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We discovered a novel somatic gene fusion, CD74–NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74–NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2–ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74–NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K–AKT pathway, and led to increased colony formation in soft agar. Thus, CD74–NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment.Significance: CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease. Cancer Discov; 4(4); 415–22. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 377

XLSX file 75K, This is an excel file containing 9 sheets, one for each of the 9 supplementary tables (S1 to S9) mentioned in the main text

PDF file 234K, This is a file containing the list of supplementary tables, the list of supplementary figures, as well as the 7 supplementary figures (S1 to S7) and their correspondent legends, mentioned in the main text

PDF file 136K,Methods: Additional information on experimental techniques and data analysis. Legends: describing supplementary figures S1-S14

PDF file 34156K, Table of genes from 16 recurrent amplified genomic regions (Figure S1), amplicon focality on TCGA copy number aberrations (Figure S2), hierarchical clustering of CLCGP copy number data (Figure S3), centrality frequencies of common cancer genes (Figure S4), genotypic cell line annotation (Figure S5), receptor signaling activation of H520 and HCC15 lines (Figure S6), experimental draft for ligand dependency (Figure S7), whole transcriptome analysis FGFR1 splicing (Figure S8), validation of gene expression by qPCR (Figure S9), tumor formation of FGFR1α cells in nude mice (Figure S10), quantification of MYC stains in murine tumor samples (Figure S11), apoptosis is accompanied by cytochrome c release and breakdown of mitochondrial potential (Figure S12), IHC scores of FGFR phosphorylation and MYC expression (Figure S13), High MYC expression identifies a second responder to FGFR inhibitory therapy (Figure S14)

Clinicopathologic parameters of mice and patients with the corresponding survival significance analysis of SCLC bearing mice treated with vehicle, IgG control, anti-VEGF monotherapy, anti-PD-L1 monotherapy or anti-VEGF/anti-PD-L1 combination therapy

Small cell lung cancer (SCLC) represents the most aggressive pulmonary neoplasm and is often diagnosed at late stage with limited survival, despite combined chemotherapies. We show in an autochthonous mouse model of SCLC that combined anti-VEGF/anti-PD-L1–targeted therapy synergistically improves treatment outcome compared with anti–PD-L1 and anti-VEGF monotherapy. Mice treated with anti–PD-L1 alone relapsed after 3 weeks and were associated with a tumor-associated PD-1/TIM-3 double-positive exhausted T-cell phenotype. This exhausted T-cell phenotype upon PD-L1 blockade was abrogated by the addition of anti-VEGF–targeted treatment. We confirmed a similar TIM-3–positive T-cell phenotype in peripheral blood mononuclear cells of patients with SCLC with adaptive resistance to anti–PD-1 treatment. Mechanistically, we show that VEGFA enhances coexpression of the inhibitory receptor TIM-3 on T cells, indicating an immunosuppressive function of VEGF in patients with SCLC during anti–PD-1-targeted treatment. Our data strongly suggest that a combination of anti-VEGF and anti–PD-L1 therapies can be an effective treatment strategy in patients with SCLC.Significance: Combining VEGF and PD-L1 blockade could be of therapeutic benefit to patients with small cell lung cancer. Cancer Res; 78(15); 4270–81. ©2018 AACR.

Supplementary data supporting an exhausted T cell phenotype in SCLC upon acquired resistance to PD1/PD-L1 blockade which is regulated by VEGF/VEGFR signaling and in addition, an immunosuppressive role of tumor-associated macrophages in the tumor microenvironment

PDF file - 66KB

PDF file - 665KB, Imaging of tumor cell proliferation using 18FFLT PET in H1975 tumors.

PDF file - 357KB, Changes of mean voxel radioactivity of 15OH2O PET and tumor growth during intermittent PTK787 treatment.

PDF file - 1341KB, Pericyte coverage and tumor cell proliferation.

PDF file - 677KB, Multimodal imaging of tumor blood flow using 15OH2O PET in xenograft (H1975) in mice using ZD6474 as anti-angiogenic agent.

BackgroundSingle-agent immunotherapy has shown remarkable efficacy in selected cancer entities and individual patients. However, most patients fail to respond. This is likely due to diverse immunosuppressive mechanisms acting in a concerted way to suppress the host anti-tumor immune response. Combination immunotherapy approaches that are effective in such poorly immunogenic tumors mostly rely on precise knowledge of antigenic determinants on tumor cells. Creating an antigen-agnostic combination immunotherapy that is effective in poorly immunogenic tumors for which an antigenic determinant is not known is a major challenge.MethodsWe use multiple cell line and poorly immunogenic syngeneic, autochthonous, and autologous mouse models to evaluate the efficacy of a novel combination immunotherapy named tripartite immunotherapy (TRI-IT). To elucidate TRI-ITs mechanism of action we use immune cell depletions and comprehensive tumor and immune infiltrate characterization by flow cytometry, RNA sequencing and diverse functional assays.ResultsWe show that combined adoptive cellular therapy (ACT) with lymphokine-activated killer cells, cytokine-induced killer cells, Vγ9Vδ2-T-cells (γδ-T-cells) and T-cells enriched for tumor recognition (CTLs) display synergistic antitumor effects, which are further enhanced by cotreatment with anti-PD1 antibodies. Most strikingly, the full TRI-IT protocol, a combination of this ACT with anti-PD1 antibodies, local immunotherapy of agonists against toll-like receptor 3, 7 and 9 and pre-ACT lymphodepletion, eradicates and induces durable anti-tumor immunity in a variety of poorly immunogenic syngeneic, autochthonous, as well as autologous humanized patient-derived models. Mechanistically, we show that TRI-IT coactivates adaptive cellular and humoral, as well as innate antitumor immune responses to mediate its antitumor effect without inducing off-target toxicity.ConclusionsOverall, TRI-IT is a novel, highly effective, antigen-agnostic, non-toxic combination immunotherapy. In this study, comprehensive insights into its preclinical efficacy, even in poorly immunogenic tumors, and mode of action are given, so that translation into clinical trials is the next step.

Summary Background Individualization of treatment in Hodgkin’s lymphoma is necessary to improve cure rates and reduce treatment side effects. Currently, it is hindered by a lack of genomic characterization and sensitive molecular response assessment. Sequencing of cell-free DNA is a powerful strategy to understand the cancer genome and can be used for extremely sensitive disease monitoring. In Hodgkin’s lymphoma, a high proportion of cell-free DNA is tumor-derived, whereas traditional tumor biopsies only contain a little tumor-derived DNA. Methods We comprehensively genotype and assess minimal residual disease in 121 patients with baseline plasma as well as 77 follow-up samples from a subset of patients with our targeted cell-free DNA sequencing platform. Findings We present an integrated landscape of mutations and copy number variations in Hodgkin’s lymphoma. In addition, we perform a deep analysis of mutational processes driving Hodgkin’s lymphoma, investigate the clonal structure of Hodgkin’s lymphoma, and link several genotypes to Hodgkin’s lymphoma phenotypes and outcome. Finally, we show that minimal residual disease assessment by repeat cell-free DNA sequencing, as early as a week after treatment initiation, predicts treatment response and progression-free survival, allowing highly improved treatment guidance and relapse prediction. Conclusions Our targeted cell-free DNA sequencing platform reveals the genomic landscape of Hodgkin’s lymphoma and facilitates ultrasensitive detection of minimal residual disease. Funding Mildred Scheel School of Oncology Aachen-Bonn-Cologne-Dusseldorf MD Research Stipend, Next Generation Sequencing Competence Network grant 423957469, Deutsche Krebshilfe grant 70112502, Deutsche Forschungsgemeinschaft (DFG) grant EN 179/13-1, the HL MRD consortium, and the Frau-Weiskam und Christel Ruranski-Stiftung.

Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8+ T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients., Kinase inhibitors are widely used to treat cancer, however patients frequently develop resistance. Here, the authors investigate adaption mechanisms during drug persistence and show that stimulation of the innate immunity sensor RIG-I enhances cancer cell death when combined with kinase inhibition.

Introduction: The Trophoblast cell surface antigen 2 (TROP2) is expressed in many carcinomas and may represent a target for treatment. Sacituzumab govitecan (SG) is a TROP2–directed antibody-drug conjugate (ADC). Nearly nothing is known about the biological effectiveness of SG in esophageal adenocarcinoma (EAC). Material and Methods: We determined the TROP2 expression in nearly 600 human EAC. In addition, we used the EAC cell lines (ESO-26, OACM5.1C, and FLO-1) and a xenograft mouse model to investigate this relationship. Results: Of 598 human EACs analyzed, 88% showed varying degrees of TROP2 positivity. High TROP2 positive ESO-26 and low TROP2 positive OACM5.1C showed high sensitivity to SG in contrast to negative FLO-1. In vivo, the ESO-26 tumor shows a significantly better response to SG than the TROP2-negative FLO-1 tumor. ESO-26 vital tumor cells show similar TROP2 expression on all carcinoma cells as before therapy initiation, FLO-1 is persistently negative. Discussion: Our data suggest that sacituzumab govitecan is a new therapy option in esophageal adenocarcinoma and the TROP2 expression in irinotecan-naïve EAC correlates with the extent of treatment response by sacituzumab govitecan. TROP2 is emerging as a predictive biomarker in completely TROP2-negative tumors. This should be considered in future clinical trials.

EGFR-driven non-small-cell lung cancer (NSCLC) patients are currently treated with TKIs targeting EGFR, such as erlotinib or osimertinib. Despite a promising initial response to TKI treatment, most patients gain resistance to oncogene-targeted therapy, and tumours progress. With the development of inhibitors against immune checkpoints, such as PD-1, that mediate an immunosuppressive microenvironment, immunotherapy approaches attempt to restore a proinflammatory immune response in tumours. However, this strategy has shown only limited benefits in EGFR-driven NSCLC. Approaches combining EGFR inhibition with immunotherapy to stimulate the immune response and overcome resistance to therapy have been limited due to insufficient understanding about the effect of EGFR-targeting treatment on the immune cells in the TME. Here, we investigate the impact of EGFR inhibition by erlotinib on the TME and its effect on the antitumour response of the immune cell infiltrate. For this purpose, we used a transgenic conditional mouse model to study the immunological profile in EGFR-driven NSCLC tumours. We found that EGFR inhibition mediated a higher infiltration of immune cells and increased local proliferation of T-cells in the tumours. Moreover, inhibiting EGFR signalling led to increased activation of immune cells in the TME. Most strikingly, combined simultaneous blockade of EGFR and anti-PD-1 (aPD-1) enhanced tumour treatment response in a transgenic mouse model of EGFR-driven NSCLC. Thus, our findings show that EGFR inhibition promotes an active and proinflammatory immune cell infiltrate in the TME while improving response to immune checkpoint inhibitors in EGFR-driven NSCLC.

Individualizing treatment is key to improve outcome and reduce long-term side-effects in any cancer. In Hodgkin lymphoma (HL), individualization of treatment is hindered by a lack of genomic characterization and technology for sensitive, molecular response assessment.Sequencing of cell-free (cf)DNA is a powerful strategy to understand an individual cancer genome and can be used to develop assays for extremely sensitive disease monitoring. In HL, a high proportion of cfDNA is tumor-derived making it a highly relevant disease model to study the role of cfDNA sequencing in cancer.Here, we introduce our targeted cfDNA sequencing platform and present the largest genomic landscape of HL to date, which was entirely derived by cfDNA sequencing. We comprehensively genotype and assess minimal residual disease in 324 samples from 121 patients, presenting an integrated landscape of mutations and copy number variations in HL. In addition, we perform a deep analysis of mutational processes driving HL, investigate the clonal structure of HL and link several genotypes to HL phenotypes and outcome. Finally, we show that minimal residual disease assessment by repeat cfDNA sequencing as early as a week after treatment initiation is feasible and predicts overall treatment response allowing highly improved treatment guidance and relapse prediction. Our study also serves as a blueprint showcasing the utility of our platform for other cancers with similar therapeutic challenges.

Objective Single-nucleotide polymorphisms in the FTO gene encoding an m6Am and an m6A demethylase are associated with obesity. Moreover, recent studies have linked a dysregulation of m6A modifications and its machinery, including FTO, to the development of several forms of cancers. However, the functional role of hepatic FTO in metabolism and the development and progression of hepatocellular carcinoma (HCC), a proteotypic obesity-associated cancer, remains unclear. Thus, we aimed to reveal the role of hepatic FTO in metabolism and in the initiation and progression of HCC in vivo. Methods We generated mice with hepatic FTO deficiency (FTOL−KO). The effect of hepatic FTO on metabolism was investigated by extensive metabolic phenotyping. To determine the impact of hepatic FTO on HCC development, FTOL−KO and Ctrl mice were subjected to long-term diethylnitrosamine (DEN)-induced HCC-development and the tumor initiation phase was examined via a short-term DEN protocol. Results In long-term DEN experiments, FTOL−KO mice exhibit increased HCC burden compared to Ctrl mice. In the tumor initiation phase, Ctrl mice display a dynamic regulation of FTO upon induction of liver damage, while this response is abrogated in FTO-deficient mice. Proteomic analyses revealed that liver damage-induced increases in FTO expression reduce CUL4A protein abundance. Functionally, simultaneous knockdown of Cul4a reverses the increased hepatocyte proliferation observed upon loss of FTO. Conclusion Collectively, our study demonstrates that hepatic FTO is dispensable for the control of energy homeostasis and glucose metabolism. However, we show a protective function of FTO in liver carcinogenesis and suggest the FTO-dependent dynamic mRNA demethylation of Cul4a in the initiation of HCC development contributes to this effect., Graphical abstract Image 1, Highlights • Hepatic FTO is dispensable for whole body metabolism. • FTO is dynamically regulated upon acute liver damage and controls proliferation. • Hepatic FTO function protects against the development of hepatocellular carcinoma (HCC). • Cul4a is a downstream target of FTO, and Cul4a knockdown reduces damage-induced proliferation in FTOL−KO livers.

Summary: Anti-angiogenic treatment targeting vascular endothelial growth factor (VEGF)-VEGFR2 signaling has shown limited efficacy in lung cancer patients. Here, we demonstrate that inhibition of VEGFR2 in tumor cells, expressed in ∼20% of non-squamous non-small cell lung cancer (NSCLC) patients, leads to a pro-invasive phenotype. Drug-induced inhibition of tumor VEGFR2 interferes with the formation of the EphA2/VEGFR2 heterocomplex, thereby allowing RSK to interact with Serine 897 of EphA2. Inhibition of RSK decreases phosphorylation of Serine 897 EphA2. Selective genetic modeling of Serine 897 of EphA2 or inhibition of EphA2 abrogates the formation of metastases in vivo upon VEGFR2 inhibition. In summary, these findings demonstrate that VEGFR2-targeted therapy conditions VEGFR2-positive NSCLC to Serine 897 EphA2-dependent aggressive tumor growth and metastasis. These data shed light on the molecular mechanisms explaining the limited efficacy of VEGFR2-targeted anti-angiogenic treatment in lung cancer patients. : Anti-angiogenic treatment targeting VEGFR2 signaling has shown limited efficacy in lung cancer patients. Volz et al. show that VEGFR2 inhibition leads to a pro-invasive phenotype in VEGFR2-positive non-small cell lung cancer cells, which is mediated by phosphorylation of EphA2-S897. Keywords: VEGFR2 inhibition, tumor cell invasion, S897 EphA2, RSK, NSCLC

FGFR1 amplification has been found in 15% of patients with breast cancer and has been postulated as a promising marker to predict response against FGFR inhibitors. However, early phase clinical trials of selective FGFR inhibitors demonstrated only limited efficacy in FGFR1-amplified breast cancer patients. We found that BGJ398, an FGFR inhibitor, effectively inhibited phosphorylation of FGFR1 and MEK/ERK signaling in FGFR1-amplified breast cancer without affecting tumor cell proliferation. However, FGFR1 knockout inhibited tumor angiogenesis in vivo. We unraveled that FGFR1 regulates the secretion of the proangiogenic vascular endothelial growth factor (VEGF) in a MAPK-dependent manner. We further found that FGF-FGFR1 signaling induces an autocrine activation of VEGF-VEGFR1 pathway that again amplifies VEGF secretion via VEGF-VEGFR1-AKT signaling. Targeting both VEGFR1 and FGFR1 resulted in synergistic anti-angiogenic treatment effects in vivo. We thus postulate synergistic treatment effects in FGFR1/VEGFR1-positive breast cancer patients by dual targeting of FGFR and VEGFR.

LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRASG12V-driven mouse model. We found that LIN28B overexpression significantly increased the number of CD44+/CD326+ tumor cells, upregulated VEGF-A and miR-21 and promoted tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) accompanied by enhanced AKT phosphorylation and nuclear translocation of c-MYC. Moreover, LIN28B accelerated tumor initiation and enhanced proliferation which led to a shortened overall survival. In addition, we analyzed lung adenocarcinomas of the Cancer Genome Atlas (TCGA) and found LIN28B expression in 24% of KRAS-mutated cases, which underscore the relevance of our model.

PURPOSE Vitamin D deficiency is described as a modifiable risk factor for the incidence of and mortality in many common cancers; however, data in Hodgkin lymphoma (HL) are lacking. PATIENTS AND METHODS We thus performed a study measuring pretreatment vitamin D levels in prospectively treated patients with HL and correlated this with clinical outcomes. A total of 351 patients from the German Hodgkin Study Group clinical trials (HD7, HD8, and HD9) were included. RESULTS Fifty percent of patients were vitamin D deficient (< 30 nmol/L) before planned chemotherapy. Pretreatment vitamin D deficiency was more common in relapsed/refractory patients than matched relapse-free controls (median baseline vitamin D, 21.4 nmol/L v 35.5 nmol/L; proportion with vitamin D deficiency, 68% v 41%; P < .001). Vitamin D–deficient patients had impaired progression-free survival (10-year difference, 17.6%; 95% CI, 6.9% to 28.4%; hazard ratio, 2.13; 95% CI, 1.84 to 2.48; P < .001) and overall survival (10-year difference, 11.1%; 95% CI, 2.1% to 20.2%; hazard ratio, 1.82; 95% CI, 1.53 to 2.15; P < .001), consistent across trials and treatment groups. We demonstrated that vitamin D status is an independent predictor of outcome and hypothesized that vitamin D status might be important for the chemosensitivity of HL. We subsequently performed experiments supplementing physiologic doses of vitamin D (calcitriol) to cultured HL cell lines and demonstrated increased antiproliferative effects in combination with chemotherapy. In an HL-xenograft animal model, we showed that supplemental vitamin D (dietary supplement, cholecalciferol) improves the chemosensitivity of tumors by reducing the rate of tumor growth compared with vitamin D or chemotherapy alone. CONCLUSION On the basis of our clinical and preclinical findings, we encourage that vitamin D screening and replacement be incorporated into future randomized clinical trials to properly clarify the role of vitamin D replacement therapy in HL.

Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is however disturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from senescent cell nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin or about its roles in the nucleus. To address this, we mapped HMGB1 binding genome-wide in different primary cells using a tailored protocol. We integrated ChIP-seq and Hi-C data with a graph theory approach to uncover HMGB1 demarcation of a subset of topologically-associating domains (TADs) that harbor genes required for paracrine senescence. Moreover, using sCLIP, knock-down and overexpression experiments, we now show that HMGB1 is abona fideRNA-binding protein (RBP) bound to senescence-relevant mRNAs and affecting splicing. HMGB1 also has an interactome rich in RBPs, many of which are implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and concertedly regulate the availability of SASP-relevant transcripts. Our findings highlight a broader than hitherto assumed role for HMGB1. It coordinates chromatin folding and RNA homeostasis as part of a feedforward loop controlling both cell-autonomous and paracrine senescence inside and outside of cells.

Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP‐dependent peri‐centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP–tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP–tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de‐clustering, prolonged multipolar mitosis, and cell death. 3D‐organotypic invasion assays reveal that CCB02 has broad anti‐invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)‐resistant EGFR‐mutant non‐small‐cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug‐resistant cancers exhibiting high incidence of centrosome amplification.

Small cell lung cancer (SCLC) represents the most aggressive pulmonary neoplasm and is often diagnosed at late stage with limited survival, despite combined chemotherapies. We show in an autochthonous mouse model of SCLC that combined anti-VEGF/anti-PD-L1–targeted therapy synergistically improves treatment outcome compared with anti–PD-L1 and anti-VEGF monotherapy. Mice treated with anti–PD-L1 alone relapsed after 3 weeks and were associated with a tumor-associated PD-1/TIM-3 double-positive exhausted T-cell phenotype. This exhausted T-cell phenotype upon PD-L1 blockade was abrogated by the addition of anti-VEGF–targeted treatment. We confirmed a similar TIM-3–positive T-cell phenotype in peripheral blood mononuclear cells of patients with SCLC with adaptive resistance to anti–PD-1 treatment. Mechanistically, we show that VEGFA enhances coexpression of the inhibitory receptor TIM-3 on T cells, indicating an immunosuppressive function of VEGF in patients with SCLC during anti–PD-1-targeted treatment. Our data strongly suggest that a combination of anti-VEGF and anti–PD-L1 therapies can be an effective treatment strategy in patients with SCLC. Significance: Combining VEGF and PD-L1 blockade could be of therapeutic benefit to patients with small cell lung cancer. Cancer Res; 78(15); 4270–81. ©2018 AACR.

Ageing-relevant processes, like cellular senescence, are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events that trigger such cascades remain elusive. We hypothesized that senescence entry by primary human cells is characterized by an early disruption of the cells' three-dimensional genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, super-resolution imaging, in silico simulations, and functional analyses on proliferating and replicatively-senescent cells from three distinct lineages. We discovered a cluster of genes involved in DNA conformation maintenance being suppressed upon senescence entry across all cell types. Of these, the abundant non-histone-like HMGB2 is depleted from nuclei before typical senescence markers appear, and is involved in loop formation. Its loss coincides with a reorganization of chromatin interactions via dramatic spatial clustering of CTCF foci. Upon HMGB2 knock-down this senescence-induced CTCF clustering is recapitulated and CTCF loops are reshuffled, as HMGB2 appears to confer local insulation both at TAD boundaries and within TADs. Our data suggest that the HMGB-mediated deregulation of genomic organization constitutes a primer for the ensuing senescent program across cell lineages.

LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRAS

We discovered a novel somatic gene fusion, CD74–NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74–NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2–ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74–NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K–AKT pathway, and led to increased colony formation in soft agar. Thus, CD74–NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. Significance: CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease. Cancer Discov; 4(4); 415–22. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 377

NKG2D, an activating receptor expressed on NK cells and T cells, is critically involved in tumor immunosurveillance. In this study, we explored the potential therapeutic utility of the NKG2D ligand ULBP2 for the treatment of colon carcinoma. To this end we designed a fusion protein consisting of human ULBP2 and an antibody-derived single chain targeting the tumor carcinoembryonic antigen (CEA). The bispecific recombinant fusion protein re-directed NK cells towards malignant cells by binding to both, tumor cells and NK cells, and triggered NK cell-mediated target cell killing in vitro. Moreover, tumor growth was significantly delayed in a syngeneic colon carcinoma mouse model in response to immunoligand treatment. The anti-tumor activity could be attributed to the stimulation of immune cells with an elevated expression of the activation marker CD69 on NK, T and NKT cells and the infiltration of CD45+ immune cells into the solid tumor. In summary, it was demonstrated that immunoligands provide specific tumor targeting by NK cells and exert anti-tumor activity in vitro and in vivo. This technology represents a novel immunotherapeutic strategy for solid tumors with the potential to be further developed for clinical applications.

The molecular mechanisms that control the balance between antiangiogenic and proangiogenic factors and initiate the angiogenic switch in tumors remain poorly defined. By combining chemical genetics with multimodal imaging, we have identified an autocrine feed-forward loop in tumor cells in which tumor-derived VEGF stimulates VEGF production via VEGFR2-dependent activation of mTOR, substantially amplifying the initial proangiogenic signal. Disruption of this feed-forward loop by chemical perturbation or knockdown of VEGFR2 in tumor cells dramatically inhibited production of VEGF in vitro and in vivo. This disruption was sufficient to prevent tumor growth in vivo. In patients with lung cancer, we found that this VEGF:VEGFR2 feed-forward loop was active, as the level of VEGF/VEGFR2 binding in tumor cells was highly correlated to tumor angiogenesis. We further demonstrated that inhibition of tumor cell VEGFR2 induces feedback activation of the IRS/MAPK signaling cascade. Most strikingly, combined pharmacological inhibition of VEGFR2 (ZD6474) and MEK (PD0325901) in tumor cells resulted in dramatic tumor shrinkage, whereas monotherapy only modestly slowed tumor growth. Thus, a tumor cell-autonomous VEGF:VEGFR2 feed-forward loop provides signal amplification required for the establishment of fully angiogenic tumors in lung cancer. Interrupting this feed-forward loop switches tumor cells from an angiogenic to a proliferative phenotype that sensitizes tumor cells to MAPK inhibition.

Summary Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CT CF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.

Positron emission tomography (PET) allows non-invasive assessment of physiological, metabolic and molecular processes in vivo. With the achievements in detector technology, spatial resolution of PET has been improved (1–2mm) enabling investigations in small experimental animals such as mice. With the development in tracer technology a variety of endogenously expressed and exogenously introduced genes can be analyzed by PET. This opens up the exciting and rapidly evolving field of molecular imaging aiming towards the non-invasive localization of the dynamics of disease specific molecular alterations and biological processes in animal models and humans in vivo. The main and most intriguing advantage of molecular imaging is the kinetic analysis of a given molecular event in the same experimental subject over time. This allows non-invasive characterization of animal models of human disease at various disease stages and after therapeutic intervention. Optical imaging (OI) is a non-invasive molecular imaging technology that uses light to interrogate cellular and molecular function in the living body. Although OI technologies are still limited by light scattering and depth of light penetration they provide a low cost and rapid alternative to the more expensive and time-consuming PET imaging. By introducing multi-modality reporter constructs (PET, fluorescence and/or bioluminescence) drawbacks of each modality can be overcome and advantages will complement one another. Thus, allowing on the one side rapid, cost-effective optical reporter imaging and on the other side tomographic as well as quantitative imaging of reporter gene expression by PET. With PET being a modality well developed for imaging in human subjects as well as small animals it provides a link from bench-to-bedside and may facilitate translation into clinical applications. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN01-04.

Methyl-L-(11)C-methionine ((11)C-MET) PET has been shown to detect brain tumors with a high sensitivity and specificity. In this study, we investigated the potential of (11)C-MET PET to noninvasively detect tumor progression in patients with gliomas. Moreover, we analyzed the relationship between changes in (11)C-MET uptake on PET and changes in various molecular immunohistochemical markers during progression of gliomas.Twenty-four patients with histologically proven glioma were investigated repeatedly with (11)C-MET PET. (11)C-MET uptake was determined for a circular region of interest. Histologic and molecular analyses for tumor progression were performed after open surgery and stereotactic biopsy, respectively.In patients with malignant progression, the mean increase in (11)C-MET uptake was 54.4% (SD, 45.5%; range, 3.1%-162.2%), whereas in patients without a change in tumor grade, mean (11)C-MET uptake did not significantly change (3.9%; SD, 13.7%; range, -24.4% to 26.3%). The difference in the change in (11)C-MET uptake between the group with malignant progression and the group without malignant progression was highly significant (P0.001). Receiver-operating-curve analysis revealed a sensitivity of 90% and a specificity of 92.3% for the detection of malignant transformation by an increase in (11)C-MET uptake of more than 14.6%. Increased (11)C-MET uptake of more than 14.6% was indicative of malignant progression in all but 3 leave-one-out iterations. A detailed immunohistochemical analysis demonstrated a significant correlation between changes in (11)C-MET uptake and the expression of vascular endothelial growth factor.These data suggest that (11)C-MET-PET represents a noninvasive method to detect malignant progression in patients with gliomas. Moreover, the increase in (11)C-MET uptake during malignant progression is reflected by an increase in angiogenesis-promoting markers as vascular endothelial growth factor.

Purpose Compartmental modelling of 3′-deoxy-3′-[18F]-fluorothymidine (18F-FLT) PET-derived kinetics provides a method for noninvasive assessment of the proliferation rate of gliomas. Such analyses, however, require an input function generally derived by serial blood sampling and counting. In the current study, 18F-FLT kinetic parameters obtained from image-derived input functions were compared with those from input functions derived from arterialized blood samples. Methods Based on the analysis of 11 patients with glioma (WHO grade II–IV) a procedure for the automated extraction of an input function from 18F-FLT brain PET data was derived. The time–activity curve of the volume of interest with the maximum difference in 18F-FLT uptake during the first 5 min after injection and the period from 60 to 90 min was corrected for partial-volume effects and in vivo metabolism of 18F-FLT. For each patient a two-compartment kinetic model was applied to the tumour tissue using the image-derived input function. The resulting kinetic rate constants K1 (transport across the blood–brain barrier) and Ki (metabolic rate constant or net influx constant) were compared with those obtained from the same data using the input function derived from blood samples. Additionally, the metabolic rate constant was correlated with the frequency of tumour cells stained with Ki-67, a widely used immunohistochemical marker of cell proliferation. Results The rate constants from kinetic modelling were comparable when the blood sample-derived input functions were replaced by the image-derived functions (K1,img and K1,sample, r = 0.95, p

Purpose We investigated the relationship between three-dimensional volumetric data of the metabolically active tumour volume assessed using [11C]-methionine positron emission tomography (MET-PET) and the area of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) enhancement assessed using magnetic resonance imaging (MRI) in patients with recurrent glioblastoma (GBM). Material and methods MET-PET and contrast-enhanced MRI with Gd-DTPA were performed in 12 uniformly pretreated patients with recurrent GBM. To calculate the volumes in cubic centimetres, a threshold-based volume-of-interest (VOI) analysis of the metabolically active tumour volume (MET uptake indexes of ≥1.3 and ≥1.5) and of the area of Gd-DTPA enhancement was performed after coregistration of all images. Results In all patients, the metabolically active tumour volume as shown using a MET uptake index of ≥1.3 was larger than the volume of Gd-DTPA enhancement (30.2 ± 22.4 vs. 13.7 ± 10.6 cm3; p = 0.04). Metabolically active tumour volumes as shown using MET uptake indexes of ≥1.3 and ≥1.5 and the volumes of Gd-DTPA enhancement showed a positive correlation (r = 0.76, p = 0.003, for an index of ≥1.3, and r = 0.74, p = 0.005, for an index of ≥1.5). Conclusion The present data suggest that in patients with recurrent GBM the metabolically active tumour volume may be substantially underestimated by Gd-DTPA enhancement. The findings support the notion that complementary information derived from MET uptake and Gd-DTPA enhancement may be helpful in developing individualized, patient-tailored therapy strategies in patients with recurrent GBM.

Somatic genetic alterations in cancers have been linked with response to targeted therapeutics by creation of specific dependency on activated oncogenic signaling pathways. However, no tools currently exist to systematically connect such genetic lesions to therapeutic vulnerability. We have therefore developed a genomics approach to identify lesions associated with therapeutically relevant oncogene dependency. Using integrated genomic profiling, we have demonstrated that the genomes of a large panel of human non–small cell lung cancer (NSCLC) cell lines are highly representative of those of primary NSCLC tumors. Using cell-based compound screening coupled with diverse computational approaches to integrate orthogonal genomic and biochemical data sets, we identified molecular and genomic predictors of therapeutic response to clinically relevant compounds. Using this approach, we showed that v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations confer enhanced Hsp90 dependency and validated this finding in mice with KRAS-driven lung adenocarcinoma, as these mice exhibited dramatic tumor regression when treated with an Hsp90 inhibitor. In addition, we found that cells with copy number enhancement of v-abl Abelson murine leukemia viral oncogene homolog 2 (ABL2) and ephrin receptor kinase and v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) kinase family genes were exquisitely sensitive to treatment with the SRC/ABL inhibitor dasatinib, both in vitro and when it xenografted into mice. Thus, genomically annotated cell-line collections may help translate cancer genomics information into clinical practice by defining critical pathway dependencies amenable to therapeutic inhibition.

Purpose To monitor the metabolic effects of an individual patient-tailored, experimental glioma therapy regimen that included repetitive multiple neurosurgical resections, radiosurgical interventions, and an adjuvant maintenance therapy based on the tyrosine kinase inhibitor imatinib in combination with the chemotherapeutic agent hydroxyurea (HU). Procedures Therapeutic effects were monitored in a 26-year-old male patient with a glioblastoma multiforme by multimodal imaging using sequential l-[methyl-(11)C]-methionine positron emission tomography (MET–PET) and MRI. The normalized MET uptake and volume of the metabolically active tumor were assessed sequentially. Results The individual patient-tailored, experimental glioma therapy caused a continuous decline of metabolically active tumor volume, associated with clinical remission over a period of more than two years. Conclusions MET–PET seems to be useful for monitoring patient-tailored, experimental glioma therapy regimens, especially when patients are treated with a multi-step therapeutic regimen. Monitoring and guidance of those experimental therapy regimens by MET–PET in a larger patient group are needed to confirm its clinical value.

Improvements of radionuclide and magnetic resonance-based imaging modalities over the past decade have enabled clinicians to noninvasively assess the dynamics of disease-specific processes at the molecular level in humans. This article will provide an overview of the recent advances in multimodal molecular neuroimaging in patients with primary brain tumors. To date, a range of complementary imaging parameters have been established in the diagnosis of brain tumors. Magnetic resonance imaging (MRI) provides mostly morphological and functional information such as tumor localization, vascular permeability, cell density, and tumor perfusion. The use of positron emission tomography (PET) enables the assessment of molecular processes, such as glucose consumption, expression of nucleoside and amino acid transporters, as well as alterations of DNA and protein synthesis. Taken together, MRI and PET give complementary information about tumor biology and activity, providing an improved understanding about the kinetics of tumor growth.