Summary Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.
Focal adhesion kinase (FAK) mediates tumor cell–intrinsic behaviors that promote tumor growth and metastasis. We previously showed that FAK also induces the expression of inflammatory genes that inhibit antitumor immunity in the microenvironment. Here, we identified a crucial, previously unknown role for the dual-function cytokine IL-33 in FAK-dependent immune evasion. In murine squamous cell carcinoma (SCC) cells, specifically nuclear FAK enhanced the expression of the genes encoding IL-33, the chemokine CCL5, and the soluble, secreted form of the IL-33 receptor sST2. The abundance of IL-33 and CCL5 was increased in FAK-positive SCC cells but not in normal keratinocytes. IL-33 associated with FAK in the nucleus, and the FAK–IL-33 complex interacted with a network of chromatin modifiers and transcriptional regulators, including TAF9, WDR82 and BRD4, which promote the activity of nuclear factor κB (NF-κB) and its induction of genes encoding chemokines, including CCL5. We did not detect secretion of IL-33 from FAK-positive SCC cells; thus, we propose that the increased production and secretion of sST2 likely sequesters IL-33 secreted by other cell types within the tumor environment, thus blocking its stimulatory effects on infiltrating host immune cells. Depleting FAK, IL-33, or sST2 from SCC cells before implantation induced tumor regression in syngeneic mice, except when CD8+ T cells were co-depleted. Our data provide mechanistic insight into how FAK controls the tumour immune environment, namely through a transcriptional regulatory network mediated by nuclear IL-33. Targeting this axis may boost antitumor immunity in patients.
SRC tyrosine kinase is frequently overexpressed and activated in late-stage, poor prognosis ovarian tumours, and preclinical studies have supported the use of targeted SRC inhibitors in the treatment of this disease. The SAPPROC trial investigated the addition of the SRC inhibitor saracatinib (AZD0530) to weekly paclitaxel for the treatment of platinum resistant ovarian cancer; however, this drug combination did not provide any benefit to progression free survival (PFS) of women with platinum resistant disease. In this study we aimed to identify mechanisms of resistance to SRC inhibitors in ovarian cancer cells. Using two complementary strategies; a targeted tumour suppressor gene siRNA screen, and a phospho-receptor tyrosine kinase array, we demonstrate that activation of MAPK signalling, via a reduction in NF1 (neurofibromin) expression or overexpression of HER2 and the insulin receptor, can drive resistance to AZD0530. Knockdown of NF1 in two ovarian cancer cell lines resulted in resistance to AZD0530, and was accompanied with activated MEK and ERK signalling. We also show that silencing of HER2 and the insulin receptor can partially resensitize AZD0530 resistant cells, which was associated with decreased phosphorylation of MEK and ERK. Furthermore, we demonstrate a synergistic effect of combining SRC and MEK inhibitors in both AZD0530 sensitive and resistant cells, and that MEK inhibition is sufficient to completely resensitize AZD0530 resistant cells. This work provides a preclinical rationale for the combination of SRC and MEK inhibitors in the treatment of ovarian cancer, and also highlights the need for biomarker driven patient selection for clinical trials.
In addition to central functions in cell adhesion signalling, integrin-associated proteins have wider roles at sites distal to adhesion receptors. In experimentally defined adhesomes, we noticed that there is clear enrichment of proteins that localise to the nucleus, and conversely, we now report that nuclear proteomes contain a class of adhesome components that localise to the nucleus. We here define a nucleo-adhesome, providing experimental evidence for a remarkable scale of nuclear localisation of adhesion proteins, establishing a framework for interrogating nuclear adhesion protein functions. Adding to nuclear FAK’s known roles in regulating transcription, we now show that nuclear FAK regulates expression of many adhesion-related proteins that localise to the nucleus and that nuclear FAK binds to the adhesome component and nuclear protein Hic-5. FAK and Hic-5 work together in the nucleus, co-regulating a subset of genes transcriptionally. We demonstrate the principle that there are subcomplexes of nuclear adhesion proteins that cooperate to control transcription.
In addition to central functions in cell adhesion signalling, integrin-associated proteins have wider roles at sites distal to adhesion receptors. In experimentally defined adhesomes, we noticed that there is clear enrichment of proteins that localise to the nucleus, and conversely, we now report that nuclear proteomes contain a class of adhesome components that localise to the nucleus. We here defined a nucleo-adhesome, providing experimental evidence for a remarkable scale of nuclear localisation of adhesion proteins, establishing a framework for interrogating nuclear adhesion protein functions. In adding to nuclear FAK's known roles in regulating transcription, we now show that nuclear FAK regulates expression of many adhesion-related proteins that localise to the nucleus and that nuclear FAK binds to the adhesome component and nuclear protein Hic-5. FAK and Hic-5 work together in the nucleus, co-regulating a subset of genes transcriptionally. We describe the first nucleo-adhesome using a squamous cancer cell model, and demonstrate the new principle that there are nuclear adhesion protein subcomplexes that cooperate to control transcription.
Introduction: 40-45% of patients with High Grade Serous Ovarian Cancer (HGSOC) will eventually relapse with platinum resistant disease. Tothill et al and TCGA are two independent gene expression datasets which have demonstrated the presence of a mesenchymal molecular subgroup, characterised by upregulation of angiogenesis regulating genes. Angiogenesis is known to be an integral pathological feature of HGSOC and anti-angiogenics have dominated the field of drug development in EOC. However, despite this, anti-angiogenic agents have failed to demonstrate a significant impact on overall survival (OS) benefit. In this study, we asked if platinum resistance could be associated with an improved response to anti-angiogenic agents and what the underlying biological rationale for this could be. Methods: A meta-analysis of 14 phase II and III clinical trials in EOC were used to investigate the association between platinum resistance and response to anti-angiogenic agents. In addition, we analysed gene expression in 12 matched pre- and post-chemotherapy EOC samples. Novel isogenic cisplatin-resistant HGSOC cell lines were established to study the development of an angiogenic phenotype. Further studies were performed in novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy. Result: In the clinical trial meta-analysis, an OS benefit for antiangiogenics was observed in platinum-resistant disease (p=0.029), whilst platinum-sensitive EOC only derived progression free survival (PFS) (p=<0.0001) benefit and not OS (p=0.125). In the 12 matched pairs of patient samples, post-platinum samples had a higher micro-vessel density (MVD) relative to their paired treatment-naïve sample (p= 0.0001). Additionally, an in vivo angiogenesis matrigel plug assay demonstrated that cisplatin-resistant EOC cell lines were associated with an increase in MVD (p=<0.0001). MVD was reduced in the platinum-resistant cells following treatment with bevacizumab (p=0.001). Ascites-derived primary cells established from platinum-resistant patients demonstrated overexpression of VEGF-A, consistent with stimulating angiogenesis. Gene expression analysis of pre- and post-platinum paired samples identified that PDGFRα (p=0.007) and PDGFRβ (p=0.005) were differentially expressed in the post-platinum therapy samples. In vitro validation in the platinum-resistant cell lines demonstrated that VEGF-A expression was regulated by PDGFRα. Discussion: We have demonstrated that previous platinum therapy for EOC is associated with an increase in tumor PDGFα and VEGF-A expression, correlating with a response to anti-angiogenic therapies. This data suggests that platinum therapy resistance may inform the selection of EOC patients for novel antiangiogenic therapies in future clinical trials. Citation Format: Aya El Helali, Nuala McCabe, Christopher Steele, Lara Dura Perez, Christina L. O'Neill, Naomi Dickson, Niamh McGivern, Caolan Harkin, Andrena McCavigan, Reinhold J. Medina, Laura A. Knight, Stephen McQuaid, Jacqueline A. James, Caroline O. Michie, Charlie Gourley, W Glenn McCluggage, Denis P. Harkin, Richard H. Wilson, Alan W. Stitt, Richard D. Kennedy. Platinum resistance in epithelial ovarian cancer is dependent on a PDGFR alpha-VEGF-A signalling mechanism that activates downstream angiogenesis pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 7.
Introduction: WEE1 kinase is a key component in maintaining the G2/M cell cycle checkpoint for pre-mitotic DNA repair, and is overexpressed in several cancer types. Novel therapeutics are currently being developed to target WEE1 kinase in cancer, however, to date no predictive biomarkers have been approved to aid patient stratification and clinical trial design. To address this, we employed a siRNA screening to identify tumour suppressor genes (TSGs) whose loss mediates sensitivity to WEE1 inhibition. Experimental procedures: U2OS cells were reverse transfected with a customised siRNA library containing 3 independent siRNAs targeting 178 tumour suppressor genes and 24 hours later treated with either DMSO control or MK-1775 (Wee1 Kinase Inhibitor). Cell viability was measured using a cell titer-glo luminescent Cell Viability Assay 72 hours post-treatment. Hits were selected based on robust z-score analysis. Those genes with 2 or more targeted siRNAs demonstrating a robust z-score of ±1 median absolute deviation (MAD) were taken forward for validation studies. Sensitive hits were selected on a z-score of <-1 and resistant hits were selected on a z-score of >1. siRNA knockdown of WEE1 was performed in multiple human cancer cell lines and confirmed by western blotting and RT-q-PCR. Basal expression levels of phosphorylated WEE1, total WEE1, FOS and JUNB were assessed by western blotting. Results: Consistent with previously published findings, the siRNA screen demonstrated that loss of BRCA2 conferred increased sensitivity to WEE1 inhibition (Aarts et al. 2015). The siRNA screen also identified an additional 12 TSGs whose loss mediated sensitivity and 14 TSGs whose loss mediated resistance to WEE1 kinase inhibition. Interestingly, we found that loss of two early response genes, FOS and JUNB conferred resistance to WEE1 inhibition. FOS and JUNB interact to form the AP1 heterodimer, and previous published work has demonstrated the presence of an AP1 binding motif on the WEE1 promoter (Kawasaki et al. 2003). Using publically available gene expression data (TCGA) we have shown a significant correlation between expression of WEE1 with FOS and JUNB in multiple cancer types. Conclusions: Using a TSG siRNA screen, we have identified that loss of JUNB and FOS confers resistance to the WEE1 inhibitor MK1775. Future studies will investigate the mechanisms by which the loss of these genes affects response to WEE1 inhibition, and will also investigate the utility of these genes as predictive biomarkers for response to WEE1 inhibition in clinical samples, thereby aiding patient stratification. Citation Format: Victoria L. Dunne, Niamh McGivern, Kienan I. Savage, Nuala McCabe, Richard Kennedy. The role of early response genes (ERG’s) as a biomarker of response to Wee1 targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3163.
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