bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3755: PDGF-induced tumor cell growth despite VEGF/EGF inhibition and absence of PDGF receptor expression in colon cancer
Authors: Moench Romana , Gasser Martin , Nawalaniec Karol , Grimmig Tanja , Cao Minghua , Luo Yueming , Hsiao Li-Li , Waaga-Gasser Ana Maria
Published: September 2020

Anti-Epithelial/Vascular Endothelial Growth Factor (EGF/VEGF)-targeted therapy plays important roles in advanced stage colorectal cancer (CRC) patients but fails to show clinical efficacy in specific subgroups which lack of clear underlying mechanisms. In vitro studies indicated that cross-talk or alternative receptor/ligand activities can bypass anti-growth factor signaling arrest and therefore may explain failure of monoclonal antibody (mAb) targeted therapy. In this study we analyzed effects of Platelet Derived Growth Factor (PDGF)-mediated bypassed signaling in CRC. We first investigated human UICC (Union for International Cancer Control ) stage I-IV tumors (n=46) from our Center and found increased PDGFRβ and VEGFR1/2 gene expression (p<0.01/p<0.001). Interestingly, further analysis on human HT29 colon cancer in vitro showed upregulated VEGFR1/2 expression upon VEGF stimulation. This was even more pronounced upon stimulation with PDGF. Inhibition of VEGFR2 and EGFR resulted in decreased HT29 cell proliferation, while additional treatment with VEGF and particularly PDGF attenuated the inhibiting effect and resulted in altered downstream signaling events. Treatment with the tyrosine kinase inhibitor Regorafenib alone, that blocks VEGFR/EGFR+PDGFR, prevented PDGF and VEGF-induced proliferation. This effect was not observed for either VEGFR2 mAb treatment with Ramucirumab or EGFR mAb therapy with Cetuximab. To conclude, PDGF-induced human colon cancer proliferation in the absence of PDGFR and simultaneously inhibited VEGFR2 and EGFR expression in HT29 colon cancer cells suggests bypassed alternative PDGF receptor partners for ligand binding on the tumor cell surface and subsequent intracellular signaling. This may explain failure of anti-VEGF/EGF-targeted monoclonal antibody therapies in CRC patient subgroups and underlines the necessity to effectively inhibit PDGF-mediated signaling events beside VEGF/EGF blockade in patients with increased PDGF expression within their tumors.

Citation Format: Romana Moench, Martin Gasser, Karol Nawalaniec, Tanja Grimmig, Minghua Cao, Yueming Luo, Li-Li Hsiao, Ana Maria Waaga-Gasser. PDGF-induced tumor cell growth despite VEGF/EGF inhibition and absence of PDGF receptor expression in colon 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 3755.
Abstract 3765: c-Kit mediated phosphorylation of Spred1 regulates Neurofibromin-Spred1 interaction
Authors: Lorenzo Claire , Young Lucy C. , Tannka Alexandra , Urisman Anatoly , McCormick Frank
Published: September 2020

Spred1 negatively regulates Ras/MAPK signaling following growth factor stimulation. Spred1 inhibits Ras by binding and localizing Neurofibromin, a RasGAP, to the plasma membrane to accelerate Ras GTPase activity. c-Kit, a receptor tyrosine kinase (RTK), is known to interact with Spred1 but the consequence of this interaction is unknown. Here we demonstrate that c-Kit signaling regulates Neurofibromin-Spred1 interaction. Stimulation with c-Kit ligand, SCF, results in a transient disruption in Neurofibromin-Spred1 binding which corresponds to increased Ras signaling, followed by restoration of Neurofibromin-Spred binding which corresponds to nearly basal levels of Ras signaling. Mass spectrometry analysis identified potential phosphorylation sites on Spred1 that correspond to the initial disruption and later restoration of Neurofibromin-Spred1 binding. Phosphomimetic and phosphodeficient mutants affect the interaction. Our findings provide a potential mechanism by which RTK signaling regulates negative feedback to allow transient activation and subsequent termination of Ras signaling.

Citation Format: Claire Lorenzo, Lucy C. Young, Alexandra Tannka, Anatoly Urisman, Frank McCormick. c-Kit mediated phosphorylation of Spred1 regulates Neurofibromin-Spred1 interaction [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 3765.
Abstract 3777: The role of IFITMs in maintaining glioma stem cells
Authors: Nita Erisa , Evers Bernard , Nekulova Marta , Morrison Gillian , Ball Kathryn
Published: September 2020

Glioblastoma (GBM), one of the most lethal and heterogeneous primary brain tumors, contains cellular hierarchies with both quiescent and self-renewing highly tumorigenic glioblastoma stem cells (GSCs). Cancer stem cells within the context of the brain likely contribute to a lack of progress in patient survival time as they appear to have an innate resistance to both chemotherapeutic drugs and radiotherapy. The aim of this study is to elucidate how constitutive expression of interferons (IFNs) and their downstream signalling pathways maintain GSCs thereby supporting treatment resistance and tumor reoccurrence. To understand the mechanism of action of a subset of IFN regulated genes, originally characterized as a signature for radiation and chemotherapy resistance (IRDS), a panel of patient derived glioma stem cells was generated and manipulated using CRISPR/Cas9 mediated gene-editing. Phenotypic changes in growth, viability and drug resistance were characterised in the edited cells and linked to molecular level events using transcriptomic and proteomic techniques. Using this approach, in conjunction with patient tissue analysis, signalling under basal as well as IFN-activated conditions was studied in the context of GSC status.

Immunohistopathology of a glioma tissue micro-array identified an increased expression of IFN induced transmembrane receptors (IFITMs), which co-localised with Nestin, around the blood vessels suggesting that IFITM positive cells were in the stem cell niche. Further analysis showed the presence of both IFITM1 and IFITM3 in GSCs and established that loss of IFITM function impacted on growth rate and cell cycle progression as well as treatment sensitivity. By using wild-type and gene-edited GSC to focus on the relationship between IFITM1 and IFITM3, we have compelling phenotypic and molecular evidence that IFN-induced changes in the localisation of IFITMs impact on the ability of stem cells to ‘escape' normal growth control mechanisms. Taking into consideration the constitutive expression of interferons in the tumor microenvironment, our study provides evidence that IFITMs regulate glioma stem cell self-renewal and are involved in maintaining a quiescent population which can escape chemotherapy.

Citation Format: Erisa Nita, Bernard Evers, Marta Nekulova, Gillian Morrison, Kathryn Ball. The role of IFITMs in maintaining 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 3777.
Abstract 3778: MBNL1 regulated alternative splicing of MAP2K7 promotes de-differentiation of cancer cells by activating JNK
Authors: Ray Debleena
Published: September 2020

Therapeutic targeting of cancer stem cells (CSCs) is key to relapse-free, lasting response to cancer treatment. Several strategies against CSCs are currently in various stages of clinical development. However, there is need for additional targets. Here, we demonstrate that MBNL1, an RNA binding protein, is a suppressor of cancer-stemness. RNAi mediated down-regulation of MBNL1 promotes tumor-initiation, self-renewal, migration and invasion by up-regulating CSC-specific splice-isoforms. Particularly, the up-regulation of MAP2K7Δexon2 splice-isoform activates JNK signaling and establishes a feedback loop that promotes cancer-stemness. Pharmacological inhibition of JNK reverses CSC phenotypes in MBNL1low/MAP2K7Δexon2high CSC-like cells and these cells also show enhanced susceptibility to JNK inhibition. Remarkably, MBNL1 is down-regulated and MAP2K7Δexon2 splice-isoform is up-regulated in several cancer types. Down-regulation of MBNL1 is correlated with poor survival in gastric, lung and breast adenocarcinoma. In triple negative breast cancer, which is a highly aggressive disease characterised by enrichment of CSC-like cells, low MBNL1 correlates with poor relapse-free and distant metastasis-free survival. Our work shows, low MBNL1 and consequently high MAP2K7Δexon2 expression, is a biomarker for cancer stemness, high JNK activity and enhanced susceptibility to JNK inhibition. Hence, we have identified a novel alternative splicing driven mechanism of cancer cell de-differentiation and have identified biomarkers that can prognosticate patients for JNKi as a strategy to target CSCs.

Citation Format: Debleena Ray. MBNL1 regulated alternative splicing of MAP2K7 promotes de-differentiation of cancer cells by activating JNK [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 3778.
Abstract 3794: Mammary cancer stem cells promote an aggressive phenotype in letrozole resistant breast cancer
Authors: Gallegos Karen Melissa , Patel Jankiben , Walker Rashidra R. , Davidson A Michael , Davenport Ian R. , Tilghman Syreeta L.
Published: September 2020

Aromatase inhibitors (AI), like letrozole, are the first-line treatment for ER+ breast cancer in post-menopausal women. Despite widespread successful usage of letrozole, resistance to therapy, tumor relapse, and metastasis remain the principal causes of death for breast cancer patients. While there are no cures for AI-resistant breast cancer, previous reports demonstrate AI resistance is associated with hormone independence, increased growth factor signaling, enhanced cellular motility and the epithelial to mesenchymal transition (EMT). Given recent evidence suggesting a convergence of EMT and cancer stem cells (CSC) and their combined implications in endocrine resistance, we hypothesized that breast cancer stem cells contribute to letrozole resistant breast cancer cells by increasing cellular motility. To test this hypothesis, ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with either MCF-7 cells stably transfected with the human aromatase gene that were long term letrozole-treated (LTLT-Ca) or letrozole sensitive, MCF-7 cells stably transfected with the human aromatase gene (AC-1). Afterwards, intratumoral putative CSC marker expression was assessed by immunohistochemistry. Results indicate LTLT-Ca tumors were CD44+/CD24+ while AC-1 tumors were CD44low/CD24low. Both cell lines were cultured either adherently (2D) or as mammospheres (3D) and CD44/CD24 protein expression was assessed by immunoblots. When both cell lines were cultured three dimensionally, they expressed higher levels of CD44 and CD24 compared to their adherent counterparts, with the most robust change occurring within the LTLT-Ca cells. In order to quantitate the breast cancer stem cell activity mammosphere formation assay was performed and LTLT-Ca cells formed mammospheres at a 3.4-fold higher index compared to AC-1 cells. Additionally, we performed targeted gene expression arrays and our results demonstrated decreased expression of genes involved in cell adhesion and tumor suppression (ie., E-cadherin, caveolin 1 and β catenin). Taken together our study demonstrated a strong correlation between AI resistance, increased cancer stem cell expression, an invasive phenotype that may be associated with a poor prognosis. This work was supported by an NIH grant awarded to SL Tilghman (1SC1GM125617).

Citation Format: Karen Melissa Gallegos, Jankiben Patel, Rashidra R. Walker, A Michael Davidson, Ian R. Davenport, Syreeta L. Tilghman. Mammary cancer stem cells promote an aggressive phenotype in letrozole resistant breast 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 3794.
Abstract 3806: Cigarette smoke extract permits survival of TET2-/- and JAK2V617F hematopoietic cells
Authors: Ramanathan Gajalakshmi , Craver Brianna , Arechavala Rebecca J. , Herman David , Kleinman Michael T. , Fleischman Angela G.
Published: September 2020

Inactivating somatic mutations in the ten-eleven translocation 2 (TET2) gene is commonly found in clonal hematopoiesis (CH) and contributes to the pathogenesis of myeloid malignancies such as myeloproliferative neoplasms (MPN), myelodysplastic syndrome and acute myeloid leukemia. Incidentally, smoking behavior is associated with CH and we hypothesize that TET2 mutant hematopoietic cells are resistant to the deleterious effects of smoke exposure.

Cigarette smoke extract (CSE) was prepared from mainstream cigarette smoke using a standard protocol and volume/volume percentage (v/v %) dilutions used for in vitro exposures. Lineage negative bone marrow cells isolated from TET2 wildtype (TET2+/+) and TET2 knockout (TET-/-) mice were exposed to CSE and cultured in methylcellulose containing semi-solid medium to assess myeloid colony formation. Cells were also grown in liquid culture under long-term culture conditions to demonstrate the effect of CSE on cell proliferation. Peripheral blood mononuclear cells from JAK2V617F positive MPN patients were treated with CSE for colony forming assays.

TET2+/+ bone marrow cells showed decreased myeloid colony formation in the presence of CSE while TET2-/- cells were not significantly affected by CSE. Similarly in liquid cultures, TET2-/- lineage negative cells expanded significantly compared to their wildtype counterparts indicating resistance to the growth suppressive effects of CSE. The JAK2V617F mutation is the most common somatic mutation found in MPN patients and we observed that the suppressive effect of CSE on myeloid colony formation was more pronounced in normal subjects than JAK2V617F positive MPN subjects.

This suggests that CH associated somatic mutations such as in TET2 promote resistance of bone marrow progenitors to environmental stressors like cigarette smoke, possibly via anti-apoptotic mechanisms. Future studies include studying the effects of inhalation exposure of combustible and electronic cigarette smoke on TET2-/- and JAK2V617F hematopoiesis in competitive bone marrow transplanted mice.

Citation Format: Gajalakshmi Ramanathan, Brianna Craver, Rebecca J. Arechavala, David Herman, Michael T. Kleinman, Angela G. Fleischman. Cigarette smoke extract permits survival of TET2-/- and JAK2V617F hematopoietic 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 3806.
Abstract 3807: Exposure to environmental chemicals leads to extensive branching and expression of cancer-promoting proteins in human mammary gland organoids
Authors: Hinrichs Johanna H. , Winkler Juliane , Liu Pengyuan , Ataii Nassim , Fisher Susan , Werb Zena
Published: September 2020

Bisphenol A (BPA) is a chemical compound used as a plasticizer in a variety of plastic goods. Environmental chemicals like BPA haven been detected in the blood of women and girls. Yet, they are thought to interact with the endocrine system, thereby having carcinogenic effects on the tissue. While BPA is removed from many commercial products because of its potential carcinogenic effect, surprisingly little is known about its replacement chemicals, such as bisphenol S (BPS) and bisphenol F (BPF). In this study, we aim to link the exposure to those environmental chemicals to changes in human mammary gland morphology and to the underlying alterations at the protein level.

We established organoid cultures from non-malignant primary human mammary gland tissue as advanced disease model. Human breast organoids were treated with physiological levels of various environmental chemicals, includingBPA, BPF, BPS, 17-β-estradiol (E2), or DMSO as vehicle control. Effects on organoid morphology were studied using brightfield and confocal microscopy. We analyzed relative protein abundances by quantitative mass spectrometry to define proteome changes between the different treatments.

The exposure to the environmental chemicals as well as E2 resulted in altered branching morphology while organoid size was not affected. It is already known that BPA has oncogenic potential. Surprisingly, in our study, BPA has the least severe effects when compared to the other bisphenols. BSF or BPS treatment lead to more extensive branching and changed morphology.On protein level, each environmental chemical resulted in distinct protein profiles which show proteins alterations independent of endocrine signaling. Moreover, the exposure to the different bisphenols resulted in the upregulation of proteins which are involved in carcinogenesis, tumor progression and metastasis in various cancer types including breast cancer and thus may support tumor development.

In this study, we identified BPS and BPF as potential carcinogens. Not only BPA, but also BPF and in particular BPS alter branching morphology of mammary organoids and influence various biological processes that are distinct from endocrine signaling. Our study highlights the desperate need of a thorough characterization of environmental chemicals to prevent the development of breast cancer caused by the exposure to potential harmful and carcinogenic substances.

Citation Format: Johanna H. Hinrichs, Juliane Winkler, Pengyuan Liu, Nassim Ataii, Susan Fisher, Zena Werb. Exposure to environmental chemicals leads to extensive branching and expression of cancer-promoting proteins in human mammary gland organoids [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 3807.
Abstract 3834: Histone methyltransferase SET8 is regulated by miR-192/-215 and induces oncogene-induced senescence via p53-dependent DNA damage in human gastric carcinoma cells
Authors: Zhang Xiaojing , Peng Yin , Hu Fan , Gao Yuli , Zhe Jin , Meltzer Stephenj
Published: September 2020

Purpose: This aim of study was to investigate the clinicopathologic significance and potential role of histone methyltransferase SET8 in the progression of gastric carcinoma (GC).

Experimental Design: SET8 was screened out by gene microarray in gastric cell lines with or without miR-192/-215 inhibitors or mimics. The effects of SET8 on cell proliferation, invasion, and metastasis were examined using EdU assays, invasion studies, subcutaneous tumor experiments, and tail-vein injection in nude mice. Immunohistochemical staining was performed to detect SET8 expression in GC tissues, and real-time PCR was performed to examine the correlations between miR-192/-215 and SET8. SA-β-Gal staining assays were performed to identify the occurrence of senescence. Senescence-associated proteins were examined by real-time PCR and Western blotting.

Results: SET8 regulated by miR-192/-215 displayed decreased expression in GC. Inhibition of miR-192/-215 resulted in weakened proliferation, migration, and invasion in GC. The 3'UTR of SET8 was targeted by miR-192/-215, and SET8 siRNA treatment successfully rescued the biological phenomenon. SET8 was found to trigger oncogene-induced senescence (OIS) in GC in vivo and in vitro, highlighting the functional mechanism of the miR-192/-215/SET8 axis in GC. During OIS, p53 and p21 were key genetic switch points. Taken together, these data show that the miR-192/-215/SET8/p53 circuit is a key regulator of GC promotion and is highly associated with proliferation, migration, and metastasis.

Conclusions: Our findings reveal that SET8 functions via the OIS-signaling pathway as a negative regulator of metastasis. This provides an experimental rationale for investigation of the OIS-signaling pathway as a potential therapeutic target in GC.

Supported by: National Nature Science Foundation of China (81772592) to Z.J.; National Nature Science Foundation of China (81871969) to X.Z.; National Natural Youth Science Foundation of China (31601028) to Y.P.; Guangdong Provincial Key Laboratory of Regional Immunity and Diseases (2019B030301009) to Z.J.

Citation Format: Xiaojing Zhang, Yin Peng, Fan Hu, Yuli Gao, Jin Zhe, Stephenj Meltzer. Histone methyltransferase SET8 is regulated by miR-192/-215 and induces oncogene-induced senescence via p53-dependent DNA damage in human gastric carcinoma 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 3834.
Abstract 3842: Histone methyltransferase G9a promotes migration, invasion of non-small cell lung cancer through enhancing FAK and NF-kB signal pathways
Authors: Sun Ting , Zhang Keqiang , Pangeni Rajendra P. , Li Wendong , Raz Dan J.
Published: September 2020

Overexpression of euchromatic histone methyltransferase 2 (EHMT2 or G9a) is associated with carcinogenesis and poor prognosis in a number of cancer types. In this study, we investigated the effect of G9a on invasion of non-small cell lung cancer (NSCLC) cells and the mechanism underlying cancer cell invasion. Knockdown of G9a was shown to drastically suppress in vitro cellular proliferation, migration, and invasion of A549 and H1299 NSCLC cells, while overexpression of G9a significantly enhanced the proliferation and invasion of these NSCLC cells. Additionally, knockdown of G9a led to significant decrease in expression of focal adhesion kinase (FAK) gene and activation of FAK pathway. UNC0638, a selective inhibitor of G9a, significantly suppressed both the proliferation, migration and invasion of these NSCLC cells, and concurrently led to a dramatic drop of global demethylation of H3K9 (H3K9me2) and suppressed the activation of FAK pathway. In addition, Defactinib, a potent FAK inhibitor, partially abolished the enhanced invasion by overexpression of G9a in these NSCLC cells. Furthermore, G9a was proved to regulate nuclear NF-κB transcriptional activity in NSCLC cells, and NF-κB inhibitor partially abolished the activated FAK signaling pathway by overexpressed G9a, which indicates that G9a-enhanced invasion and activation of FAK pathway may be mediated by NF-κB transcriptional activity. Notably, immunohistochemistry analysis further revealed that activation of FAK pathway was associated with overexpressed G9a in about 150 cases of NSCLC tissues. Altogether, these data have strongly demonstrated that G9a may promote invasion of NSCLC cells by upregulating FAK protein and pathway via enhancing NF-κB transcriptional activity, indicating a therapeutic implication of targeting these two pathways in NSCLC with overexpressed G9a.

Citation Format: Ting Sun, Keqiang Zhang, Rajendra P. Pangeni, Wendong Li, Dan J. Raz. Histone methyltransferase G9a promotes migration, invasion of non-small cell lung cancer through enhancing FAK and NF-kB signal pathways [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 3842.
Abstract 3854: Prevalence and prognostic role of FoxP3regulatory T lymphocytes in cancer: A tissue microarray study on >20,000 cancers
Authors: Mandelkow Tim , Bady Elena , Blessin Niclas , Hube-Magg Claudia , Simon Ronald , Sauter Guido , Fraune Christoph , Lennartz Maximilian , Möller Katharin , Weidemann Sören , Luebke Andreas , Höflmayer Doris
Published: September 2020

Regulatory FoxP3+ lymphocytes function as suppressors of T-cell activity. The clinical impact of high FoxP3+ cell density in cancers is not fully understood, as some studies have linked high FoxP3+ cell density to good prognosis and others to poor prognosis in tumor cohorts with associated clinical data. While some data suggest that these variable data are due to biological differences between tumor entities, it is also possible that methodological differences have caused these discrepancies. This study was undertaken to analyze the density of FoxP3+ cells in various different cancer types by employing standardized methods. Tissue microarrays and large sections made from >20,000 prostate, breast, colorectal, ovarian, pancreatic, bladder and stomach cancers were analyzed together with various normal and inflamed tissues by conventional brightfield FoxP3 immunohistochemistry. Samples were also analyzed by fluorescent multiplex immunohistochemistry to assess the fraction of Ki67+ FoxP3+ cells. Our results indeed suggested a variable role of FoxP3+ cells in different tumor types. High FoxP3+ density was linked to high Gleason grade (p=0.0003) and early biochemical recurrence (p<0.0001) in 16923 prostate cancers, but to low tumor stage (p=0.027) and prolonged survival (p=0.0029) in 1341 breast cancers, and to low tumor stage (p<0.0001) in 744 colorectal cancers. No significant associations were found to tumor phenotype in 549 ovarian, 574 pancreatic, 549 bladder and 346 stomach cancers. Multiplex fluorescence IHC analysis of FoxP3 and Ki67 revealed comparable fractions of proliferating FoxP3+ cells in healthy tissues (average 12.3%, range 5.8-18.5%) and inflammatory conditions (average 7.6%, range 2.6-17.2%). Interestingly, the rate of Ki67+FoxP3+ cells was markedly higher in 36 bladder cancers (average 14.2%, range 0-49.3%) suggesting active expansion of FOXP3+ cells in cancer.

Our data demonstrate an inverse prognostic impact of the FoxP3+ cell density in prostate and breast cancers. The increased proliferation rate of immune-regulatory FoxP3+ cells in some bladder cancer is interesting in the light of the variable response of these tumors to immune checkpoint inhibitors.

Citation Format: Tim Mandelkow, Elena Bady, Niclas Blessin, Claudia Hube-Magg, Ronald Simon, Guido Sauter, Christoph Fraune, Maximilian Lennartz, Katharin Möller, Sören Weidemann, Andreas Luebke, Doris Höflmayer. Prevalence and prognostic role of FoxP3regulatory T lymphocytes in cancer: A tissue microarray study on >20,000 cancers [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 3854.
Abstract 3870: Patterns of CD112R expression in normal lymphatic tissues, inflammation and cancer
Authors: Blessin Niclas Christian , Mandelkow Tim , Bady Elena , Hube-Magg Claudia , Sauter Guido , Simon Ronald , Fraune Christoph , Lennartz Maximilian , Weidemann Sören A. , Möller Katharina , Höflmayer Doris
Published: September 2020

CD112R is an inhibitory immune checkpoint receptor and a putative target for novel immune therapies, but little is known about its molecular epidemiology in healthy and diseased tissues. To study the prevalence and expression level of CD112R+ immune cells, we analyzed more than 200 samples of normal lymphatic, inflamed and cancerous tissues in a microenvironment tissue microarray format (4mm tissue spot diameter) and large sections using fluorescent multiplex immunohistochemistry.

CD112R expression was detected at variable intensity levels in 47% of CD8+ cytotoxic lymphocytes, 49% of CD4+ T helper cells, 30% of FOXP3+ regulatory T helper cells and in 25% of CD56+ natural killer cells, but no expression was seen in CD11c+ dendritic cells and CD68+ macrophages. All analyzed compartments across normal and diseased tissues showed a small subset (CD8: 9±18%, CD4: 5±15%, FOXP3: 2±5%) of immune cells with supramaximal CD112R expression. The highest fraction of cells with supramaximal CD112R expression was found in the subset of CD8+ cytotoxic T cells in the Peyer's patches of ileum (62%), the intergranuloma area of lymph node sarcoidosis (27%) and in ovarian cancer (37%). In cancerous tissues, the density and the fraction cytotoxic T cells with supramaximal CD112R expression was highly variable and ranged from 5% in bladder cancer to 3% in lung cancer and 36% in ovarian cancer. A high variability of the number of cells with supramaximal CD112R expression was also seen within every tumor entity.

In summary, our analysis shows that CD112R expression is abundant in various subsets of immune cells but identifies a small fraction of cells with exceedingly high CD112R levels. The widespread occurrence of CD112R+ cytotoxic T cells in the cancer microenvironment may suggest considerable opportunities for checkpoint inhibitors targeting CD112R.

Citation Format: Niclas Christian Blessin, Tim Mandelkow, Elena Bady, Claudia Hube-Magg, Guido Sauter, Ronald Simon, Christoph Fraune, Maximilian Lennartz, Sören A. Weidemann, Katharina Möller, Doris Höflmayer. Patterns of CD112R expression in normal lymphatic tissues, inflammation and 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 3870.
Abstract 3935: Runt related transcription factor-1 (runx1) plays a central role in vessel co-option of colorectal cancer liver metastases
Authors: Rada Miran , Kapelanski-Lamoureux Audrey , Petrillo Stephanie , Tabariès Sébastien , Siegel Peter , Lazaris Anthoula , Metrakos Peter
Published: September 2020

Colorectal cancer liver metastasis (CRCLM) has two major morphological growth pattern subtypes including desmoplastic (DHGP) and replacement (RHGP). The DHGP tumors depend on angiogenesis for their growth. Conversely, RHGP tumors are non-angiogenic and obtain their blood supply through vessel co-option, which the cancer cells hijacking pre-existing blood vessels of the surrounding liver tissue. Therefore, anti-angiogenic therapies have shown modest efficacy in CRCLM patients with RHGP lesions. In this study, we found overexpression of Runt Related Transcription Factor-1 (RUNX1) in the cancer cells of RHGP lesions. Upregulation of RUNX1 is positively correlated with cancer cell motility and epithelial mesenchymal transition (EMT) in vitro. Our results also showed that RUNX1 regulated by Transforming Growth Factor Beta-1 (TGFβ1) through TGFβ Receptor II (TGFβRII). Significantly, RUNX1 knockdown induced conversion RHGP lesions to DHGP in vivo. Collectively, this study suggests RUNX1 as a potential target therapy to overcome resistance to anti-angiogenic therapy in CRCLM.

Citation Format: Miran Rada, Audrey Kapelanski-Lamoureux, Stephanie Petrillo, Sébastien Tabariès, Peter Siegel, Anthoula Lazaris, Peter Metrakos. Runt related transcription factor-1 (runx1) plays a central role in vessel co-option of colorectal cancer liver metastases [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 3935.
Abstract 396: The bile acid receptor FXR suppress colitis-induced colon cancer
Authors: Fu Ting , Evans Ronald
Published: September 2020

Colorectal cancer (CRC) is the third most common cancer and a major cause of cancer related death. Colitis-associated colorectal carcinoma (CAC) is a type of CRC that develops in patients with inflammatory bowel disease (IBD), and is associated with chronic, idiopathic intestinal inflammation. While the underlying cause(s) are poorly understood, environmental factors including high fat diets and aberrant intestinal immune responses contribute to disease progression. Bile acid (BA) overproduction is implicated in diarrhea associated with IBD and CAC. As early sensors of dietary status, BAs are critical mediators of gut inflammation and immunity, in part through their ability to affect the activity of the Farnesoid X Receptor (FXR). FXR is considered the master regulator of BA homeostasis, also a key modulator of the pro- and anti-inflammatory responses in the intestinal epithelium, whose function severely compromised in both CAC patients and in AOM/DSS mouse model. Here, we show that inflammation-induced epithelial abnormalities altered the BAs profile, given the role of the crypt-villus architecture in establishing a BA gradient necessary for homeostasis. In addition, AOM/DSS treatment enhanced immune cell infiltration, as well as induced the formation of massive tumors in the colon. Given that intestinally-restricted FXR agonist FexD can reduced tumorigenesis in APCmin/+ mice, we also treated AOM/DSS mice with FexD. Notably, FexD activated FXR signaling pathway and restored BA homeostasis. Further, it reduced fecal bleeding, intestinal permeability, and decreased serum levels of the late carcinogenesis markers CEA and CA19. Treatment of FexD also resulted in a smaller spleen and reduced pro-inflammatory cytokine levels such as IL17 and IL6. The present study highlights the role of FXR in inflammation induced colon cancer, opening new therapeutic avenues for the treatment of CAC.

Citation Format: Ting Fu, Ronald Evans. The bile acid receptor FXR suppress colitis-induced colon 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 396.
Abstract 397: Myeloma derived mitochondrial DNA activates TLR9 driven pro-tumoral expansion
Authors: Jibril Aisha , Mistry Jayna J. , Kumar Prakrit , Moore Jamie A. , Hellmich Charlotte , Gomez Cesar , Bowles Kristian , Rushworth Stuart A.
Published: September 2020

Multiple myeloma (MM) is reported to cause 85,000 global deaths per year which is estimated to double by 2040. MM is an incurable malignancy of antibody (Ig) secreting differentiated B cells (plasma cells) characterized by the accumulation and localisation of tumor cells in the bone marrow microenvironment (BMM). Mitochondrial genome (mtDNA) contains CpG DNA repeats and CpG DNA has been shown to activate and induce proliferation of memory B cells to secrete Ig. Here we determine if malignant plasma cells maintain their proliferative advantage by releasing mtDNA into the tumour microenvironment which acts as feedback mechanism to induce further proliferation and Ig production by the malignant plasma cell.

Human MM cell lines U266 and MM1S were engrafted into NSG immunocompromised mice. Blood sample from control and engrafted mice were collected at various times and plasma was isolated and spun at 15,000g to remove cell debris and large extracellular vesicles. Human and mouse mtDNA was determine by real-time PCR. Results show that human mtDNA can be detected in the plasma from mice engrafted with U266 and MM1S at 2 weeks and 3 weeks post injection of cells. Next, CpG oligonucleotides were used to determine if MM cells can be activated by mtDNA CpG repeats. Primary MM, U226 and MM1S cells were all treated with CpG and CpG control for 4 and 24 hours. Real-time PCR was then used to detect the pro-survival signature showing that IL-1beta, IL-6 and IL-8 were all upregulated by CpG. We then analysed primary MM and MM cell lines for TLR9 expression by flow cytometry and showed that all samples tested had high levels of cytosolic TLR9 expression. Using TLR9 antagonist (ODN TTAGGG (A151)) we show that primary MM cells and MM cell lines have reduced IL-6 and IL-8 expression and decreased cell proliferation. Finally, U266 and MM1S transduced with TLR9 knockdown lentivirus had reduced engraftment, as measured by bioluminescence, in NSG mice.

Here we show that MM release mtDNA in vivo and this can induce a tumour directed proliferative response through the engagement of TLR9. This process is necessary for optimum tumor growth and forms part of the malignant phenotype of MM.

Citation Format: Aisha Jibril, Jayna J. Mistry, Prakrit Kumar, Jamie A. Moore, Charlotte Hellmich, Cesar Gomez, Kristian Bowles, Stuart A. Rushworth. Myeloma derived mitochondrial DNA activates TLR9 driven pro-tumoral expansion [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 397.
Abstract 4025: New candidate therapy for BAP1-mutant cancer identified using novel screen
Authors: OWENS DAWN A. , Kuznetsov Jeffim N. , Lopez Andy , Kurtenbach Stefan , Bilbao Daniel , Roberts Evan R. , Volmar Claude-Henry , Wahlestedt Claes R. , Brothers Shaun P. , Harbour J. William
Published: September 2020

Inactivation of the oncogene BAP1 is associated with poor prognosis in many cancers, including uveal melanoma (UM). Therapies for mediating the detrimental effects of BAP1 mutations have yet to be developed. Previously, we found that BAP1 depletion in Xenopus causes a striking developmental phenotype and a global dysregulation of differentiation markers across the genetic landscape. This was caused by impaired promoter assembly at commitment genes, marked by decreased H3K27ac levels. Inhibition of HDAC4 protein rescues the affected BAP1-depleted phenotype and genetic regulations, further emphasizing the role of H3K27ac in BAP1 function. Pairing these findings with a lab-devised multitiered screening process, we identified an epigenetic compound that successfully reverses the detrimental effects of the BAP1 deficient phenotype back to phenotypically normal conditions. This multitiered screening process tests prospective compounds for the ability to mediate transcriptional aberrations caused by BAP1 loss in UM cells (phase 1), rescue the BAP1 loss phenotype in Xenopus (phase 2), and provide significant inhibition of UM tumor growth in a mouse model (phase 3). These findings reveal a new application for a promising epigenetic compound as a therapeutic and reversing agent for BAP1 mutant UM. Furthermore, our streamlined process for the testing of promising therapeutic agents can accelerate the testing of such agents and getting them to clinical trials in a timely manner.

Citation Format: DAWN A. OWENS, Jeffim N. Kuznetsov, Andy Lopez, Stefan Kurtenbach, Daniel Bilbao, Evan R. Roberts, Claude-Henry Volmar, Claes R. Wahlestedt, Shaun P. Brothers, J. William Harbour. New candidate therapy for BAP1-mutant cancer identified using novel screen [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 4025.
Abstract 4082: Hsp90B protects MAST1 from CHIP-mediated ubiquitination and degradation providing cisplatin resistance
Authors: Pan Chaoyun , Li Jie , Boese Austin , Kang Sumin
Published: September 2020

Microtubule-associated serine/threonine kinase 1 (MAST1) plays a key role in cisplatin resistance of human cancers. However, the mechanism underlying MAST1 regulation remains elusive. We identified the hsp90B as a direct MAST1 binding partner essential for its stabilization via a proteomics screen. Targeting hsp90B in cancer cells sensitizes cisplatin treatment mainly through MAST1 destabilization. Mechanistically, hsp90B protects MAST1 from ubiquitination at lysine 317 and 545 by the E3 ubiquitin ligase CHIP and prevents proteasomal degradation. The role of the hsp90B-MAST1-CHIP signaling axis in cisplatin resistance was clinically validated in cancer patients. Furthermore, a combination of hsp90 inhibitor and MAST1 inhibitor lestaurtinib further inhibited MAST1 activity and consequently potentiated the cisplatin effect in a patient-derived xenograft model. Our study not only reveals the mechanism of MAST1 regulation in tumors but also demonstrates a promising combinatorial therapy to overcome cisplatin resistance in human cancers.

Citation Format: Chaoyun Pan, Jie Li, Austin Boese, Sumin Kang. Hsp90B protects MAST1 from CHIP-mediated ubiquitination and degradation providing cisplatin resistance [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 4082.
Abstract 4093: Drug combinations that can overcome resistance to PARP inhibitor therapy for BRCA1-associated breast cancer
Authors: Moustafa Dina Mohamed Abd El Aziz , Elwahed Maha Abd , Said Hanaa El , Parvin Jeffrey
Published: September 2020

Triple negative breast cancer (TNBC) represents approximately 10–15% of all breast cancers and has a poor outcome as it lacks a receptor target for therapy. Poly (ADP-ribose) polymerases inhibitors (PARPi) drugs have demonstrated some effectiveness in treating BRCA1 or BRCA2 mutated breast and ovarian cancers but resistance to PARPi is common. Published results found that resistance to Olaparib, a PARPi, can be due to downregulation of EMI1 and the consequent upregulation of the RAD51 recombinase. Using a tissue culture-based cell viability assay, we extended those observations to another PARPi and to other chemotherapy drugs that affect DNA repair or the cell cycle. As we expected, EMI1 downregulation resulted in resistance to another PARPi drug, Talazoparib. EMI1 downregulation also led to resistance to other cytotoxic drugs, Cisplatin and CHK1 inhibitor. Surprisingly, EMI1 downregulation also led to resistance to a MEK inhibitor, though this inhibitor blocks cells in G1 phase of the cell cycle and would not be expected to be sensitive to EMI1 levels. Notably, increasing the RAD51 protein expression can mimic the EMI1 downregulation and lead to resistance to different PARPis and the other cytotoxic drugs. This result suggests that the key downstream effect of EMI1 downregulation is increasing the concentration of RAD51 protein in the cell. We found that combining CHK1 inhibitor with Olaparib results in cell death in a dose specific manner. This combination therapy may be a means to overcome the PARPi resistance in BRCA1-deficient TNBC cells.

Citation Format: Dina Mohamed Abd El Aziz Moustafa, Maha Abd Elwahed, Hanaa El Said, Jeffrey Parvin. Drug combinations that can overcome resistance to PARP inhibitor therapy for BRCA1-associated breast 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 4093.
Abstract 4113: Discriminatory 11-plex assay designed to monitor estrogen receptor mutations using a novel, melt-analysis capable digital PCR platform
Authors: Bernier Jennifer N. , Takach Johanna E. , Wang Tiffany , Gallagher Steve , Lin Robert , Zayac Andrew , Hung Paul , Lesho Matthew , Whitman Douglas
Published: September 2020

In a large fraction of ER+/HER2- metastatic breast cancer (MBC) patients, treatment with aromatase inhibitors fails due to emerging resistance. A key mechanism of this resistance is associated with a limited set of missense mutations in the gene encoding for the estrogen receptor (ESR1). These mutations are primarily found in the ligand binding domain of the protein. They result in a constitutively active estrogen receptor, are infrequently found in primary tumors (<5%) and instead likely coincide with the emergence of an endocrine resistant phenotype. The occurrence of ESR1 mutations following treatment has varied from study to study depending on patient selection and method of detection, but prevalence may be as high as 50%. Because these patients require second line therapies as soon as possible, ESR1 mutations are an area of intense focus in research and clinical trials for ER+/HER2- breast cancer. Studies utilizing droplet digital PCR (ddPCR) assays have correlated an increase of ESR1 mutations in patient liquid biopsy samples with therapeutic resistance approximately 8 months prior to conventional methods, however the number of individual mutations evaluated in these studies was limited due to an inability to accurately and sensitively identify more than one individual mutated SNP allele within a single reaction. We have developed a multiplex digital PCR assay that utilizes melt-based probes to detect and discriminate between 10 unique, clinically relevant ESR1 mutations in circulating tumor DNA (ctDNA) samples while simultaneously quantifying total cell-free DNA (cfDNA). Designed for use with an innovative, array-based dPCR system, the assay requires a hands-on time of only 10-15 minutes and a total run time of 2.5 hours. This assay will allow breast cancer researchers to further refine our understanding of the role ESR1 mutations play in personalized treatment strategies for MBC.

Citation Format: Jennifer N. Bernier, Johanna E. Takach, Tiffany Wang, Steve Gallagher, Robert Lin, Andrew Zayac, Paul Hung, Matthew Lesho, Douglas Whitman. Discriminatory 11-plex assay designed to monitor estrogen receptor mutations using a novel, melt-analysis capable digital PCR platform [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 4113.
Abstract 4134: Characterization of ATRX loss combined with IDH1 R132H mutation in gliomas
Authors: Garbarino Jennifer , Bindra Ranjit , Jensen Ryan
Published: September 2020

Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) is mutated frequently in gliomas and is a potential therapeutic target. ATRX functions as a histone chaperone that helps incorporate histone variant H3.3 into the genome. There is some evidence of its involvement in both nonhomologous-end-joining (NHEJ) and replication stress, but a distinct role for ATRX in DNA repair has not been determined. Our lab has found that another common mutation in gliomas in the isocitrate dehydrogenase (IDH1) gene, leads to deficiency in homologous recombination (HR). We hypothesize that modelling these common mutations in glioma cell lines will to confer sensitivity to combinations of DNA repair inhibitor and damaging agents. To test this, we have created multiple cell lines with a CRISPR knockout of ATRX as well as lines that overexpress the IDH1 R132H in the presence of doxycycline. This will allow comparison of the double mutation and each mutation individually in different glioma cell lines for their DNA repair capacity. Currently, we have found a retention of H2AX foci in these cells after hydroxyurea treatment. A luciferase reporter based DNA repair assay has shown a decrease in HR capacity and no change in NHEJ in ATRX KO cells compared to WT. Additionally, we have begun screening DNA repair inhibitors and DNA damaging agents to identify compounds that specifically target ATRX KO cells or the double mutant cell lines. These preliminary screens have shown sensitivity to Chk1 inhibitors and temozolomide, which is currently being validated. We have also performed a focused DNA repair CRISPR screen to assess which DNA repair genes cause sensitivity or resistance ATRX KO cells. We infected both WT and ATRX KO cells with our pooled CRISPR library and harvested genomic DNA at multiple time points. Next generation sequencing was then used to identify sgRNAs that dropped out of the population compared to control guides. Sensitive hits from these screens were underrepresented in the ATRX KO population compared to WT and resistant hits were overrepresented in ATRX KO. These hits are the in the process of being validated. This work is continuing to expand on the hypothesis that ATRX is an important targetable mutation in low and high grade gliomas. Further understanding its mechanism of action as well as its action in combination with IDH1 R132H will help understand these cancers and lead to more targeted treatments for patients.

Citation Format: Jennifer Garbarino, Ranjit Bindra, Ryan Jensen. Characterization of ATRX loss combined with IDH1 R132H mutation in 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 4134.