Norman Woller

Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this ‘secretory phenotype’ can have pro- as well as anti-tumorigenic effects. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as ‘senescence surveillance’), which depends on an intact CD4+ T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of NrasG12V. We also found that CD4+ T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

Telomerase-Dependent Virotherapy Overcomes Resistance of Hepatocellular Carcinomas against Chemotherapy and Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand by Elimination of Mcl-1

Hepatocellular carcinomas (HCC) are drug-resistant tumors that frequently possess high telomerase activity. It was therefore the aim of our study to investigate the potential of telomerase-dependent virotherapy in multimodal treatment of HCC. In contrast to normal liver, HCC xenografts showed high telomerase activity, resulting in tumor-restricted expression of E1A by a telomerase-dependent replicating adenovirus (hTERT-Ad). Neither tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or chemotherapy alone nor the combined treatment with both agents resulted in significant destruction of HCC cells. Application of hTERT-Ad at low titers was also not capable to destroy HCC cells, but telomerase-dependent virotherapy overcame the resistance of HCC against TRAIL and chemotherapy. The synergistic effects are explained by a strong down-regulation of Mcl-1 expression through hTERT-Ad that sensitizes HCC for TRAIL- and chemotherapy-mediated apoptosis. To investigate whether down-regulation of Mcl-1 alone is sufficient to explain synergistic effects observed with virotherapy, Mcl-1 expression was inhibited by RNA interference. Treatment with Mcl-1-siRNA significantly enhanced caspase-3 activity after chemotherapy and TRAIL application, confirming that elimination of Mcl-1 is responsible for the drug sensitization by hTERT-Ad. Consistent with these results, heterologous overexpression of Mcl-1 significantly reduced the sensitization of hTERT-Ad transduced cells against apoptosis-inducing agents. Chemotherapy did not interfere with quantitative hTERT-Ad production in HCC cells. Whereas hTERT-Ad virotherapy alone was only capable to inhibit the growth of Hep3B xenografts, virochemotherapy resulted in vast destruction of the drug-resistant HCC. In conclusion our data indicate that telomerase-dependent virotherapy is an attractive strategy to overcome the natural resistance of HCC against anticancer drugs by elimination of Mcl-1.

Viral Infection of Tumors Overcomes Resistance to PD-1-immunotherapy by Broadening Neoantigenome-directed T-cell Responses

There is evidence that viral oncolysis is synergistic with immune checkpoint inhibition in cancer therapy but the underlying mechanisms are unclear. Here, we investigated whether local viral infection of malignant tumors is capable of overcoming systemic resistance to PD-1-immunotherapy by modulating the spectrum of tumor-directed CD8 T-cells. To focus on neoantigen-specific CD8 T-cell responses, we performed transcriptomic sequencing of PD-1-resistant CMT64 lung adenocarcinoma cells followed by algorithm-based neoepitope prediction. Investigations on neoepitope-specific T-cell responses in tumor-bearing mice demonstrated that PD-1 immunotherapy was insufficient whereas viral oncolysis elicited cytotoxic T-cell responses to a conserved panel of neoepitopes. After combined treatment, we observed that PD-1-blockade did not affect the magnitude of oncolysis-mediated antitumoral immune responses but a broader spectrum of T-cell responses including additional neoepitopes was observed. Oncolysis of the primary tumor significantly abrogated systemic resistance to PD-1-immunotherapy leading to improved elimination of disseminated lung tumors. Our observations were confirmed in a transgenic murine model of liver cancer where viral oncolysis strongly induced PD-L1 expression in primary liver tumors and lung metastasis. Furthermore, we demonstrated that combined treatment completely inhibited dissemination in a CD8 T-cell-dependent manner. Therefore, our results strongly recommend further evaluation of virotherapy and concomitant PD-1 immunotherapy in clinical studies.

Antitumoral Immune Response by Recruitment and Expansion of Dendritic Cells in Tumors Infected with Telomerase-Dependent Oncolytic Viruses

Virotherapy can potentially be used to induce tumor-specific immune responses and to overcome tumor-mediated tolerance mechanisms because apoptotic tumor cells are exposed together with viral danger signals during oncolysis. However, insufficient numbers of dendritic cells (DC) present at the site of oncolysis can limit a tumor-specific immune response and the resulting therapeutic benefit. We investigated MHC class I peptide-specific immune responses against model antigens ovalbumin (OVA) and hemagglutinin (HA) in mouse tumor models that support efficient replication of the oncolytic adenovirus hTert-Ad. Virotherapy resulted in peptide-specific cytotoxic T-cell responses against intracellular tumor antigens. Triggering of DC and T-cell infiltration to the oncolytic tumors by macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) and Fms-like tyrosine kinase-3 ligand (Flt3L) enhanced both antitumoral and antiviral immune responses. Although immune-mediated clearance of the virus can restrict therapeutic efficacy of virotherapy, MIP-1alpha/FLT3L-augmented hTert-Ad virotherapy inhibited local tumor growth more effectively than virotherapy alone. In agreement with the hypothesis that immune-mediated mechanisms account for improved outcome in MIP-1alpha/FLT3L virotherapy, we observed systemic antitumoral effects by MIP-1alpha/FLT3L virotherapy on uninfected lung metastasis in immunocompetent mice but not in nude mice. Furthermore, MIP-1alpha/FLT3L virotherapy of primary tumors was strongly synergistic with tumor DC vaccination in inhibition of established lung metastasis. Combined viroimmunotherapy resulted in long-term survival of 50% of treated animals. In summary, improvement of cross-presentation of tumor antigens by triggering of DC and T-cell infiltration during virotherapy enhances antitumoral immune response that facilitates an effective viroimmunotherapy of primary tumors and established metastases.

Oncolytic Viruses as Anticancer Vaccines

Oncolytic virotherapy has shown impressive results in preclinical studies and first promising therapeutic outcomes in clinical trials as well. Since viruses are known for a long time as excellent vaccination agents, oncolytic viruses are now designed as novel anticancer agents combining the aspect of lysis-dependent cytoreductive activity with concomitant induction of antitumoral immune responses. Antitumoral immune activation by oncolytic virus infection of tumor tissue comprises both, immediate effects of innate immunity and also adaptive responses for long lasting antitumoral activity, which is regarded as the most prominent challenge in clinical oncology. To date, the complex effects of a viral tumor infection on the tumor microenvironment and the consequences for the tumor-infiltrating immune cell compartment are poorly understood. However, there is more and more evidence that a tumor infection by an oncolytic virus opens up a number of options for further immunomodulating interventions such as systemic chemotherapy, generic immunostimulating strategies, dendritic cell-based vaccines, and antigenic libraries to further support clinical efficacy of oncolytic virotherapy.

Genetic predisposition and environmental danger signals initiate chronic autoimmune hepatitis driven by CD4+ T cells

Autoimmune hepatitis (AIH) is defined as a chronic liver disease with loss of tolerance against liver tissue eventually leading to cirrhosis if left untreated. 80%‐90% of patients can be treated with a life‐long immunosuppression. Unfortunately, there are strong drug‐related side effects and steroid‐refractory patients. Therefore, there is a need for a model system to investigate the complex immunopathogenesis of this chronic disease and subsequently to develop new therapeutic interventions. We developed a new model of experimental murine AIH (emAIH) by a self‐limited adenoviral infection with the hepatic autoantigen formiminotransferase cyclodeaminase (FTCD). After an initial transient hepatitis there was a chronic evolving AIH, finally leading to portal and lobular fibrosis. We could show that the genetic predisposition provided by the NOD background was essential for creating a fertile field for the development of liver‐specific autoimmunity. However, a strong environmental trigger was additionally necessary to initiate the disease. Besides the break of humoral tolerance, T‐cell tolerance against hepatic self‐antigens was also broken and CD4+ T cells were identified as essential drivers of the disease. As the disease was successfully treated with prednisolone and budesonide, the model will be helpful to develop and test new therapeutic interventions. Conclusion: We developed a new murine AIH model closely resembling AIH in patients that explains the mechanisms of AIH pathophysiology. In addition, emAIH provides options to test therapeutic alternatives for patients not achieving remission, with reduced side effects of chronic nonspecific immunosuppression. (Hepatology 2013;58:718–728)

Virus-induced tumor inflammation facilitates effective DC cancer immunotherapy in a Treg-dependent manner in mice

Vaccination using DCs pulsed with tumor lysates or specific tumor-associated peptides has so far yielded limited clinical success for cancer treatment, due mainly to the low immunogenicity of tumor-associated antigens. In this study, we have identified intratumoral virus-induced inflammation as a precondition for effective antitumor DC vaccination in mice. Administration of a tumor-targeted DC vaccine during ongoing virus-induced tumor inflammation, a regimen referred to as oncolysis-assisted DC vaccination (ODC), elicited potent antitumoral CD8+ T cell responses. This potent effect was not replicated by TLR activation outside the context of viral infection. ODC-elicited immune responses mediated marked tumor regression and successful eradication of preestablished lung colonies, an essential prerequisite for potentially treating metastatic cancers. Unexpectedly, depletion of Tregs during ODC did not enhance therapeutic efficacy; rather, it abrogated antitumor cytotoxicity. This phenomenon could be attributed to a compensatory induction of myeloid-derived suppressor cells in Treg-depleted and thus vigorously inflamed tumors, which prevented ODC-mediated immune responses. Consequently, Tregs are not only general suppressors of immune responses, but are essential for the therapeutic success of multimodal and temporally fine-adjusted vaccination strategies. Our results highlight tumor-targeting, replication-competent viruses as attractive tools for eliciting effective antitumor responses upon DC vaccination.

Protein Transduction Domains Fused to Virus Receptors Improve Cellular Virus Uptake and Enhance Oncolysis by Tumor-Specific Replicating Vectors

ABSTRACT Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. Protein transduction domains (PTDs) have been shown to deliver proteins, antisense oligonucleotides, liposomes, or plasmid DNA into cells. In our study, we investigated the role of several PTD motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirus-adenovirus receptor (CARex) to attach PTDs to adenoviral fiber knobs. CARex-Tat and CARex-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Dose-dependent competition of CARex-PTD-mediated infection using CARex and inhibition experiments with heparin showed that binding of CARex-PTD to both adenoviral fiber and cellular glycosaminoglycans is essential for the improvement of infection. CARex-PTD-treated adenoviruses retained their properties after density gradient ultracentrifugation, indicating stable binding of CARex-PTD to adenoviral particles. Consequently, the mechanism of CARex-PTD-mediated infection involves coating of the viral fiber knobs by CARex-PTD, rather than placement of CARex domains on cell surfaces. Expression of CARex-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CARex-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Together, our study shows that CARex-PTDs facilitate gene transfer in nonpermissive cells and improve viral uptake at reduced titers and infection times. The data suggest that PTDs fused to virus binding receptors may be a valuable tool to overcome natural tropism of vectors and could be of great interest for gene therapeutic approaches.

Selectivity of Oncolytic Viral Replication Prevents Antiviral Immune Response and Toxicity, but Does Not Improve Antitumoral Immunity

Oncolytic infection elicits antitumoral immunity, but the impact of tumor-selective replication on the balance between antiviral and antitumoral immune responses has not yet been investigated. To address this question, we constructed the highly tumor-selective adenovirus Ad-p53T whose replication in target tumor cells is governed by aberrant telomerase activity and transcriptional p53 dysfunction. Telomerase-dependent or nonselective adenoviruses were constructed as isogenic controls. Following infection of mice with the nonselective adenovirus, viral DNA and mRNA levels correlated with strong stimulation of innate immune response genes and severe liver toxicity, whereas telomerase-/p53-specific replication did not trigger innate immunity and prevented liver damage. Compared to telomerase-dependent or unselective viral replication, telomerase-/p53-specific virotherapy significantly decreased antiviral CD8-specific immune responses and antiviral cytotoxicity in vivo. Consistent with our hypothesis, telomerase-selective replication led to intermediate results in these experiments. Remarkably, all viruses efficiently lysed tumors and induced a therapeutically effective tumor-directed CD8 cytotoxicity. In immunocompetent mice with extended lung metastases burden, treatment of subcutaneous primary tumors with Ad-p53T significantly prolonged survival by inhibition of lung metastases, whereas unselective viral replication resulted in death by liver failure. In summary, the degree of tumor selectivity of viral replication marginally influences antitumoral immune responses, but is a major determinant of antivector immunity and systemic toxicity.

p53-dependent antiviral RNA-interference facilitates tumor-selective viral replication

RNA-interference (RNAi) is a potent tool for specific gene silencing. In this study, we developed an adenovirus for conditional replication in p53-dysfunctional tumor cells that uses p53-selective expression of a microRNA-network directed against essential adenoviral genes. Compared to a control virus that expressed a scrambled microRNA-network, antiviral RNAi selectively attenuated viral replication in cells with transcriptionally active p53, but not in p53-dysfunctional tumor cells where both viruses replicated equivalently. Since these results were confirmed by an in vivo comparison of both viruses after infection of p53-knockout and normal mice, we could demonstrate that attenuated replication was indeed a result of p53-selective exhibition of antiviral RNAi. Addressing the therapeutic applicability, we could show that the application of RNAi-controlled virus efficiently lysed p53-dysfunctional tumors in vitro and in vivo but resulted in drastically reduced load of virus-DNA in the liver of treated mice. We have generated a broadly applicable adenovirus for selective destruction of p53-dysfunctional tumors and thereby demonstrate that virus-encoded RNAi-networks represent an efficient and versatile tool to modify viral functions. RNAi-networks can be applied to all transcriptionally regulated DNA-viruses to remodulate viral tropism and thus provide means to generate specifically replicating vectors for clinical applications.