Novel Pharmacological Approaches to Target Mitochondrial Metabolism and DNA Damage Response in Colorectal Cancer and their Synergism with Established Chemotherapeutics

Colorectal cancer (CRC) is one of the most frequently diagnosed malignant diseases worldwide and occurs at an alarmingly increasing rate especially in young adults. Ongoing progress in the field of cancer pharmacology has provided new treatment options in recent years, particularly in the form of immunotherapy. Yet, standard chemotherapy has been based on cytostatic agent 5-fluorouracil (5-FU) for over five decades with the more recent addition of leucovorin (LV), oxaliplatin (OXA) and irinotecan (IT). Established chemotherapeutic approaches mainly rely on the induction of DNA damage, which particularly affects rapidly growing cancer cells. The altered tumor metabolism and the DNA repair machinery have more recently been identified as potential drug targets in CRC. The assessment of novel anticancer drugs for CRC with a focus on induction of DNA damage, inhibition of DNA repair and disruption of mitochondrial metabolism as potential mechanisms constituted the objective of this work. For this purpose, this PhD thesis aimed to evaluate the applicability of antitumor agents which are already clinically used in the treatment of other cancer entities, including the metabolic inhibitor devimistat and the poly (ADP-ribose)-polymerase inhibitors (PARPi) olaparib and veliparib. In addition, the biological activity of novel chemical compounds for CRC treatment was analyzed. The investigated substances were either derived from natural sources (merosesquiterpenes isolated from marine sponges) or chemically synthesized as potential PARPi. CRC cell lines with varying mutational status were applied as surrogates representing the diversity of this disease and experiments with transient genetic knockdown or isogenic knockout cell lines were performed to further detail the pharmacological mode of action of the tested compounds. In addition, murine intestinal tumor organoids and primary organoids as well as patient-derived short-term cultures were used to elucidate the tumor cell specificity of the observed effects. An array of methods was utilized to investigate the underlying cell death mechanisms, including flow cytometry, confocal microscopy, western blot and gene expression analysis. The cytotoxicity of the mitochondrial disruptor devimistat and of PARPi in combination with established CRC chemotherapeutic 5-FU, OXA and IT was analyzed in CRC cell lines to identify a potential synergy. Finally, devimistat was applied either as monotreatment or in combination with IT in a murine xenograft model to evaluate the therapeutic efficacy in vivo. At first, the tumor cell specific cytotoxicity of the metabolic inhibition by devimistat was revealed, independent of genetic and epigenetic alterations in CRC cell lines and murine tumor organoids. A reduced oxygen consumption rate (OCR), attenuated mitochondrial activity and induction of reactive oxygen species (ROS) were identified as underlying mechanisms, resulting in p53-independent induction of CRC cell death. Synergistic anticancer activity was achieved by combination treatment with the chemotherapeutic agents IT and 5-FU, as demonstrated by a Combenefit-model and Chou-Talalay analyses. Mechanistically, synergism was based on downregulation of antiapoptotic Bcl-2 proteins and posttranslational accumulation of the proapoptotic protein Bim, as demonstrated by transient genetic knockout experiments. Antitumor efficacy and synergistic activity with IT were confirmed by applying human CRC cells in a xenograft mouse model. In the second study, a compound library comprised of 11 merosesquiterpenes isolated from marine sponges was analyzed and ilimaquinone (IQ), dactylospontriol (DS) and smenospongine (SP) were identified as the most cytotoxic compounds in a panel of three human CRC cell lines. On the mechanistic level, all three compounds induced DNA strand breaks and upregulated the DNA damage response (DDR) irrespective of the mutational status of p53, resulting in cell cycle arrest and activation of the mitochondrial apoptosis pathway. Furthermore, merosesquiterpenes induced pronounced cytotoxicity in murine intestinal tumor organoids, underlining their potential in CRC treatment. In the third study, the applicability of established and novel PARPi in the treatment of DNA repair deficient and proficient CRC was investigated. Therefore, the two novel compounds X17613 and X17618, which inhibit PARP-1 activity in sub-micromolar concentrations, were identified based on an in silico and in vitro screening of a library of novel 3,4bifunctionalized and -bridged indole compounds. In contrast to clinically applied PARPi olaparib and veliparib, both compounds showed no cytotoxicity in PARP-1-deficient and -proficient CRC cell line pairs. In accordance, absence of үH2AX formation and low PARP-1 trapping activity compared to olaparib were identified after treatment with X17613 and X17618. In the last step, sensitization of BRCA2 (breast cancer type 2 susceptibility protein)-deficient CRC cells to IT was demonstrated for X17613. In conclusion, our research assessed the potential of novel therapeutic approaches for CRC treatment, including mitochondrial disruption by the clinically applied metabolic inhibitor devimistat, inhibition of DNA repair by novel PARP-1 inhibitors and DNA damage induction by marine sponge toxins. Our results provide insight into the underlying molecular mechanisms, the potential synergistic activity with clinically applied drugs and the impact of common CRC mutations on sensitivity to guide further development of new therapeutic approaches.