bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3438: Hypoxia inhibits BMP4-induced astrocytic differentiation in glioma stem cells
Authors: Wu Ronald S. , Liedtke Elisabeth , Mansouri Sheila , Brazier Samantha , Guilhamon Paul , Park Nicole I. , Chen Eric , Lupien Mathieu , De Carvalho Daniel D. , Dirks Peter G. , Zadeh Gelareh , Wouters Bradley G.
Published: September 2020

The tumor microenvironment is an important contributor to malignancy in glioblastoma (GBM) by influencing stemness properties in glioma stem cells (GSCs) and their capacity for differentiation into various cell types. Tumor hypoxia is a common feature of the GBM microenvironment and can influence cell state through a variety of processes including the inhibition of oxygen-dependent lysine histone demethylases (KDMs) and subsequent epigenetic changes required for differentiation or maintenance of the stem cell state. However, the precise mechanisms underlying hypoxic restriction of differentiation to specific neural lineages is unclear. Here, we show that hypoxia suppresses astrocytic differentiation induced by bone morphogenetic protein 4 (BMP4) in patient-derived GSCs. In 8/10 patient derived GSC lines, BMP4 induced expression of astrocytic markers, such as glial fibrillary acidic protein, and this induction was repressed by hypoxia in 7/10 lines. Interestingly, significant anti-proliferative effects of BMP4 induced differentiation were only observed in 3/10 lines and was rescued in only one GSC line by hypoxia, suggesting that these processes are uncoupled. Although hypoxia may directly limit the activity of oxygen-dependent KDMs important in this process, levels of S-2-hydroxyglutarate (S-2HG), an endogenous KDM inhibitor, were also increased in 5/8 GSC lines under hypoxia. Exogenous treatment with S-2HG or a small molecule KDM6 family inhibitor, phenocopied the repressive effects of hypoxia on BMP4 induced differentiation. In patient tumors, hypoxic areas surrounding pseudopalisading necrosis showed high expression of hypoxia markers, and these hypoxia markers were anti-correlated with markers of astrocytic differentiation. Together, these results demonstrate that the hypoxic tumor microenvironment in GBM, through multiple mechanisms limiting oxygen-dependent KDM activity, promote maintenance of the malignant stemness state by inhibiting astrocytic differentiation. This study implicates tumour hypoxia as a key regulator of stemness and a therapeutic target to promote differentiation in GBM. Treatment strategies that target hypoxic cells are urgently needed to overcome these challenges.

Citation Format: Ronald S. Wu, Elisabeth Liedtke, Sheila Mansouri, Samantha Brazier, Paul Guilhamon, Nicole I. Park, Eric Chen, Mathieu Lupien, Daniel D. De Carvalho, Peter G. Dirks, Gelareh Zadeh, Bradley G. Wouters. Hypoxia inhibits BMP4-induced astrocytic differentiation in glioma stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3438.
Abstract 3453: Cancer Cell Line Factory: A systematic approach to create next-generation cancer model at scale
Authors: Tseng Yuen-Yi , AI-Jazrawe Mushriq , Deasy Rebecca , Keskula Paula , Johnson Grace , Hong Andrew , Chatterji Priya , Vasquez Francisca , Bass Adam , Van Hare Barbara , Sandak David , Ligon Keith , Boehm Jesse
Published: September 2020

Precision cancer medicine is based on the ability to predict the dependencies of a given tumor from its molecular makeup. Despite successes in multiple common cancers, such prediction remains challenging for the majority of rare and understudied tumors given the absence of laboratory model systems in which to discover and/or validate therapeutic hypotheses. Here, we describe our efforts to address this challenge systematically with the ultimate goal of making it possible to learn how to predict ex vivo growth requirements for cancer samples based on technical, clinical and genomic properties of the starting tumor material. Over the last 5 years, we have processed nearly 2,000 tumor biospecimens and created over 375 genomically-confirmed patient-derived cell lines, organoids and neurosphere cultures, with >10% of these representing rare cancers. To make this possible, we have implemented three key workflows including (1) direct-to-patient sample sourcing, (2) a tissue cryopreservation and genomic credentialing system to ensure quality prior to model creation, and (3) a systematic empirical approach to screening rich medias and variations on organoid technologies ex vivo (HYBRID). We have begun performing genome-wide CRISPR viability screens in these cultures as part of our larger activities to generate a systematic laboratory-based functional map of cancer dependencies (a ‘Cancer Dependency Map'). The novel organoid, spheroid and cell line models created as part of this effort are being made publically available to the scientific community. Looking ahead, as the barriers to culturing rare tumors are overcome, we expect that preclinical functional genomics data will be useful for difficult-to-treat tumors without existing molecularly guided standard-of-care regimens.

Citation Format: Yuen-Yi Tseng, Mushriq AI-Jazrawe, Rebecca Deasy, Paula Keskula, Grace Johnson, Andrew Hong, Priya Chatterji, Francisca Vasquez, Adam Bass, Barbara Van Hare, David Sandak, Keith Ligon, Jesse Boehm. Cancer Cell Line Factory: A systematic approach to create next-generation cancer model at scale [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3453.
Abstract 347: Neogenin as an anti-invasive target for diffuse intrinsic pontine gliomas
Authors: Sesen Julie , Driscoll Jessica , Smith Edward
Published: September 2020

Introduction. Diffuse Intrinsic Pontine Glioma (DIPG) is a rare high-grade tumor of the brainstem that occurs almost exclusively in children. The significant mortality rates of DIPG stem largely from its capacity to invade adjacent normal brain. We have previously published studies identifying the axon guidance factor netrin-1 and its receptor neogenin as key drivers regulating brain tumor invasion in medulloblastoma and glioblastoma. Here we expand this work to investigate the role of netrin-1 and neogenin in DIPG and identify them as putative biomarkers and governors of tumor invasion.

Methods. Urine of 49 DIPG patients and 42 age- and sex-matched controls was collected. Netrin-1 expression was quantified by ELISA. Results were subjected to univariate and multivariate statistical analyses and compared between clinical cohorts. In vitro experiments with established DIPG cell lines utilized RTqPCR, Western blot and flow cytometry to quantify expression of netrin-1 and neogenin. The effect of neogenin blockade on viability and invasion was assessed by flow cytometry (n=3) and transwell chamber assays (n=3) respectively.

Results. Patient samples revealed a significantly low level of urinary netrin-1 compared to matched controls (p<0.005). This result was corroborated with cell lines in vitro. Interestingly, its receptor neogenin was upregulated in these cells when compared to control glial cells when quantified by mRNA, protein and FACS analysis. Blockade of neogenin in DIPG cell lines resulted in an increase of cell death and a significant decrease of cell invasion.

Conclusion. Expanding on previous work, this data suggests that urinary levels of netrin-1 may have utility as a non-invasive biomarker for DIPG. Our data demonstrates the vital role of neogenin as a regulator of tumor invasion and survival in DIPG. The blockade of neogenin in DIPG cells also demonstrates the potential utility of neogenin as a cell-surface druggable target, capable of reducing tumor invasion and increasing tumor cell death.

Citation Format: Julie Sesen, Jessica Driscoll, Edward Smith. Neogenin as an anti-invasive target for diffuse intrinsic pontine gliomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 347.
Abstract 3501: Lower perceived colorectal cancer risk is associated with less aerobic physical activity in Latino adults
Authors: Agrawal Pooja , Yanez Jocelyn , Murillo Rosenda
Published: September 2020

INTRODUCTION: Engaging in aerobic physical activity (PA) has been linked to reduced risk of adverse health outcomes, such as colorectal cancer (CRC). Despite known benefits of engaging in aerobic PA, Latino adults have a low prevalence of aerobic PA compared to other racial/ethnic groups. Limited research has examined the relationship between perceived risk of CRC and aerobic PA among Latinos. Therefore, the purpose of our study was to examine perceived risk of CRC and aerobic PA among US Latino adults.

METHODS: We used cross-sectional 2015 National Health Interview Survey (NHIS) data on Latino participants ≥18 years of age (n=5,457). Perceived CRC risk was assessed based on self-reported measures to which participants responded whether they considered themselves at less than average risk, average risk, or higher than average risk for CRC. Aerobic PA was measured based on self-reported minutes of moderate-to-vigorous aerobic activity per week, which were categorized into none (0 min/week), some aerobic PA (>0 min/week of activity but less than recommendation), and meeting the aerobic activity guideline (≥150 min of moderate-vigorous PA or ≥75 min of vigorous PA or 150 min of moderate PA/week). Multinomial logistic regression models that accounted for complex survey design of NHIS were used to estimate the associations between perceived risk of CRC (ref= less than average risk) with aerobic PA (ref= none). All models were adjusted for age, sex, education, insurance, and nativity.

RESULTS: In adjusted models, compared with individuals who perceived themselves at higher than average risk, those who perceived themselves at less than average risk for CRC were significantly less likely to engage in some aerobic activity (Odds Ratio [OR]: 0.63, 95% Confidence Interval [CI]: 0.40-0.99). In addition, those who perceived themselves at average risk for CRC were also significantly less likely to engage in some aerobic activity (OR 0.60, 95% CI: 0.37-0.96).

CONCLUSION: These findings suggest that perceived CRC risk is associated with aerobic activity among Latino adults. CRC prevention efforts should consider the role of perception of CRC risk in the promotion of physical activity among Latinos.

Citation Format: Pooja Agrawal, Jocelyn Yanez, Rosenda Murillo. Lower perceived colorectal cancer risk is associated with less aerobic physical activity in Latino adults [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3501.
Abstract 3527: Paraneoplastic glomerulonephropathies associated with solid tumor: A review of published literature
Authors: Zhang Xiaojie , Paul Asit K.
Published: September 2020

Background: Paraneoplastic glomerulonephropathies (PGN) are paraneoplastic syndromes associated with solid tumors and hematologic malignancies. PGN is difficult to differentiate from other forms of glomerulonephropathies and standard management of glomerulonephropathies is often ineffective. A delay in diagnosis may lead to significant morbidity and mortality. To date, no retrospective or prospective data on PGN have been published. We reviewed clinical variables, treatment, and outcome in published case reports of PGN associated with solid tumors over the past three decades.

Methods: We conducted a search to identify published case reports on solid tumors associated with PGN in English PubMed-indexed journals. Keywords used included: “solid tumor”, “lung cancer”, “gastrointestinal cancer”, “gynecological cancer”, “genitourinary cancer”, “paraneoplastic nephropathy,” “paraneoplastic glomerulopathy,” “minimal change disease,” “IgA nephropathy,” “membranoproliferative,” “membranous,” “nephropathy” and “glomerulonephropathy”. Published articles from January 1st, 1987 to November 30,2019 were screened. Included were the patients who had laboratory and/or biopsy proven nephrotic syndrome in association with a solid tumor diagnosis.

Results: We identified 95 patients who fulfilled the inclusion criteria. The median age of the patients were 58 years. The majority of the patients were male (65%). Among them, 37 (39%) were diagnosed with PGN prior to the diagnosis of cancer. Membranous nephropathy (41%) was the most common subtype, followed by minimal change disease (17%), focal segmental glomerulonephritis (14%) and IgA nephropathy (11%). Genitourinary cancers (36%) followed by lung cancers (30%), gastrointestinal cancers (15%) and gynecological cancers (13%) are the common cancers associated with PGN. Among the patients, 44 (46%) had non-metastatic tumors, 39 (41%) had metastatic tumors and 12 (13%) were unspecified. Treatment information were available for 79 of 95 (83%) patients. Overall, PGN improved in 76% of patients who received definitive or systemic therapy with or without steroid compared to 57% of patients who were treated with steroid alone. Definitive and systemic treatment alone led to improvement of PGN in 24 of 29 (83%) patients without metastasis and 13 of 17 (76%) patients with metastasis.

Conclusions: PGNs associated with solid tumors are rare and can happen in both non-metastatic and metastatic cancers. Diagnosis of PGN might precede the diagnosis of cancer. Definitive or systemic anticancer therapies improved PGN in most cases and were more effective than steroid alone.

Citation Format: Xiaojie Zhang, Asit K. Paul. Paraneoplastic glomerulonephropathies associated with solid tumor: A review of published literature [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3527.
Abstract 3530: Investigating aneuploidy, CIN and mechanisms of DNA content reduction in cancer
Authors: Mandelia Mara , Drosopoulos Kostas , Linardopoulos Spiros
Published: September 2020

Chromosomal instability (CIN) is a form of genomic instability that refers to a dynamic state in which the cells are constantly changing the number and/or the structure of their chromosomes. CIN leads to aneuploidy, a state in which a cell's set of chromosomes deviates from the euploid set. Causes of aneuploidy include errors during DNA replication, mitosis and cell fusion events. Furthermore, aneuploidy has been shown to promote tumorigenesis and tumor progression, but also to reduce cell fitness or induce cell death, especially when high levels of aneuploidy are present. Nevertheless, antimitotic drugs that induce high levels of aneuploidy, such as Paclitaxel, are not always effective in the clinic.

In this study, we aim to investigate the mechanisms by which cancer cells tolerate aneuploidy, to elucidate the mechanisms by which cell fusion can lead to aneuploidy and resistance to paclitaxel and to identify new targets in aneuploidy and CIN.

Towards this direction, aneuploid clones of pseudo-diploid cancer cells were generated after treatment with anti-mitotic drugs and single cell sorting and were characterized by cell cycle profile, metaphase spreads and CGH or SNP profile. Furthermore, CIN was induced by inhibition of CENP-E or ATM or impairment of the SWI/ SNF complex and quantified by induction of chromosome missegregation and micronuclei formation.

Interestingly, several near tetraploid clones that were isolated after anti-mitotic drug treatment, reduced their DNA content to near diploid during propagation in culture. Thus, we hypothesize that cancer cells may utilize specific mechanisms to change their DNA content and maintain tolerable levels of aneuploidy. By time-lapse microscopy, we observed that cells treated with Paclitaxel, can increase their DNA content by abortive mitosis, cytokinesis failure or by undergoing cell fusion. Interestingly, these multinucleated cells could reduce their DNA content by undergoing independent mitosis, independent nucleus catastrophe or independent nucleus separation (cell exodus). We identify cell exodus, as a potential mechanism by which cells can recover after treatment with Paclitaxel. Inhibition of ROCK pathway, that stabilizes actin filaments and prevents cellular contractility, reduced cell fusion and exodus events and delayed cell recovery after paclitaxel treatment.

Finally, in order to identify new targets in CIN and aneuploidy, we setup sgRNA loss of function screening conditions in the generated CIN and aneuploid cell models and we aim to identify genes that are essential for the survival of aneuploid and/or CIN cancer cells.

Citation Format: Mara Mandelia, Kostas Drosopoulos, Spiros Linardopoulos. Investigating aneuploidy, CIN and mechanisms of DNA content reduction in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3530.
Abstract 3563: Fatty acid binding protein 7 regulates beige fat-like differentiation of breast cancer cells and thermogenesis
Authors: Kawashima Masahiro , Bensaad Karim , Zois Christos , Barberis Alessandro , Bridges Esther , Lagerholm Christoffer , Dmitriev Ruslan I. , Toi Masakazu , Papkovsky Dmitri B. , Buffa Francesca , Harris Adrian L.
Published: September 2020

Cancer cells alter the metabolism in their microenvironment to sustain their survival, growth and progression. Heat is an essential factor that can affect metabolism. However, it is unclear whether cancer cells can alter their temperature by regulating heat production. Non-shivering thermogenesis is known to takes place in inducible beige adipocytes expressing the mitochondrial thermogenic protein, uncoupling protein 1 (UCP1). UCP1 dissipates the proton gradient of the mitochondrial inner membrane and converts its energy into heat. We found that the inhibition of a hypoxia-inducible protein, fatty acid binding protein 7 (FABP7), triggered beige fat-like differentiation and induced thermogenesis in breast cancer cells. We established the breast cancer cell line with a stable knockdown of FAPB7, using lentivirus transduction particles. UCP1-expressing cells were found in 21 ± 5% of the knockdown cells while they were found in only 4 ± 1.7% of the control cells: p = 0.004). UCP1 expression was maximized in the knockdown cells when cultured in hypoxia. A novel thermo-sensitive fluorescent probe confirmed that the cellular temperature of the knockdown cells was 2°C higher than the controls. In addition, the knockdown cells exhibited the increased mitochondrial depolarization, increased proton leak and increased respiration rate compared to controls, suggesting they had the similar energy profile with beige adipocytes. The knockdown cells became more sensitive to hypoxia and radiation, and the clinical manifestation of breast cancer patients differed depending on the expression levels of both UCP1 and FABP7. These observations suggested a missing link between hypoxia-related fatty acid metabolism and cellular thermogenesis which might be a novel therapeutic target for breast cancer.

Citation Format: Masahiro Kawashima, Karim Bensaad, Christos Zois, Alessandro Barberis, Esther Bridges, Christoffer Lagerholm, Ruslan I. Dmitriev, Masakazu Toi, Dmitri B. Papkovsky, Francesca Buffa, Adrian L. Harris. Fatty acid binding protein 7 regulates beige fat-like differentiation of breast cancer cells and thermogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3563.
Abstract 361: Pleckstrin-2 as a prognostic factor and mediator of gastric cancer progression
Authors: Sun Bo , Xiang Jian-Bin , Xuan Yi , Chen Zong-You , Gu Xiao-Dong , Xu Da-Zhi
Published: September 2020

Background: Pleckstrin-2 (PLEK2) is a crucial mediator of cytoskeletal reorganization. However, the potential roles and clinical significance of PLEK2 in gastric cancer are still unknown.

Methods: We aimed to illustrate the function of PLEK2 in gastric cancer. Pearson's correlation analysis was utilized to test the clinical impacts of the levels of PLEK2 in gastric cancer patients. Loss-of-function approaches were used to estimate the potential roles played by PLEK2 in modulating gastric cancer proliferation, self-renewal, and tumourigenicity. Immunoblotting and bioinformatics approaches were used to monitor the effect of PLEK2 on epithelial-mesenchymal transition (EMT) signalling pathways.

Results: In gastric cancer tissue samples examined by western blot and real-time PCR, there was an increased level of PLEK2 compared with that in matched non-tumour samples. Kaplan-Meier plotter analysis revealed that gastric cancer patients with higher PLEK2 levels had substantially poorer overall survival (p=0.0028) compared with gastric cancer patients with lower PLEK2 levels. The upregulation or downregulation of PLEK2 in gastric cancer cell lines effectively enhanced or inhibited cell proliferation and pro-invasive behaviour, respectively. Additionally, we also found that PLEK2 enhanced EMT concomitantly with a reduction in E-cadherin and an elevation in Vimentin.

Conclusions: Our findings demonstrated that PLEK2 plays a potential role in gastric cancer and may act as a prognostic factor for gastric cancer.

Citation Format: Bo Sun, Jian-Bin Xiang, Yi Xuan, Zong-You Chen, Xiao-Dong Gu, Da-Zhi Xu. Pleckstrin-2 as a prognostic factor and mediator of gastric cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 361.
Abstract 3689: Mutation spectrum of MEF cells transformed by overexpression of novel activating KRAS mutation candidates in lung adenocarcinoma
Authors: Abe Jiro , Tanuma Nobuhiro , Nomura Miyuki , Ito Shin , Kasugai Isao , Sato Ikuro , Tamai Keiichi , Mochizuki Mai , Yamaguchi Kazunori , Shima Hiroshi , Okada Yoshinori , Yasuda Jun
Published: September 2020

Lung adenocarcinoma (LUAC) is a unique lung cancer subtype that is responsive to several therapeutic agents. The KRAS gene is the second most frequently mutated gene in LUAC and the majority of KRAS mutations are one of three classical activating mutations (G12, G13, and Q61). Recently, we have characterized other types of “minor” KRAS mutation (A66T, A66V, and G75E) identified among LUAC patients and found these three mutants had tumor-forming activity in mouse embryonic fibroblasts in an allograft model. RNA-Seq analysis revealed that mouse embryonic fibroblasts overexpressing these three minor KRAS mutations have distinct expression profiles compared with overexpression of the wild type but similar expression profiles compared with overexpression of the classical KRAS mutants, G12V. In this study, we have searched candidate somatic genetic alterations in the RNA-seq data and will discuss the impact of the mutations in KRAS carcinogenesis.

Citation Format: Jiro Abe, Nobuhiro Tanuma, Miyuki Nomura, Shin Ito, Isao Kasugai, Ikuro Sato, Keiichi Tamai, Mai Mochizuki, Kazunori Yamaguchi, Hiroshi Shima, Yoshinori Okada, Jun Yasuda. Mutation spectrum of MEF cells transformed by overexpression of novel activating KRAS mutation candidates in lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3689.
Abstract 3707: Transcriptional downregulation of microRNA 199a-1 in the tumor microenvironment confers chemoresistance and stemness in ovarian cancer cells
Authors: Hassan Ayon A. , Yang Zhenyu , Shum Ho-Cheung , Lai Hung-Cheng , Wong Alice S.
Published: September 2020

The tumor microenvironment is actively involved in cancer progression and is becoming an attractive therapeutic target. It is increasingly clear that both biotic and abiotic factors in the microenvironment play a crucial role in disease progression. Using a three-dimensional microfluidic platform that mimics the peritoneum, we previously demonstrated that physiologically relevant shear-stress can induce chemoresistance and stemness in ovarian cancer cells. Importantly, this mechanical stimulus resulted in downregulation of microRNA (miR)199a-3p, suggesting a previously unknown mechanosensitive property of this miRNA. In testing other microenvironmental factors known to be involved in ovarian cancer progression in the regulation of miR-199a-3p, we found that hepatocyte growth factor (HGF), which is abundant in malignant ascites, could decrease miR-199a-3p expression in a similar manner to shear stress. Both shear stress and HGF induced this downregulation through a transcriptional regulation of primary mir-199a-1, but not primary mir-199a-2, via a PI3/Akt signaling pathway. Using metanalysis, there was also a decrease of miR-199a-3p in patients' tumor samples, and an increase of Akt in high-risk group. Moreover, ectopic overexpression of primary mir-199a-1 could sensitize ovarian cancer cells to cisplatin and reduce their ability to form multicellular spheroids, confirming a role for this miRNA in chemoresistance and stemness. Taken together, our findings suggest a causal involvement of downregulation of primary mir-199a-1, and ultimately its mature counterpart miR-199a-3p, in the tumor microenvironment mediated induction of stemness and chemoresistance, and represents a potential therapeutic target for the treatment of ovarian cancer (This work is supported by HMRF 05163536).

Citation Format: Ayon A. Hassan, Zhenyu Yang, Ho-Cheung Shum, Hung-Cheng Lai, Alice S. Wong. Transcriptional downregulation of microRNA 199a-1 in the tumor microenvironment confers chemoresistance and stemness in ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3707.
Abstract 3722: Extracellular arginine starvation imposes DNA replication fork stall and permits DNA damage tolerance
Authors: Wang Yi-Chang , Kelso Andrew A. , Chen Yi-Hsuan , Hsieh Chi-An , Chen Wei-Kai , Stark Jeremy M. , Kung Hsing-Jien , Ann David K.
Published: September 2020

Regional nutrients deprivation, which is frequently encountered in many solid tumors due to poor vascularization, induces metabolic rewiring and genetic dysregulation to support the demands of proliferating cancer cells. Numerous chemotherapies exert their S-phase dependent cytotoxicity by producing deleterious effects on DNA to kill proliferating tumor cells. How regional nutrients deprivation affects the response of cancer cells to chemotherapy, especially DNA damaging genotoxicity, and what is the key nutrient dominates these effects is an active area of interest across tumor biology. Of all the amino acids we examined, only arginine is regional deprived in tumor core and indispensable for S-phase progression in multiple breast cancer cells and non-tumorigenic breast epithelial cells. We found that arginine deprivation stalls DNA replication in an mTORC1 inactivation, insufficient metabolites, or the integrated stress response pathway independent manner. We also found that low levels of newly synthesized Histone H4 marks (H4K5ac and H4K12ac), which were reported to be associated with adverse outcomes in various cancers including basal-like and HER2-positive breast cancer, are positively correlated with arginine bioavailability and could serve as surrogate markers of arginine sufficiency. Mechanistically, extracellular arginine is required for the translation of DNA-synthesis-dependent histone H4, but not H3, to promote DNA replication. Knockdown of arginyl-tRNA-transferase, like arginine deprivation, recapitulates the loss of histone H4 lysine 5 and 12 acetylation marks and compromises replication fork speed. Notably, one round of arginine deprivation and addition did not cause an increase in γH2AX, a hallmark of nuclear DNA double-stand breaks. However, multiple rounds of arginine fluctuation promotes the emergence of ATR-Chk1 checkpoint independent-nuclear p53-binding protein 1 bodies in the subsequent G1-phase of daughter cells and induces DNA damage tolerance and resistance to genotoxic agents. Thus, intratumoral variation in the arginine microenvironment synchronizes DNA replication with therapeutic response.

Citation Format: Yi-Chang Wang, Andrew A. Kelso, Yi-Hsuan Chen, Chi-An Hsieh, Wei-Kai Chen, Jeremy M. Stark, Hsing-Jien Kung, David K. Ann. Extracellular arginine starvation imposes DNA replication fork stall and permits DNA damage tolerance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3722.
Abstract 3737: Mutant isocitrate dehydrogenase driven metabolic reprogramming results in therapeutic vulnerability to lactate dehydrogenase inhibition
Authors: Bi Aiwei , Xu Jun , Jin Nan , Lan Xiaojing , Tang Shuai , Shou Matthew , Liu Jia , Ding Jian , Geng Meiyu , Lin Shuhai , Huang Min
Published: September 2020

Mutant isocitrate dehydrogenase (mIDH), with a gain-of-function in the production of an oncometabolite (R)-2-hydroxyglutarate (2-HG), is considered as a promising target for cancers carrying IDH mutations. IDH1 mutations, typically involving an amino acid substitution in the active site of the enzyme in codon 132, have been identified in a broad spectrum of solid tumors. The massive production of 2-HG remodels the metabolic network in cancer cells and results in epigenetic alterations facilitating malignant phenotypes. Of note, while the pharmacological inhibition of mIDH1 effectively leads to the remarkable reduction in the intracellular 2-HG level, it seems not associated with the sufficient suppression of solid tumor growth. We hence asked whether it is possible to identify extra metabolic vulnerability in IDH1 mutant tumors. To this end, we performed 13C isotope tracing analysis of glucose and glutamine, two major nutrient supply for 2-HG production, in solid tumor cell lines including HT1080 cells with spontaneous IDH1 mutation and IDH1 mutant glioma cell lines expressing mIDH1. [U-13C6]-glucose tracking enabled us to discover that IDH1 mutated tumors exhibit increased glycolysis and decreased tricarboxylic acid (TCA) metabolism, which can be reversed by mIDH1 inhibition. Meanwhile, cell mitochondrial stress test indicated decreased oxygen consumption rate (OCR) compared with the wildtype counterpart. These data suggest that 2-HG production paralyzes TCA metabolism and subsequent oxidative phosphorylation but enhances aerobic glycolysis, thus reprogramming tumor cells prone to Warburg effect. In line with this hypothesis, lactate dehydrogenase (LDH) inhibition that shut down lactate production increased 2-HG levels in IDH1 mutant cells. Further, LDH inhibition obtained the improved response in IDH1 mutant tumors while combined inhibition of mIDH1 abolished this effect. Together, we discovered that mutant IDH1 impaired TCA cycle by production of millimolar level 2HG which may lead to more dependency on glycolysis.

Citation Format: Aiwei Bi, Jun Xu, Nan Jin, Xiaojing Lan, Shuai Tang, Matthew Shou, Jia Liu, Jian Ding, Meiyu Geng, Shuhai Lin, Min Huang. Mutant isocitrate dehydrogenase driven metabolic reprogramming results in therapeutic vulnerability to lactate dehydrogenase inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3737.
Abstract 375: In vivo metabolic autofluorescence imaging of macrophage heterogeneity across normal and cancerous tissue
Authors: Heaster Tiffany , Esbona Karla , Sondel Paul M. , Skala Melissa C.
Published: September 2020

Tissue microenvironmental conditions affect macrophage metabolism and function. However, standard methods to measure macrophage function require tissue dissociation (e.g. flow cytometry) or tissue fixation (e.g. immunohistochemistry), which remove the cells from their native microenvironment and prevent in vivo monitoring. Autofluorescence imaging enables non-invasive, dynamic imaging of cell metabolism in live, intact samples by measuring fluorescence from metabolic co-enzymes, NAD(P)H and FAD, naturally present in all cells.

Here, we test autofluorescence imaging to assess macrophage function in vivo within normal skin and tumor microenvironments. Transgenic mice harboring a floxed mCherry fusion protein controlled by the Csf1r promoter were crossed with Lyz2-Cre mice to generate a model of mCherry-expressing macrophages. Multiphoton wavelength mixing was used to simultaneously image mCherry, NAD(P)H, and FAD fluorescence in spectrally distinct channels. NAD(P)H and FAD fluorescence intensities and lifetimes were quantified for tissue-resident macrophages in the mouse ear dermis and tumor-associated macrophages in Panc02 pancreatic tumors engrafted on the flank.

Spatial distributions of macrophages were visualized at each tissue site. Dermal macrophages were predominantly localized around hair follicles in the ear, while tumor-associated macrophages were randomly distributed throughout the tumor. Co-localization of mCherry and FAD signal indicated high FAD intensities within macrophages across both tissues. Population density modeling of autofluorescence variables on a single cell level in each tissue site indicate metabolic heterogeneity of macrophages within normal and cancerous tissues. Immunofluorescence of standard macrophage markers (CD86/CD206) further characterize diversity in macrophage phenotypes across both tissue microenvironments. These studies demonstrate metabolic autofluorescence imaging as a novel non-invasive tool to assess tissue-specific macrophage metabolism at the single cell level in vivo. Ultimately, this imaging approach could provide insight on the role of macrophage plasticity in tumor growth and treatment response in vivo.

Citation Format: Tiffany Heaster, Karla Esbona, Paul M. Sondel, Melissa C. Skala. In vivo metabolic autofluorescence imaging of macrophage heterogeneity across normal and cancerous tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 375.
Abstract 3754: PPP6C regulation of ERK signaling in melanoma
Authors: Cho Eunice , Lou Hua Jane , Turk Benjamin E.
Published: September 2020

Melanoma, the deadliest form of skin cancer, is characterized by aberrant hyperactivation of the ERK mitogen-activated protein kinase signaling pathway. Inhibitors of BRAF and MEK1/2, both members of this pathway, have shown clinical efficacy and are used in combination to treat BRAFV600E melanoma, the most common melanoma genomic subtype. However, not all BRAFV600E melanomas respond to these inhibitors, and those that do respond eventually acquire resistance. To better understand mechanisms of drug susceptibility in BRAFV600E melanoma, we performed a loss-of-function screen to identify kinases and phosphatases that modulate sensitivity to two clinical MEK1/2 inhibitors. In this screen, we identified PPP6C, the catalytic subunit of protein phosphatase 6 (PP6), as a factor promoting sensitivity to MEK1/2 inhibition. PPP6C is a potential tumor suppressor mutated in 7-12% of melanomas, and has roles in cell cycle regulation and DNA damage repair. shRNA-mediated knockdown and CRISPR/Cas9-mediated knockout of PPP6C in BRAF mutant melanoma cells both lead to hyperactivation of MEK1/2 and ERK1/2, resulting in decreased sensitivity to pathway inhibitors. This finding was confirmed in a panel of BRAFV600E melanoma cell lines. Through in vitro biochemical and cell-based studies, we have established that PP6 directly dephosphorylates MEK1/2. As with complete loss of PPP6C, cells expressing most recurrent cancer-associated PPP6C mutants similarly have hyperactive ERK signaling. Our studies support a novel negative regulatory role for PPP6C in ERK signaling that contributes to its activity as a tumor suppressor and promotes pathway inhibitor sensitivity in BRAFV600E melanoma.

Citation Format: Eunice Cho, Hua Jane Lou, Benjamin E. Turk. PPP6C regulation of ERK signaling in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3754.
Abstract 3758: Impact of ANXA7 I1 expression on PDGFRA and MET endosomal trafficking in glioblastoma
Authors: White Zachary B. , Hurt Centdrika Dates , Rajbhandari Rajani , Nair Sindhu , Nozell Susan , Bredel Markus
Published: September 2020

Purpose/Objective(s): Glioblastomas (GBM) are highly malignant tumors that arise from astrocytes. GBMs rely on signaling through receptor tyrosine kinases (RTK) to ensure tumorigenicity. EGFR, PDGFRA and MET are distinct RTKs; each is capable of binding a ligand, which promotes receptor dimerization and kinase activation. Annexin A7 (ANXA7), a protein involved in membrane binding and vesicle trafficking, is spliced into 2 isoforms - isoforms 1 (I1) and isoform 2 (I2). A normal adult brain will express both isoforms, however, in GBM, I1 expression is prevented. We determined that I1, but not I2, is tumor suppressive, in part, because it promotes the endocytic degradation of EGFR. Because I1 is typically not expressed in GBM, EGFR signaling persists unabated and tumors thrive. Recently, we extended these observations and determined that I1 also impacts additional RTKs, including PDGFRA and MET. By understanding how ANXA7 I1 impacts PDGFRA and MET signaling, we hope to target multiple tumorigenic RTKs and improve therapy for patients with GBM.

Materials/Methods: To understand how and when I1 downregulates PDGFRα and MET, U251-malignant glioma (MG) cells, which only express I2 (P), were modified to express I1. Cells were serum starved overnight, and then stimulated with PDGFAA and HGF(20 ng/ml), which activate the aforementioned receptors respectively. As a positive control, cells were stimulated with EGF to activate EGFR. Total protein was then collected and analyzed by western blot analyses. Next, we sought to determine whether I1 was competent to down-regulate several RTKs coincidentally. To do this, P and I1 cells were serum starved, stimulated with EGF, PDGFAA or both, and total protein levels were evaluated.

Results: The levels of activated EGFR, PDGFRα and MET are dramatically reduced in those cells that express I1 as compared to cells expressing I2 (P). Upon activation with both PDGFAA and EGF, in cells expressing I1 (I1), levels of PDGFRα and EGFR were markedly reduced 30 minutes post-stimulation as compared to cells expressing I2.

Conclusion: Our results suggest I1 may be a master regulator of RTK endocytosis and degradation. Further investigations of the impact of I1 on endocytic trafficking patterns using immunofluorescence and confocal microscopy are underway. Collectively, understanding how I1 functions and targets multiple tumorigenic RTKs will provide the foundation for future selective therapies that restore I1 expression in GBM and reduce tumorigenicity.

Citation Format: Zachary B. White, Centdrika Dates Hurt, Rajani Rajbhandari, Sindhu Nair, Susan Nozell, Markus Bredel. Impact of ANXA7 I1 expression on PDGFRA and MET endosomal trafficking in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3758.
Abstract 376: Intravital microscopy reveals HER2-dependent and -independent effects of myeloid cells on the single-cell biodistribution of trastuzumab
Authors: Li Ran , Weissleder Ralph , Miller Miles A.
Published: September 2020

Advents in single-cell profiling have revealed that tumor tissues are highly heterogenous, and this cell-to-cell heterogeneity can greatly affect therapeutic efficacy. This is especially true for monoclonal antibody (mAb) therapies that exhibit variability in both delivery and action within solid tumors. Moreover, recent studies have highlighted the importance of tumor microenvironment in determining mAb responses. Yet, it has been traditionally difficult to analyze the kinetics of mAb biodistribution and delivery at a single-cell resolution, as well as how mAb interacts with tumor-associated stromal cells in vivo. To address these issues, we developed a high-resolution intravital microscopy (IVM) pipeline to study single-cell pharmacokinetics and pharmacodynamics of mAb in a tumor consists of cancer cells with variable target expression. We applied this imaging strategy to track the in vivo binding of trastuzumab (Tzm), a HER2-targeting mAb, to fluorescently-labeled HER2 in the heterogenous tumor microenvironment. We observed efficient binding of Tzm to HER2+ cancer cells, and the amount of Tzm on cancer cells correlated with HER2 expression level. We also discovered a large accumulation of Tzm in tumor-associated macrophages (TAMs). Time-lapse IVM imaging further revealed an increase in Tzm uptake in TAMs over the period of 72 hrs, with concomitant decrease in Tzm level on the HER2+ cancer cells. Further controlled experiments demonstrated that the baseline ability of TAMs to take up Tzm was not dependent on HER2 expression in the surrounding cancer cells, yet HER2 over-expression can increase the levels of Tzm in TAMs. All in all, these results revealed crucial roles that tumor-associated stromal cells play in mAb delivery and action. They also demonstrated the utility of IVM in dissecting mAb behaviors in heterogenous tumor microenvironment, which can significantly impact our understanding of mAb treatment efficacy.

Citation Format: Ran Li, Ralph Weissleder, Miles A. Miller. Intravital microscopy reveals HER2-dependent and -independent effects of myeloid cells on the single-cell biodistribution of trastuzumab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 376.
Abstract 3761: HSK26784: An oral PROTAC-BTK degrader for multiple B lymphocyte derived malignancies
Authors: Qian Meilin , Ye Fei , Zhang Chen , Wang Jianming , Zhang Yuanli , Cui Yangyang , Li Linli , Gou Xiaoli , Ni Jia
Published: September 2020

PROteolysis TArgeting Chimeras (PROTACs) have emerged as a new and promising modality utilizing an event-driven MOA, whereby protein levels could be modulated by PROTAC-induced degradation by hijacking the ubiquitin-proteasome system. Starting from 2001, many target proteins have been studied, such as BET and kinase families as well as nuclear receptors. Bruton's tyrosine kinase (BTK) is an essential component of BCR pathway, which plays an important role in development, activation, proliferation, and survival of B lymphocytes. Ibrutinib is an irreversible inhibitor of BTK, and shows promising activity against multiple B lymphocyte derived malignancies. However, there remains a big concern about the development of Ibrutinib induced resistance by BTK C481S mutant. In our study, HSK26784 was found as a novel orally available BTK-PROTAC molecule, which could selectively degrade the BTK proteins. HSK26784 selectively degraded BTK proteins in Mino cell lines, with a half degradation concentration (DC50) of 22.9 nM. HSK26784 also effectively induced BTK degradation in mouse spleen, with a DC50 of 3.8 mg/kg. In addition, HSK26784 significantly inhibit the expression of CD69 on human PBMC cells after ex vivo BCR stimulation. HSK26784 could inhibit cell proliferation in many malignant B cell lines. In Ibrutinib sensitive cell lines, the inhibitory activity of HSK26784 is similar to that of Ibrutinib. While in the Ibrutinib non-sensitive cell lines, HSK26784 was more potent than Ibrutinib. Importantly, in the xenograft models, HSK26784 could also inhibit the tumor growth in a dose dependent manner. And HSK26784 achieved much better therapeutic effects than Ibrutinib. HSK26784 could inhibit the kinase activity of BTK wt and BTK C481S mutant, with IC50 of 11 nM and 14 nM respectively. It indicated that the efficacy of HSK26784 in BTK wt and BTK C481S mutant was comparable. HSK26784 showed good orally bioavailability in different species. The oral bio availabilities in mice, rats and dogs were 15.4 %, 11.8 % and 12.8 % respectively. In conclusion, HSK26784 is a promising BTK PROTAC degrader with better efficacy than Ibrutinib in multiple B lymphocytes.

Citation Format: Meilin Qian, Fei Ye, Chen Zhang, Jianming Wang, Yuanli Zhang, Yangyang Cui, Linli Li, Xiaoli Gou, Jia Ni. HSK26784: An oral PROTAC-BTK degrader for multiple B lymphocyte derived malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3761.
Abstract 3772: Comprehensive characterization of the phosphoproteome of gastric cancer from endoscopic biopsy specimens
Authors: ADACHI Jun , Abe Yuichi , Hirano Hidekazu , Shoji Hirokazu , Gunji Daigo , Honda Kazufumi , Boku Narikazu , Tomonaga Takeshi
Published: September 2020

Cancer phosphoproteomics can provide insights regarding kinases that can be targeted for therapeutic applications. Monitoring the phosphoproteomics in cancer is expected to play a key role in optimizing treatments with kinase inhibitors. Clinical phosphoproteomics in surgical tissues and patient-derived models has been studied intensively. However, the reported data may not accurately reflect the phosphosignaling status in patients due to the effect of ischemia occurring during surgery or changes in the characteristics of cancer cells when establishing the models. In contrast, endoscopic biopsies have an advantage for clinical phosphoproteomics because they can be rapidly cryo-preserved. We aimed to develop a highly sensitive method for phosphoproteomics in endoscopic biopsies of gastric cancer.Three tumor/normal biopsies from gastric cancer tissue were obtained by endoscopy, and subjected to our optimized phosphoproteomics. Phosphopeptides were enriched with an immobilized metal affinity chromatography, and labeled with Tandem Mass Tag reagent. Quantified phosphosites were compared between the pairs of tumor/normal biopsies within same patient. Cancer-specific activated pathways and kinases were identified by pathway enrichment analysis and kinase-substrate enrichment analysis.Our protocol enabled the identification of more than 10,000 class-1 phosphosites from endoscopic biopsies. A comparison between samples from cancer tissue and normal mucosa demonstrated differences in the phosphosignaling, including biomarkers of response to DNA damage. Finally, cancer-specific activation of DNA damage response signaling was validated by additional phosphoproteomics of other patients and western blotting of gastric cancer/normal cells. In summary, our pioneering approach will facilitate more accurate clinical phosphoproteomics in endoscopic biopsies, which can be applied to monitor the activities of therapeutic kinases and, ultimately, can be a useful tool to precision medicine.

Citation Format: Jun ADACHI, Yuichi Abe, Hidekazu Hirano, Hirokazu Shoji, Daigo Gunji, Kazufumi Honda, Narikazu Boku, Takeshi Tomonaga. Comprehensive characterization of the phosphoproteome of gastric cancer from endoscopic biopsy specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3772.