Application of Skepinone-L, a p38α MAP (mitogen-activated-protein) kinase inhibitor, to overcome antiviral resistances to immunovirotherapy of colon, hepatocellular and renal carcinomas

Abstract

Oncolytic virotherapy is a biological approach to fight tumor cells and supposedly might be of help for more and more cancer patients in the future. For the clinical setting of virotherapy, it is of great interest to get knowledge on the application possibilities of each and every virotherapeutic compound. Accordingly, it is important to understand (i) the phenomena of antiviral resistances and oncolytic futility as well as (ii) the mechanisms which enable the permissivity and oncolytic success of each and every virotherapeutic compound. For this purpose, a well-characterized oncolytic measles vaccine virus (MeV) that has been demonstrated to be oncolytically effective in most but not all cell lines of the NCI-60 tumor cell panel (Noll et al. 2013b) was investigated in more detail. In previous studies an intact type I IFN response had been identified as a main factor for resistances of tumor cell lines against oncolytic measles viruses (Allagui et al. 2017). Further studies have shown that the p38alpha MAPK pathway is activated by type I IFNs and that this pathway is also involved in regulating type I IFN-dependent antiviral responses (Jiang et al. 2015). In this context, we formulated the hypothesis that p38alpha MAPK inhibitors might have an impact on measles vaccine virus-based virotherapy:

- Blocking of the p38alpha MAPK signaling pathway might (i) inhibit the type I IFN response in tumor cells, thus leading to (ii) increased replication of MeV-GFP in those "IFN-depleted" tumor cells, which ultimately (iii) could enhance also the immunotherapeutic antitumoral effect of MeV-GFP.

To investigate this hypothesis, the highly effective p38alpha MAPK inhibitor Skepinone-L was studied in four tumor cell lines picked from the NCI-60 tumor cell panel:

- HCT-15 (of colorectal carcinoma (CRC) origin) and ACHN (of renal cancer origin), both of which had been demonstrated to be highly resistant to MeV-based oncolysis (Noll et al. 2013b);

- HCT-116 and HT-29 (of either CRC origin), both of which had been demonstrated to be highly permissive to MeV-based oncolysis (Noll et al. 2013b);

- Hep3B (of hepatocellular carcinoma origin), which is not part of the NCI-60 tumor cell panel and which was demonstrated in this work to be partially resistant / semi-permissive to MeV-based oncolysis.

Tumor cells were incubated with defined concentrations of Skepinone-L and 24 hours later infected with MeV-GFP and after 3 hours post-treated with Skepinone-L . Oncolytic effects were determined by the Sulforhodamine B (SRB) cell viability assay as well as by xCELLigence® Real-Time Cell Analysis. As a result, MeV-GFP-mediated oncolysis enhanced by Skepinone-L was detected only in the three CRC cell lines (HCT-15, HT-29, HCT-116), but not in the renal (ACHN) nor in the hepato¬cellular carcinoma cell line (Hep3B). Interestingly, a decrease of IFN-beta release could be detected in HCT-15 and HT-29 cells treated with Skepinone-L/MeV-GFP indicating an effect of Skepinone-L on MeV-GFP-triggered humoral immune response via p38alpha MAPK signaling. Data for the other three tumor cell lines (HCT-116, ACHN, Hep3B) regarding the IFN release were not obtained within the scope of this thesis work. In conclusion, resistance phenomena against oncolytic virotherapeutics are complex, multifactorial and vary between tumor cell lines. The experiments performed here show that the p38alpha MAPK signaling pathway is modulating the IFN-release of two CRC cell lines (HCT-15 and HT-29) which might explain the impact on the oncolytic efficacy of MeV-GFP. The lack of oncolytic benefits of other tested tumor cell lines (like ACHN) are indicating that there might be other signaling pathways leading to the induction of IFN-beta, which need to be considered and addressed in further studies. Although numerous virotherapeutic compounds are currently being tested in preclinical and clinical studies, it remains important to understand the phenomena of resistance caused by the tumor`s own immune response to potentially find combinational treatment options that increase the efficacy of virotherapeutics being in clinical use.