Analysis and characterization of chemo- and radiation therapy sensitizing strategies in tumours with focus on effects of phenothiazines on DNA damage response signalling

Cancer is, despite rapid progress in development of new targeted therapies, still mainly treated with surgery, chemotherapy and/or radiation therapy. The last two mentioned conventional therapies often meet general obstacles such as normal cell toxicity and tumour cell resistance. Further efforts are, hence, required to increase efficacy of both radiotherapy and chemotherapy in order to increase patients survival. In this thesis, we analysed and characterised strategies to improve chemotherapy and radiation therapy sensitivity in tumours, with focus on lung cancer. Particle radiation therapy offers an opportunity to overcome tumours' resistance to conventional photon irradiation. Unfortunately, treatment planning systems describing parameters for ion irradiations, used in particle therapy centres worldwide, are still based on the survival data from conventional radiation. In Paper I, we used two mathematical models, the linear-quadratic (LQ) and the repairable-conditionally repairable damage (RCR), to compare the effects of high LET accelerated ions with those elicited by conventional low LET photons, in different types of tumour cells. We show that the data on response to low LET irradiation can be used to create models that can predict cellular response to high LET with radiobiological parameters assessed with the RCR model. Moreover, results achieved with the RCR but not with LQ model, suggest that tumour cells with high DNA repair capacity can benefit from radiation therapy with accelerated particles. Another cancer treatment strategy presented in this thesis is focused on the use of phenothiazines, alone or as sensitizers to chemotherapy. Phenothiazines are drugs clinically used for psychiatric disorders, but which also have been shown to possess cytotoxic activity in various regimens and tumours. In Paper II, we uncovered that monotherapy with phenothiazines caused decreased cell viability and cell death of small cell lung cancer (SCLC) cells. Furthermore, we showed that lysosomal dysfunction induced by phenothiazines was responsible for the observed higher responsiveness of SCLC cells. Our studies presented a new context of use and activity of phenothiazines in tumour cells and allow for a potential treatment opportunity for SCLC. In Paper III, we showed that phenothiazines interfere with DNA damage response (DDR) machinery in tumour cells by inhibition of one of the DNA double strand breaks (DBSs) repair pathways the nonhomologous end joining (NHEJ). We demonstrated that this phenothiazine-mediated inhibition of DNA repair was associated with increased chromatin-centred DNA-PK/ATM signalling, resulting in augmented substrate phosphorylation and protracted checkpoint arrest. This novel tumour cell selective feature of phenothiazines preferentially caused chemosensitization to genotoxic agents that induce DNA DSBs, but also opens up for possible combinations with DNA repair inhibitors. In Paper IV, in silico gene expression analysis suggested similarities in mode of action between phenothiazines and epigenetic signalling modulators. Accordingly, we showed that phenothiazines can be used to treat SCLC or the epigenetically deregulated tumour cells, neuroblastoma (NB) and acute myeloid leukemia (AML), either alone or in combination with chromatin-modifying drugs. The cytotoxicity, cell death signalling and hyperactivation of DNA repair signals observed with phenothiazines in tumour cells, were comparable in magnitude with the effect of the chromatin-modifying drugs, pan-HDACi panobinostat or the BRD4 antagonist JQ1. Moreover, the model phenothiazine compound trifluoperazine, TFP, was also found to prolong phosphorylation of DNA-PKcs in chromatin fractions of SCLC cells. The tandem treatment with TFP and either panobinostat or JQ1 was also demonstrated to increase cytotoxicity and triggered both apoptotic and autophagic cell death signalling in SCLC and NB cells. Thus, our findings in Paper IV suggest a novel therapeutic utility of phenothiazines and chromatin-modifying drugs in cancer therapy. LIST OF SCIENTIFIC PAPERS I. Mohanty C. *, Zielinska-Chomej K. *, Edgren M. , Hirayama R. , Murakami T. , Lind B. , Toma-Dasu I. Predicting the Sensitivity to Ion Therapy Based on the Response to Photon Irradiation Experimental Evidence and Mathematical Modelling. Anticancer Research 2014; 34: 2801-2806 * equal contribution II. Zong D. , Zielinska-Chomej K. , Juntti T. , Mork B. , Lewensohn R. , Haag P., Viktorsson K. Harnessing the Lysosome-Dependent Antitumor Activity by Phenothiazines in Human Small Cell Lung Cancer. Cell Death and Disease 2014; 5, e1111; doi:10.1038/cddis.2014. 56 III. Zong D. , Zielinska-Chomej K. , Haag P., Yakymovych I. , Lewensohn R. , Viktorsson K. Phenothiazines Trigger Hyperactivation of ChromatinAssociated DNA-PK and ATM Signaling and Offer a Novel Approach to Chemotherapy Sensitization. Manuscript IV. Zielinska-Chomej K. , Zong D. , Lewensohn R. , Viktorsson K. , Haag P. Phenothiazines Modulate Chromatin-Related Epigenetic Processes in Tumour DNA Damage Response and Offer a Novel Strategy for Cancer Therapy. Manuscript