Bettina Fleischmann-Mundt

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.

Adjuvant gemcitabine therapy improves survival in a locally induced, R0‐resectable model of metastatic intrahepatic cholangiocarcinoma

Complete surgical tumor resection (R0) for treatment of intrahepatic cholangiocarcinoma (ICC) is potentially curative, but the prognosis remains dismal due to frequent tumor recurrence and metastasis after surgery. Adjuvant therapies may improve the outcome, but clinical studies for an adjuvant approach are difficult and time‐consuming for rare tumor entities. Therefore, animal models reflecting the clinical situation are urgently needed to investigate novel adjuvant therapies. To establish a mouse model of resectable cholangiocarcinoma including the most frequent genetic alterations of human ICC, we electroporated Sleeping Beauty‐based oncogenic transposon plasmids into the left liver lobe of mice. KRas‐activation in combination with p53‐knockout in hepatocytes resulted in formation of a single ICC nodule within 3‐5 weeks. Lineage tracing analyses confirmed the development of ICC by transdifferentiation of hepatocytes. Histologic examination demonstrated that no extrahepatic metastases were detectable during primary tumor progression. However, formation of tumor satellites close to the primary tumor and vascular invasion were observed, indicating early invasion into normal tissue adjacent to the tumor. After R0‐resection of the primary tumor, we were able to prolong median survival, thereby observing tumor stage‐dependent local recurrence, peritoneal carcinomatosis, and lung metastasis. Adjuvant gemcitabine chemotherapy after R0‐resection significantly improved median survival of treated animals. Conclusion: We have developed a murine model of single, R0‐resectable ICC with favorable characteristics for the study of recurrence patterns and mechanisms of metastasis after resection. This model holds great promise for preclinical evaluation of novel multimodal or adjuvant therapies to prevent recurrence and metastasis after R0‐resection. (Hepatology 2013;53:1031–1041)

Perioperative, Spatiotemporally Coordinated Activation of T and NK Cells Prevents Recurrence of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and disseminating cancer resistant to therapy, including checkpoint immunotherapies, and early tumor resection and (neo)adjuvant chemotherapy fails to improve a poor prognosis. In a transgenic mouse model of resectable PDAC, we investigated the coordinated activation of T and natural killier (NK) cells in addition to gemcitabine chemotherapy to prevent tumor recurrence. Only neoadjuvant, but not adjuvant treatment with a PD-1 antagonist effectively supported chemotherapy and suppressed local tumor recurrence and improved survival involving both NK and T cells. Local T-cell activation was confirmed by increased tumor infiltration with CD103+CD8+ T cells and neoantigen-specific CD8 T lymphocytes against the marker neoepitope LAMA4-G1254V. To achieve effective prevention of distant metastases in a complementary approach, we blocked the NK-cell checkpoint CD96, an inhibitory NK-cell receptor that binds CD155, which was abundantly expressed in primary PDAC and metastases of human patients. In gemcitabine-treated mice, neoadjuvant PD-1 blockade followed by adjuvant inhibition of CD96 significantly prevented relapse of PDAC, allowing for long-term survival. In summary, our results show in an aggressively growing transgenic mouse model of PDAC that the coordinated activation of both innate and adaptive immunity can effectively reduce the risk of tumor recurrence after surgery, facilitating long-term remission of this lethal disease.Significance: Coordinated neoadjuvant and adjuvant immunotherapies reduce the risk of disease relapse after resection of murine PDAC, suggesting this concept for future clinical trials. Cancer Res; 78(2); 475-88. ©2017 AACR.