrichment analysis in primary AML samples nominated STAT5 (a downstream target of activated FLT3 signaling) as a putative regulator of MYC gene expression. STAT5 is highly bound to the MYC blood super-enhancer and FLT3 inhibition results in a loss of both STAT5 binding and MYC blood super-enhancer chromatin accessibility. In contrast, LSD1 inhibition suppresses MYC target genes by accumulation of repressive H3K9me1 marks. We validated these findings in 72 primary AML samples, including 19 FLT3-ITD positive AML samples. The combination improved responses in the vast majority of patient samples, and high MYC regulon activity was a predictor of response. Gene expression profiling in treated primary AML samples confirmed that dual FLT3 and LSD1 inhibition activates PU.1 target genes and suppresses MYC target genes. Finally, single cell ATAC-seq on primary AML blasts treated ex vivo with combined FLT3 and LSD1 shifted cells from a MYC super-enhancer high to MYC super-enhancer low state. Collectively, these data provide preclinical rationale for the investigation of dual FLT3 and LSD1 inhibition in clinical trial.

Citation Format: William M. Yashar, Brittany M. Smith, Jake VanCampen, Garth L. Kong, Jommel Macaraeg, Daniel J. Coleman, Brian J. Druker, Julia E. Maxson, Theodore P. Braun. Dual targeting of FLT3 and LSD1 disrupts the MYC super-enhancer complex in acute myeloid leukemia [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 3269.
Abstract 3274: Targeting NSD family histone methyltransferase activity with irreversible inhibitors
Authors: Howard Christina , Zari Sergei , Li Shuangjiang , Huang Huang , Kim EunGi , Park Se Ra , Purohit Trupta , Miao Hongzhi , Zhang Xiaotian , Nikolaidis Caroline , Li Hao , Ndoj Juliano , Cho HyoJe , Grembecka Jolanta , Cierpicki Tomasz
Published: June 2022

Nuclear SET domain containing (NSD) family member proteins are histone methyltransferases that mono- and di-methylate histone 3 at lysine 36 (H3K36) to alter gene expression in a context-dependent manner. NSD1 and NSD3 are two members of this family which share greater than 80 percent sequence similarity in the catalytic SET domain. NSD1 is found as a fusion with NUP98 in aggressive cases of pediatric leukemia, and the lysine methyltransferase (KMTase) activity of NUP98-NSD1 drives bone marrow progenitor proliferation. Similarly, high levels of enzymatically active NSD3 mRNA are correlated with reduced disease-free survival in breast cancer and catalytic NSD3 expression is sufficient to rescue tumor growth in a xenograft model of triple negative breast cancer. Selective and potent inhibitors of NSD1 and NSD3 KMTase activity are needed to further investigate the therapeutic potential of targeting this function in leukemia and/or solid cancers. Here, we report the development of irreversible small molecule inhibitors of the NSD1 and NSD3 SET domains. We utilized a NMR-based fragment screen of the NSD1 SET domain to identify the hit BT1, which binds reversibly to the SET domain of NSD1. BT1 was extensively optimized into a series of potent irreversible ligands. Mass spectrometry and structural studies revealed that these compounds react with a cysteine near the autoinhibitory loop, providing a rational for the strong inhibition of KMTase activity we observe in biochemical assays. These inhibitors also bind to the NSD1 and NSD3 SET domains in cells and exhibit potent anti-proliferative effects against leukemia cells containing NUP98-NSD1 and breast cancer cells with high levels of NSD3 expression. In summary, we present novel irreversible inhibitors of NSD1 and NSD3 KMTase activity which represent important chemical probes that can be used to investigate the therapeutic potential of targeting NSD family member catalytic activity in cancers.

Citation Format: Christina Howard, Sergei Zari, Shuangjiang Li, Huang Huang, EunGi Kim, Se Ra Park, Trupta Purohit, Hongzhi Miao, Xiaotian Zhang, Caroline Nikolaidis, Hao Li, Juliano Ndoj, HyoJe Cho, Jolanta Grembecka, Tomasz Cierpicki. Targeting NSD family histone methyltransferase activity with irreversible inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3274.
Abstract 3325: ERK inhibitor increases cancer stem cell population in NSCLC through Slug-Mediated epithelial-to-mesenchymal transition
Authors: Cai Shurui , Li Na , Bai Xuetao , Banerjee Ananya , Kousalya Lavudi , Wang Qi-en
Published: June 2022

The aberrant activation of the Mitogen-activated protein kinase pathway (MAPK/ERK pathway) has been found to play an important role in many cancers including non-small-cell lung cancer (NSCLC). Multiple inhibitors targeting this pathway have been developed to treat cancer patients harboring the aberrant MAPK pathway genes. ERK inhibitors (ERKi) have been shown to be effective in a variety of solid-tumor malignancies, but like other MAPK inhibitors, they rarely confer complete and durable responses. Therefore, it is important to define the mechanism underlying potential tumor relapse following ERKi treatment. In this study, we found that ERKi treatment expanded the cancer stem cell (CSC) population in various NSCLC cell lines and a NSCLC patient-derived xenograft (PDX) harboring aberrant MAPK signaling. Such expansion of the CSC population is mainly caused by enhanced epithelial-to-mesenchymal transition (EMT)-mediated cancer cell dedifferentiation. Mechanistically, we revealed that ERK inhibition induces EMT via pSTAT3-mediated upregulation of EMT-transcription factor Slug. Specifically, we found that ERK inhibition increased phosphorylation of STAT3 at Y705, which further increased the expression of miR-204. Increased miR-204 then downregulated expression of SPDEF, a transcriptional repressor of Slug, thereby derepressing Slug expression. Moreover, we found that a low expression of Slug is necessary for maintaining stemness properties of CSCs. Thus, ERKi treatment-increased Slug must be downregulated to allow these cells to fully acquire stemness, and this process is mediated by a Slug-miR-200c negative feedback loop. Finally, we demonstrated that the JAK/STAT pathway inhibitor Ruxolitinib can block the ERK inhibitor-induced EMT and CSCs expansion, providing a feasible combination inhibitor approach for treating NSCLC patients. Overall, these studies provide a better understanding of the tumor relapse following ERKi treatment, and define the role of Slug in de novo production of CSCs and their maintenance.

Citation Format: Shurui Cai, Na Li, Xuetao Bai, Ananya Banerjee, Lavudi Kousalya, Qi-en Wang. ERK inhibitor increases cancer stem cell population in NSCLC through Slug-Mediated epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3325.
Abstract 3358: Targeting p63 isoforms in cancer to restore their anti-tumorigenic activity
Authors: Ozdemir E. Sila , Gomes Michelle , Fischer Jared
Published: June 2022

p63 plays a central role in limb formation and epithelial differentiation. p63, reported to correlate with cancer formation, is rarely mutated in cancers. Different p63 isoforms can have anti-tumorigenic or pro-tumorigenic activity. TA isoforms act as tumor suppressor by promoting cell death, while deltaN isoforms are strong driver for squamous/squamous-like cancers including lung, head and neck, cervical, skin and pancreas. A large number of tumors are addicted to deltaN isoforms. Furthermore, cancer associated p53R175H mutant gains ability to interact with TAp63 isoforms and inhibit their anti-tumorigenic function, while indirectly promoting tumorigenic function of deltaN isoforms. However, the exact mechanism of p53R175H-p63 interaction is unclear. The changes in the balance of p63 isoform activity are likely to be crucial to understanding the transition between normal cell and tumor formation. Here, we modelled p53R175H-p63 complex using homology modelling, flexible/unbiased docking and molecular dynamics simulations. Moreover, we used our models to design peptides to disrupt p53R175H-p63 interaction and restore anti-tumorigenic function of TAp63 isoforms. Also, we studied deltaN isoform oligomerization and designed peptides to inhibit their oligomerization to reduce their tumorigenic activity. We showed that some of our peptides promoted cell death in a p63 enriched cancel cell line. We characterized our peptides performing kinetic binding assays to validate binding of our peptides to their designated targets. In this study, our computational and experimental analyses presented a detailed model for the p53R175H-p63 interaction. We also provided a framework for potential therapeutic peptides to outweigh the balance in the advantage of anti-tumorigenic p63 activity.

Citation Format: E. Sila Ozdemir, Michelle Gomes, Jared Fischer. Targeting p63 isoforms in cancer to restore their anti-tumorigenic activity [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 3358.