Abstract 1044 Mitomycins Interstrand crosslinks with opposite stereochemical configuration differently impact cellular repair pathways

Our study objectives are to identify critical cellular pathways triggered by the presence of DNA interstrand crosslinks (ICLs) produced by mitomycins and to identify how the structure of mitomycins ICLs determines the cellular signaling in the absence of a functioning p53. This study includes mitomycin C (MC), an anti-cancer drug currently used in the clinics and decarbamoyl mitomycin C (DMC), a derivative of mitomycin C (MC) lacking a carbamoyl group. These compounds form highly cytotoxic interstrand crosslinks (ICLs) that share common structural features. MC forms the α-ICL (trans) with DNA and DMC, the β-ICL (cis). Representative conformations of the two ICLs indicate that the level of DNA perturbation induced by each ICL increases in the order α<β with the α-ICL inducing minimal DNA perturbation. Previous research has also shown that both drugs differ in their cytotoxicity and p53 signaling. Since ICLs are the lesions primarily responsible for the cytotoxicity of mitomycins, this indicates that the two ICLs trigger very diverse biochemical cellular mechanisms, mediated by specific mutations present in cancer cells, a hallmark of modern cancer therapy. We generated site-specific ICL adducts (i.e. duplex oligonucleotides containing a single interstrand crosslink at a specific site) formed by mitomycins and transfected them into two cell lines with different p53 status: WT p53 MCF-7 and mutant p53 K562 cell lines. We used a PanCancer expression array to identify similarities and differences between the changes in gene expression induced by the two ICLs . The array included 770 targets involved in tumorigenesis related pathways as well as targets related to immune response and tumor microenvironment. Finally, we conducted bioinformatics analysis using IPA on the main pathways related to cell cycle control, cell proliferation and DNA damage response. Our results show that exposure to the β and α-ICL causes changes in gene expression encoding proteins involved in integrity of cells and tissues as well as others responsible for the maintenance of genome stability and cellular response to DNA damage. The expression of several DNA repair genes was differently impacted by each ICL: the α-ICL uniquely impacted the level of gene expression encoding proteins in the Fanconi Anemia (FA) pathway whereas the β-ICL impacted the level of gene expression encoding proteins in the MMR, NHEJ and MMEJ pathways. Using bioinformatics (Ingenuity Pathway Analysis, IPA), we also found that the α-ICL and β-ICL signaling were markedly different in both MCF-7 (WT TP53) and K562 (mutant TP53) cells. Significantly, the NER pathway was activated (z>1.75) by the α-ICL in TP53 WT MCF-7 cells (but not in TP53 mutant K562 cells). In contrast, we found that the β-ICL strongly deactivated (z<-2) that same pathway in TP53 mutant K562 cells. The study presented here identifies critical DNA repair pathways triggered by the presence of ICLs produced by mitomycins and pinpoints structural ICL features determining the cellular outcome in the presence or the absence of a functioning TP53. Knowledge derived from our research will allow the development of therapeutic strategy based on DNA damage repair inhibition. This research was possible through funding from the National Institute of Health, award number 1R16GM149514 to E.C.