RNAi im Hochdurchsatzverfahren - Identifikation potentieller Angriffspunkte zur Überwindung der Toleranz gegenüber dem niedermolekularen Wirkstoff RITA in H460 NSCLC-Zellen

The small-molecule compound RITA was first described as a potential anti-cancer drug in 2004. It was initially characterized as a p53 wild-type reactivator whose function is based on a blockade of the p53-MDM2 interaction. However, in subsequent years, RITA was also shown to act on p53-mutated cancer cells, and more recent research ultimately suggests that RITA works p53-independent as a DNA cross-linker. In the present work, RITA-tolerant cell clones of a WT-p53-bearing, primarily RITA-sensitive NSCLC cell line (H460) were investigated by RNAi. The aim was to find genes whose knockdown can restore RITA sensitivity. Due to the assumed genotoxic properties of RITA, a siRNA library of DNA damage response genes was tested. RAD18 was identified as the "hit". Resensitization of the tolerant H460 cells to RITA was achieved both by short-term siRNA and long-term shRNA knockdown of RAD18, which demonstrates a reversible mechanism of tolerance. The fact that RAD18 is a gene with functions in DNA damage response and tolerance also supports the description of RITA as a primary DNA cross-linker/substance with primarily genotoxic activity. In addition, Wanzel et al. (2016) were able to show that the knockdown of FancD2, like that of RAD18, leads to a resensitization of the tolerant cells to RITA. The literature has already discussed a connection between FancD2 (a gene of the Fanconi-Anemia-Pathway) and RAD18. Thus, it can be assumed that RAD18 plays a role in the tolerance mechanism via its connection to the FA-pathway in repairing DNA double-strand breaks. Furthermore, a microarray-analysis was performed to compare the RITA-tolerant and parenteral H460 cells to identify genes with increased expression in the tolerant cells. The genes SOX2, INHBA and FST were validated as upregulated. However, no clear resensitization to RITA could be achieved by knockdown of these genes using RNAi with siRNAs. But the strongly increased expression of SOX2 adds another stem cell marker to the characteristics of the tolerant cells (besides sphere formation and CD133 positivity, which have been shown already). The results show that the RITA-tolerant cells are a subpopulation of the H460 cells, which exhibit stem cell characteristics and a potentially reversible RITA tolerance based on an altered DNA damage response. Here a comparison to the so-called DTPs (drug tolerant persisters) can be drawn. DTPs are cancer cells in a transient slow-cycling state, that show resistance to apoptosis and characteristics of tumour stem cell. They are clinically associated with therapy resistance and recurrence, so that there is a great interest in targeting these cells. RAD18, in connection with the FA-pathway, has been identified as a potential target here.