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Background and AimsHigh expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma (HCC). In addition, Hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to HCC progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process.MethodsSeveral hepatic cell culture and mouse models were used to study functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing and PI4KIIIα specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein.ResultsHigh PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased-phosphorylation of paxillin and cofilin. This led to morphological alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane (PM) phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]- enriched, invadopodia-like structures which regulate cytoskeletal reorganization by promoting Akt2 phosphorylation.ConclusionsPI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to progression of liver cancer and identify promising targets for therapeutic intervention.IMPACT AND IMPLICATIONSUnderstanding mechanistically how high PI4KIIIα expression are associated with poor clinical outcomes of liver cancer is important to develop pharmaceutical interventions. Our study sheds light on the importance of the two lipid kinases PI4KIIIα and PIK3C2γ as well as the contribution of HCV on liver cancer progression, unraveling the signaling pathway governing this process. This preclinical study contributes to better understanding the complex connection of phospholipids, cytoskeleton and liver cancer and suggests strategies to improve therapeutic outcomes by targeting important signaling molecules.Graphical abstract

Purpose: The underlying molecular mechanisms of thymic epithelial malignancies (TEMs) are poorly understood. Consequently, there is a lack of efficacious targeted therapies and patient prognosis remains dismal, particularly for advanced TEMs. We sought to investigate protumorigenic mechanism relevant to this understudied cancer.Experimental Design: Recently established cell lines derived from thymic epithelial tumors were used as a model system. The antitumor activity of specific heat shock protein 90 (Hsp90) inhibitors was investigated by an analysis of cell viability, cell cycle, and apoptosis using MTT-assays and flow cytometry. Western blotting was used to investigate the altered expression of Hsp90 clients. Pharmacological inhibitors against select Hsp90 clients, as well as RNAi, were employed to test the relevance of each client independently. Tissue microarray analysis was performed to match the in vitro findings with observations obtained from patient-derived samples.Results: Hsp90 inhibition significantly reduces cell viability of thymic carcinoma cells, induces cell cycle arrest and apoptosis, and blocks invasiveness. Hsp90 inhibition triggers the degradation of multiple oncogenic clients, for example insulin-like growth factor 1 receptor (IGF-1R), CDK4, and the inactivation of PI3K/Akt and RAF/Erk signaling. Mechanistically, the IGF/IGF-1R–signaling axis contributes to the establishment of the antiapoptotic phenotype of thymic cancer cells. Finally, IGF-1R is overexpressed in advanced TEMs.Conclusions: We have unraveled a novel protumorigenic mechanism in TEMs, namely Hsp90-capacitated overexpression of IGF-1R, which confers apoptosis evasion in malignant thymic epithelial cells. Our data indicate that Hsp90 inhibition, which simultaneously blocks multiple cancer hallmarks, represents a therapeutic strategy in TEMs that may merit evaluation in clinical trials. Clin Cancer Res; 17(8); 2237–49. ©2011 AACR.

Supplementary Figures S1-S8; Supplementary Methods; Supplementary Tables S1-S3.

BackgroundCancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor intrinsic mechanisms of resistance to cytotoxic T-cell (TC) attack. Thus, a deeper understanding of these mechanisms is needed to develop successful immunotherapies.MethodsTo identify novel genes that protect tumor cells from effective TC-mediated cytotoxicity, we performed a genetic screening in pancreatic cancer cells challenged with tumor-infiltrating lymphocytes and antigen-specific TCs.ResultsThe screening revealed 108 potential genes that protected tumor cells from TC attack. Among them, salt-inducible kinase 3 (SIK3) was one of the strongest hits identified in the screening. Both genetic and pharmacological inhibitions of SIK3 in tumor cells dramatically increased TC-mediated cytotoxicity in several in vitro coculture models, using different sources of tumor and TCs. Consistently, adoptive TC transfer of TILs led to tumor growth inhibition of SIK3-depleted cancer cells in vivo. Mechanistic analysis revealed that SIK3 rendered tumor cells susceptible to tumor necrosis factor (TNF) secreted by tumor-activated TCs. SIK3 promoted nuclear factor kappa B (NF-κB)nuclear translocation and inhibited caspase-8 and caspase-9 after TNF stimulation. Chromatin accessibility and transcriptome analyses showed that SIK3 knockdown profoundly impaired the expression of prosurvival genes under the TNF–NF-κBaxis. TNF stimulation led to SIK3-dependent phosphorylation of the NF-κB upstream regulators inhibitory-κB kinase and NF-kappa-B inhibitor alpha on the one side, and to inhibition of histone deacetylase 4 on the other side, thus sustaining NF-κB activation and nuclear stabilization. A SIK3-dependent gene signature of TNF-mediated NF-κB activation was found in a majority of pancreatic cancers where it correlated with increased cytotoxic TC activity and poor prognosis.ConclusionOur data reveal an abundant molecular mechanism that protects tumor cells from cytotoxic TC attack and demonstrate that pharmacological inhibition of this pathway is feasible.

Little is known about the function of most non-coding RNAs (ncRNAs). The majority of long ncRNAs (lncRNAs) is expressed at very low levels and it is a matter of intense debate whether these can be of functional relevance. Here, we identified lncRNAs regulating the viability of lung cancer cells in a high-throughput RNA interference screen. Based on our previous expression profiling, we designed an siRNA library targeting 638 lncRNAs upregulated in human cancer. In a functional siRNA screen analyzing the viability of lung cancer cells, the most prominent hit was a novel lncRNA which we called Viability Enhancing LUng Cancer Transcript (VELUCT). In silico analyses confirmed the non-coding properties of the transcript. Surprisingly, VELUCT was below the detection limit in total RNA from NCI-H460 cells by RT-qPCR as well as RNA-Seq, but was robustly detected in the chromatin-associated RNA fraction. It is an extremely low abundant lncRNA with an RNA copy number of less than one copy per cell. Blocking transcription with actinomycin D revealed that VELUCT RNA was highly unstable which may partially explain its low steady-state concentration. Despite its extremely low abundance, loss-of-function of VELUCT with three independent experimental approaches in three different lung cancer cell lines led to a significant reduction of cell viability: Next to four individual siRNAs, also two complex siPOOLs as well as two antisense oligonucleotides confirmed the strong and specific phenotype. In summary, the extremely low abundant lncRNA VELUCT is essential for regulation of cell viability in several lung cancer cell lines. Hence, VELUCT is the first example for a lncRNA that is expressed at a very low level, but has a strong loss-of-function phenotype. Thus, our study proves that at least individual low-abundant lncRNAs can play an important functional role.

Over the past years, CRISPR/Cas9 mediated genome editing has developed into a powerful tool for modifying genomes in various organisms. In high-throughput screens, CRISPR/Cas9 mediated gene perturbations can be used for the systematic functional analysis of whole genomes. Discoveries from such screens provide a wealth of knowledge about gene to phenotype relationships in various biological model systems. However, a database resource to query results efficiently has been lacking. To this end, we developed GenomeCRISPR (http://genomecrispr.org), a database for genome-scale CRISPR/Cas9 screens. Currently, GenomeCRISPR contains data on more than 550 000 single guide RNAs (sgRNA) derived from 84 different experiments performed in 48 different human cell lines, comprising all screens in human cells using CRISPR/Cas published to date. GenomeCRISPR provides data mining options and tools, such as gene or genomic region search. Phenotypic and genome track views allow users to investigate and compare the results of different screens, or the impact of different sgRNAs on the gene of interest. An Application Programming Interface (API) allows for automated data access and batch download. As more screening data will become available, we also aim at extending the database to include functional genomic data from other organisms and enable cross-species comparisons.

CRISPR base editing is a potentially powerful technology that enables the creation of genetic mutations with single base pair resolution. By re-engineering both DNA and protein sequences, we developed a collection of constitutive and inducible base editing vector systems that dramatically improve the ease and efficiency by which single nucleotide variants can be created. This new toolkit is effective in a wide range of model systems, and provides a means for efficientin vivosomatic base editing.

CRISPR base editing enables the creation of targeted single-base conversions without generating double stranded breaks. However, the efficiency of current base editors is very low in many cell types. We re-engineered the sequences of BE3, BE4Gam, and xBE3 by codon optimization and incorporation of additional nuclear localization sequences. Our collection of optimized constitutive and inducible base-editing vector systems dramatically improves the efficiency by which single nucleotide variants can be created. The re-engineered base editors enable target modification in a wide range of mouse and human cell lines, and intestinal organoids. We also show that the optimized base editors mediate efficient in vivo somatic editing in the liver of adult mice.

The A kinase anchor protein 12 (AKAP12) is a central mediator of protein kinase A and protein kinase C signaling. Although AKAP12 has been described to act as a tumor suppressor and its expression is frequently down-regulated in several human malignancies, the underlying molecular mechanisms responsible for the AKAP12 reduction are poorly understood. We therefore analyzed the expression of AKAP12 and its genetic and epigenetic regulatory mechanisms in human hepatocarcinogenesis. Based on tissue microarray analyses (n = 388) and western immunoblotting, we observed a significant reduction of AKAP12 in cirrhotic liver (CL), premalignant lesions (DN), and hepatocellular carcinomas (HCCs) compared to histologically normal liver specimens (NL). Analyses of array comparative genomic hybridization data (aCGH) from human HCCs revealed chromosomal losses of AKAP12 in 36% of cases but suggested additional mechanisms underlying the observed reduction of AKAP12 expression in hepatocarcinogenesis. Quantitative methylation analysis by MassARRAY of NL, CL, DN, and HCC tissues, as well as of various tumorigenic and nontumorigenic liver cell lines revealed specific hypermethylation of the AKAP12α promoter but not of the AKAP12β promoter in HCC specimens and in HCC cell lines. Consequently, restoration experiments performed with 5-aza-2′deoxycytidine drastically increased AKAP12α mRNA levels in a HCC cell line (AKN1) paralleled by AKAP12α promoter demethylation. As hypermethylation is not observed in CL and DN, we investigated microRNA-mediated posttranscriptional regulation as an additional mechanism to explain reduced AKAP12 expression. We found that miR-183 and miR-186 are up-regulated in CL and DN and are able to target AKAP12. Conclusion: In addition to genetic alterations, epigenetic mechanisms are responsible for the reduction of the tumor suppressor gene AKAP12 in human hepatocarcinogenesis. (HEPATOLOGY 2010;.)

The inhibition of heat shock protein 90 (Hsp90) has emerged as a promising antineoplastic strategy in diverse human malignancies. Hsp90 has been predicted to be involved in hepatocellular carcinoma (HCC) development; however, its role in hepatocarcinogenesis remains elusive. Using chemically distinctive Hsp90 inhibitors, we show that Hsp90 capacitates the aberrant expression and activity of crucial hepatocarcinogenesis-driving factors (e.g., insulin-like growth factor receptor 1, hepatocyte growth factor receptor, protein kinase B, v-raf-1 murine leukemia viral oncogene homolog 1, and cyclin-dependent kinase 4). In vitro, Hsp90 inhibition with both geldanamycin analogs (17-allylamino-17-desmethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-desmethoxygeldanamycin (17-DMAG)) and the non-quinone compound 8-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine (PU-H71) reduced the viability of various HCC cell lines, induced the simultaneous degradation of numerous hepatocarcinogenic factors, and caused substantial cell cycle arrest and apoptosis. In contrast, nontumorigenic hepatocytes were less susceptible to Hsp90 inhibition. Because conventional geldanamycin-derivate Hsp90 inhibitors induce dose-limiting liver toxicity, we tested whether novel Hsp90 inhibitors lacking the benzoquinone moiety, which has been deemed responsible for hepatotoxicity, can elicit antineoplastic activity without causing significant liver damage. In HCC xenograft mouse models, PU-H71 was retained in tumors at pharmacologically relevant concentrations while being rapidly cleared from nontumorous liver. PU-H71 showed potent and prolonged in vivo Hsp90 inhibitory activity and reduced tumor growth without causing toxicity. Conclusion: Hsp90 constitutes a promising therapeutic target in HCC. Non-quinone Hsp90 inhibitors exhibit tumor-specific accumulation and exert potent antineoplastic activity without causing significant hepatotoxicity. (HEPATOLOGY 2009.)

Targeting COX-2, a key-enzyme of the prostaglandin metabolism, for the treatment of cancer has been in the focus of researchers for about a decade. However, only recently has this topic been related to hepatocellular carcinoma (HCC). HCC is one of the most common cancers and a growing health problem worldwide. At present, only few promising treatment options are available, accentuating the urgent need for novel therapeutic approaches. Since the first report of COX-2 overexpression in HCC, several findings support the notion that selective COX-2 inhibition proves to be beneficial in this malignancy. This review focuses on recent discoveries regarding the pro-tumorigenic potential of COX-2 in HCC and the functional effects of COX-2 inhibition on molecular mechanisms of this malignancy. Of clinical interest, promising data from in vivo experiments and case studies suggest a beneficial effect of COX-2 inhibitors for HCC- therapy. Detailed analysis of COX-2- activated pathways and related mechanisms may enable the evaluation and design of even more specific and combinatorial treatment approaches in the future.

Recent studies have shown that inhibition of cyclooxygenases (e.g. COX-2) exerts antitumorigenic effects on hepatocellular carcinomas (HCCs), which are to a significant extent due to the abrogation of PGE(2) synthesis. PGE(2) acts via differentially regulated prostaglandin receptors (EP(1-4)). Our study was designed to investigate the expression pattern of EP-receptors in HCCs and to evaluate the therapeutic potential of selective EP-receptor antagonists. Using tissue microarrays including a total of 14 control livers, 17 liver cirrhoses, 22 premalignant dysplastic nodules (DNs) and 162 HCCs with different histological grades, the expression of COX-2, mPGES-1 and -2 and EP(1-4)-receptors was analyzed. Western immunoblot analyses were performed to confirm the expression in HCC cell lines. The effects of EP(1-4)-receptor antagonism on cell viability and apoptosis were investigated using MTT-assays and FACS-analyses, respectively. COX-2, mPGES-1 and -2 and EP(1-4)-receptors were expressed in all HCC tissues. COX-2 expression was highest in DNs and declined with loss of HCC-differentiation. With respect to COX-2 expression, a converse expression of EP(1-3) -receptors and mPGES-1 and -2 was found in DNs compared to HCCs. Selectively antagonizing EP(1)- and EP(3)-receptors reduced the viability of HCC cells in a dose-dependent manner, which was associated with apoptosis induction. Our results suggest a differential regulation of EP-receptor subtype expression with dedifferentiation of HCCs in which a converse expression pattern for COX-2 in comparison to EP(1-3)-receptors occurs. Of clinical interest, selectively antagonizing EP(1)- and EP(3)-receptors may provide a novel systemic therapeutic approach to the treatment of HCCs.

Background Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes. Results Here we describe CRISPR library designer (CLD), an integrated bioinformatics application for the design of custom single guide RNA (sgRNA) libraries for all organisms with annotated genomes. CLD is suitable for the design of libraries using modified CRISPR enzymes and targeting non-coding regions. To demonstrate its utility, we perform a pooled screen for modulators of the TNF-related apoptosis inducing ligand (TRAIL) pathway using a custom library of 12,471 sgRNAs. Conclusion CLD predicts a high fraction of functional sgRNAs and is publicly available at https://github.com/boutroslab/cld. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0915-2) contains supplementary material, which is available to authorized users.

BACKGROUND: Being one of the most treatment-resistant cancer types, pancreatic ductal adenocarcinoma (PDAC) is characterized by its ability to escape immune surveillance by developing many immunological obstacles. These include a plethora of mechanisms that either dampen immune cell functionality, or foster tumor cell resistance towards immune attack. Immunotherapeutic strategies, such as immune checkpoint blockade, have proven clinical success in many cancer entities, but showed little clinical benefit in PDAC patients, emphasizing the need to identify more key players that could radically improve immunotherapy. AIM: We aim to systematically identify the whole arsenal of tumor-associated immune modulators by performing a high-throughput RNAi screen and subsequently validate novel therapeutic targets, whose blockade could potentially enhance anti-tumor immune response in PDAC patients. METHODS: We generated a luciferase-expressing PANC-1 cell line and knocked down 2514 genes using a siRNA library. Our library includes G-protein coupled receptors, protein kinases and 1117 surface proteins. We co-cultured HLA-A201+ matched tumor infiltrating lymphocytes (TILs) derived from a PDAC patient with the transfected tumor cells. We then measured the remaining luciferase intensity of the tumor cells as an estimation of TIL-mediated cytotoxicity. In order to exclude genes whose knock-down affected cell viability per se, we cultivated tumor cells with the siRNA library in the absence of TILs. RESULTS: We identified 155 candidate genes whose knock-down enhances TIL-mediated killing more efficiently than PD-L1 down-regulation. 35% of these genes are surface molecules and are most likely to directly mediate tumor immune evasion. Beside novel undescribed genes, our list contains well characterized immune modulators, supporting the reliability of our approach. Of note 13 of our hits were also found in a related melanoma screen and might play a role in the regulation of immune surveillance of different tumor entities. Among our candidates, 4 hits were chosen for further validation. We confirmed the expression of our selected candidates in several tumor cell lines and assessed the siRNA on-target effect using several non-overlapping siRNA sequences targeting the same hit. Transfection of PANC-1 with different siRNA sequences showed knock-down of the target gene as assessed via qPCR. Additionally we observed increased T-cell mediated killing as measured via luciferase-based killing assay and Chromium release assay. CONCLUSION: We set up a robust and systematic method to unravel novel key players of pancreatic cancer immune surveillance. Further functional validation of our candidate genes will prove their potential to be used as relevant therapeutic targets in the clinic. Citation Format: Antonio Sorrentino, Ayse Nur Menevse, Tillmann Michels, Nisit Khandelwal, Marco Breinig, Isabel Poschke, Valentina Volpin, Sabrina Wagner, Rienk Offringa, Michael Boutros, Philipp Beckhove. RNAi discovery platform to identify novel genes that prevent immune surveillance in pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2339.

Pancreatic endocrine tumors (PET) represent a heterogenous group of neoplasms. Although surgical resection is considered a safe and effective treatment for many PET, therapeutic options for inoperable and progressive PET are limited. The expression of heat-shock protein (HSP) 90 was investigated in 120 clinically and pathomorphologically well-characterized PET from 84 patients using immunohistochemistry. In addition, in 19 snap–frozen PET and in three healthy pancreatic tissues, we performed immunoblot analyses, and in 15 snap–frozen PET and in three healthy pancreatic tissues, we investigated the expression of HSP90 isoforms by means of semiquantitative RT-PCR. Functional tests were conducted using the human pancreas carcinoid cell line BON and the mouse insulinoma cell line β-TC-3. HSP90 was expressed in 95% of the PET patients. The transcript levels of the HSP90 isoforms HSP90α, HSP90β, glucose-related protein 94, and TNF receptor-associated protein 1 were significantly increased in PET compared with non-neoplastic pancreatic tissues. The treatment of the cell lines BON and β-TC-3 with the HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin resulted in significant, dose-dependent reduction of cell viability, cell cycle arrest, and increased apoptosis. Furthermore, HSP90 inhibition induced the degradation and inactivation of several oncogenetic HSP90 client proteins in a time- and dose-dependent manner. HSP90 inhibitors increased the therapeutic effects of doxorubicin and 5-fluorucacil in BON and β-TC-3 cells. HSP90 is expressed in the vast majority of PET and its inhibition reveals significant treatment effects in vitro. Thus, HSP90 qualifies as a promising new target.

Anorectal melanomas (AMs) are very rare and highly malignant tumors that are often diagnosed in advanced stages. After the differentiation between cutaneous melanoma (CM) and AM on the molecular level based on the presence of BRAF mutations, further modes of differentiation opened up, such as the recently discovered immunohistologically relevant protein deleted in malignant brain tumors 1 (DMBT1). Over the past several years, increasingly specific therapies have been developed on the basis of new therapy principles. Tyrosin kinase receptors such as Her2 and EGFR have been awarded a large role in this context. The goal of this study was to examine AMs for a possible expression or overexpression of these markers. Expression analyses of Her2 and EGFR were performed immunohistologically on 25 primary AMs. An overexpression of Her2 (score: 3+) was found in one AM from a 68-year-old female patient among these samples. In contrast, EGFR expression was not found in any of the AMs. The results presented here show that isolated cases of AM may benefit from an additive Her2-directed therapy, as the overexpression of Her2 was found in one of our AM patients.

There is an ongoing search for new therapeutic targets in invasive non-resectable thymic tumours because of the low response rates in current chemotherapeutic treatment modalities. In this study, the possibility that platelet-derived growth factor receptor A (PDGFRA) and/ or PDGFRB may represent potential therapeutic targets in epithelial tumours of the thymus was investigated.Tissue samples were obtained by thymectomy from 36 different patients with epithelial tumours of the thymus (26 thymomas types A, AB, B1-3 and 10 thymic carcinomas). Normal thymi from three young children were used as controls. The PDGFRA and PDGFRB protein expressions as well as the mutational statuses of exons 12, 14 and 18 of the PDGFRA gene were analyzed.All the subtypes of thymomas and the thymic carcinomas showed staining for PDGFRA, but no mutations in the known mutational hotspots were identified. Only about one third of the tumours stained for PDGFRB. PDGFRA and PDGFRB protein staining were slightly positively correlated.PDGFRA may represent a potential therapeutic target in thymic tumours.

Pancreatic endocrine tumors represent morphologically and biologically heterogeneous neoplasms. Well-differentiated endocrine tumors (benign or of uncertain behavior) can be distinguished from well-differentiated and poorly differentiated endocrine carcinomas. Although many well-differentiated endocrine carcinomas show rather low rates of tumor growth, more than two-thirds of pancreatic endocrine carcinomas display distant metastases at the time of diagnosis. As the currently applied therapies beyond surgery only achieve partial or complete response rates of approximately 15%, additional chemotherapeutic targets are needed, especially in the therapy of inoperable and progressive pancreatic endocrine carcinomas.The expression of epidermal growth factor receptor (EGFR) and cyclooxygenase (COX)-2 were investigated in 110 clinically and pathomorphologically well-characterized pancreatic endocrine tumors, using immunohistochemistry and immunoblot analyses. Functional tests were performed using the human pancreas carcinoid cell line BON and the mouse insulinoma cell line beta-TC-3.The expression of EGFR correlated significantly with the grade of malignancy, increasing from low rates of expression in benign tumors and tumors of uncertain behavior to high rates of expression in well- and poorly differentiated endocrine carcinomas. The expression of COX-2 was independent of the malignant potential, but was more frequently expressed in primary tumors than in metastases. The treatment of the human pancreas carcinoid cell line BON and the mouse insulinoma cell line beta-TC-3 with EGFR and COX-2 inhibitors (monotherapy and combined therapy) resulted in a significant, dose-dependent reduction of cell viability coupled with increased apoptosis.Our results suggest that EGFR and COX-2 may represent useful additional chemotherapeutic targets in pancreatic endocrine tumors.

Thymomas and thymic carcinomas are peculiar epithelial tumors of the anterior mediastinum. They may show aggressive clinical behavior and are a paradigm for the interaction between the tumor and the immune system. So far, adequate functional studies enabling a better understanding of this malignancy have not been performed, since human thymoma/thymic carcinoma cell lines have not been available. Here, the authors describe the establishment, characterization and functional analyses of epithelial cell lines from a Type B1-thymoma and a poorly differentiated thymic carcinoma. By Fluorescence-activated cell sorting (FACS) analyses, both cell lines were aneuploid. The aneuploid cell fraction of the thymic carcinoma cell line was characterized by a high proliferation index of 55.9%, in contrast to a lower proliferation rate of the aneuploid cell fraction of the thymoma (19.7%). Array-based comparative genomic hybridization (aCGH) and conventional cytogenetic analysis of the thymoma revealed only minor imbalances whereas the thymic carcinoma was characterized by a complex karyotype in the hyperdiploid range that was readily defined with multicolor FISH (mFISH). Application of a selective COX-2 inhibitor reduced cell viability in both cell lines in a dose-dependent manner. In conclusion, these first cell lines of a thymoma and a CD5-positive thymic carcinoma are useful tools for further in vitro studies of cellular, molecular and genetic aspects of the disease and for functional tests to evaluate new therapeutic targets.

Clinical trials with immune-checkpoint blockade antibodies have bolstered the importance of immune therapy as the standard of care for cancer patients, but it has also simultaneously highlighted the heterogeneity in patient responses to such treatment. These heterogeneities can be explained to a certain extent by the lack of tumor-specific expression of the targeted immune checkpoint molecules. But in many cases it can also be conceived that tumors either develop resistance to a targeted immune-checkpoint node by circumventing it or more than one player is involved in a concerted action to subvert the T cell response. In either scenario, we lack a holistic understanding of the putative genes in the tumor genome that could functionally suppress the immune response. To bridge this gap, we employed a high-throughput RNAi-mediated knockdown of upto 2800 genes (~50% associated with surface molecules) in MCF7 (breast), M579-A2 (melanoma) and PANC-1 (pancreatic) tumor cell lines and co-cultured them with either antigen-specific T cell clones or respective patient-derived and tumor-specific infiltrating lymphocytes (TILs) to assess the impact on anti-tumor immunity using a luciferase-based readout. Primary hit-list was further subjected to a secondary screen based on multi-cytokine profiling of the T cells. Our investigation revealed a few salient caveats of tumor-mediated immune suppression. Firstly, we discovered a family of orphan receptors, which were never attributed to the immune system before, to actively suppress the T cells in a manner comparable to the currently defined immune-checkpoint molecules, such as PD-L1. Secondly, the trans-versatility of these novel molecules across the tumor types was highly limited, with only 3-14 common molecules being involved in two or more tumor types. This leads us to our third observation that there exists a complex organ-specific orchestration of peripheral immune tolerance, which needs to be taken into account when devising immune-checkpoint blockade therapies. Amongst the key immunosuppressive candidate genes, CCR9 was validated to directly subvert T cell responses in melanoma, breast and pancreatic cancer in the in vitro and in vivo tumor models. Additionally we have verified TiMi1, an orphan G-protein coupled receptor, to mediate strong immunosuppression in melanoma and pancreatic cancer against the respective TILs. Knockdown of both CCR9 and TiMi1, either via siRNAs or shRNAs, in tumor cells significantly increased Th1 cytokine secretion by TILs along with elevated tumor lysis in vitro and in vivo xenotransplanted mouse models. Moreover, they both induce a highly immunosuppressive genetic signature in the encountering TILs. While TiMi1 appeared to modulate calcium-dependent signaling, CCR9 regulated STAT signaling in T cells leading to an immunosuppressed phenotype. Overall, these candidates represent attractive targets for cancer immunotherapy either through function blocking antibodies or small molecules. In conclusion, we here report an effective genetic screen strategy in multiple tumor types that has the potential to uncover novel modifiers of anti-tumor immunity. Extensively validated candidates from these screens are attractive targets for cancer immunotherapy that will allow us to further expand our limited arsenal of immune-checkpoint inhibitors, with the overall goal of increasing patient responses to such treatments. Citation Format: Nisit Khandelwal, Tillmann Michels, Marco Breinig, Antonio Sorrentino, Isabel Poschke, Rienk Offringa, Michal Lotem, Michael Boutros, Philipp Beckhove. Genetic knockdown screens across tumor types unravel a diverse tumor “immune-modulatome” landscape. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A070.

Immunotherapeutic treatment of melanoma achieved major progress in recent years leading for the first time to improved survival. However, since melanoma cells employ various suppressive mechanisms in order to evade recognition and destruction by immune effector cells many patients still do not benefit from immunotherapy. These mechanisms are far more diverse than reflected by currently used immune modulatory drugs. In this study, we established and utilized a novel high throughput RNAi screening to identify new immune checkpoint molecules in melanoma using antigen-specific patient-derived tumor infiltrating lymphocytes (TILs) in conjunction with primary HLA-matched melanoma cells. Using this approach, we screened a siRNA library targeting more than 1200 surface receptors and kinases to explore novel targets for immunotherapy. Briefly, HLA-A2 and luciferase positive M579-A2-luc melanoma cells were reversely transfected with the siRNA library and then co-cultured with MART1- and gp100-specific TILs to validate the TIL-mediated tumor lysis. Local regression models (LOESS) were applied to generate a hit list of 48 candidates that negatively regulated CTL cytotoxicity. Interestingly, four candidates of a related breast cancer screen were among the top hits. To streamline the discovery process for large scale molecule libraries, we established a secondary screen assaying multiple T cell activation markers, including effector cytokines. One of the strongest candidates from our primary and secondary screening is TiMi1 (name altered), a cell surface receptor belonging to the class of GPCRs. We found that knock-down of TiMi1 increased TIL-mediated killing of M579-A2-luc without affecting their viability. TiMi1 knock-down increased TIL activity measured by production of type 1-associated cytokines (e.g. IFN γ and TNF-α), reduced TC apoptosis and increased markers associated with raised activity and cytotoxicity (4-1BB and CD107a). We were able to verify the immune checkpoint function of TiMi1 in melanoma patients using an autologous set of melanoma cells and TILs. Phosphoplex analysis in T cells revealed an involvement of the transcription factor CREB in the mode of action of TiMi1. Preliminary experiments suggest that TiMi1 inhibits anti-tumor immune responses in pancreatic (PDAC) and colorectal (CRC) cancers as well. In summary, we established a novel antigen-specific screening approach for immune checkpoints expressed in melanoma and were able to identify TiMi1 as a promising candidate. Moreover, TiMi1 inhibits T cell responses in melanoma, PDAC and CRC and might be an interesting target for immunotherapy. Our novel high-throughput screening offers a systematic platform to uncover the “immune-modulatome” of cancer and subsequently discover novel targets for immunotherapy. Since the presented work is considered for patent protection, some gene targets are masked in the presented study. Citation Format: Tillmann Michels, Christina A. Hartl, Nisit Khandelwal, Marco Breinig, Antonio Sorrentino, Christina Mäder, Ludmila Umansky, Isabel Poschke, Rienk Offringa, Michael Boutros, Galit Eisenberg, Michal Lotem, Philipp Beckhove. TiMi1 is a novel immune-checkpoint in solid tumors identified via a tumor-infiltrating lymphocyte (TIL)-based RNAi screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 254. doi:10.1158/1538-7445.AM2015-254

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) accounts for 95% of pancreatic cancers and constitutes the fourth leading cause of cancer related death worldwide. In contrast to other malignancies, PDAC is highly resistant to chemotherapy and radiotherapy. Additionally, few immunotherapies are currently available because this malignancy was thought to be poorly immunogenic. Recent studies have shown that infiltration of immune cells in biopsies of PDAC patients correlates with improved clinical outcome. Nevertheless, tumor cells can elude the immune system through several inhibitory mechanisms including the expression of immune checkpoints. These are a plethora of molecules that can either boost or dampen the T-cell receptor (TCR) signaling. Some of these molecules, such as PD-L1, have been successfully used as therapeutic targets of novel anticancer drugs. AIM: We hypothesize that many immune checkpoint molecules on tumor cells remain undiscovered and we performed a high-throughput RNAi screen to unravel the whole arsenal of immune modulators. METHODS: We generated a luciferase-expressing PANC-1 cell line and knocked down 2514 genes using a siRNA library. Our library included G-protein coupled receptors, protein kinases and 1117 surface proteins. We co-cultured HLA-A201+ matched tumor infiltrating lymphocytes (TILs) derived from a PDAC patient with the transfected tumor cells. We then measured the remaining luciferase intensity of the tumor cells as an estimation of TIL-mediated cytotoxicity. In order to exclude genes whose knock-down affected cell viability per se, we cultivated tumor cells with the siRNA library in the absence of TILs. Data were analyzed with the cellHTS2 R package. RESULTS: Our screen revealed 155 candidate genes whose knock-down enhances TIL-mediated killing more efficiently than PD-L1 down-regulation. 35% of these genes are surface molecules and are most likely to directly mediate tumor immune evasion. Beside novel undescribed immune checkpoints, our list contains well characterized immune modulators, supporting the reliability of our approach. Of note 13 of our hits were also found in a related melanoma screen and might play a role in the regulation of immune surveillance of many solid tumors. Among our candidates, TONI1 was one of the most prominent hits. So far, we confirmed the role of TONI1 in inhibiting TIL-mediated killing both in chromium release and luciferase based kill assays. Additionally we detected increased T-cell activity upon TONI1 down-regulation, as measured with interferon-γ ELISPOT and TNF-α ELISA. Since the presented work is considered for patent protection, some gene targets are masked. CONCLUSION: We set up a robust and systematic method to identify novel immune checkpoints for pancreatic cancer. Further functional validation of our candidate genes will prove their use as therapeutic targets. Citation Format: Antonio Sorrentino, Tillmann Michels, Ayse Nur Menevse, Nisit Khandelwal, Marco Breinig, Isabel Poschke, Rienk Offringa, Michael Boutros, Philipp Beckhove. Identification of novel immune checkpoints as potential therapeutic targets in pancreatic ductal adenocarcinoma (PDAC) using RNAi screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 245. doi:10.1158/1538-7445.AM2015-245

Use of transcription activator-like effector nucleases (TALENs) is a promising new technique in the field of targeted genome engineering, editing and reverse genetics. Its applications span from introducing knockout mutations to endogenous tagging of proteins and targeted excision repair. Owing to this wide range of possible applications, there is a need for fast and user-friendly TALEN design tools. We developed E-TALEN (http://www.e-talen.org), a web-based tool to design TALENs for experiments of varying scale. E-TALEN enables the design of TALENs against a single target or a large number of target genes. We significantly extended previously published design concepts to consider genomic context and different applications. E-TALEN guides the user through an end-to-end design process of de novo TALEN pairs, which are specific to a certain sequence or genomic locus. Furthermore, E-TALEN offers a functionality to predict targeting and specificity for existing TALENs. Owing to the computational complexity of many of the steps in the design of TALENs, particular emphasis has been put on the implementation of fast yet accurate algorithms. We implemented a user-friendly interface, from the input parameters to the presentation of results. An additional feature of E-TALEN is the in-built sequence and annotation database available for many organisms, including human, mouse, zebrafish, Drosophila and Arabidopsis, which can be extended in the future.