| Περίληψη: | Precise and complete genome duplication is critical for a dividing somatic cell to ensure the inheritance of an accurate copy of parental DNA by the offspring. However, multiple DNA barriers commonly arise and disrupt the regular progression of replisomes along replication, posing a threat to timely completion of DNA synthesis within a single cell cycle. DNA licensing controls replication to ensure complete genome duplication while restricting single origin firing to once-per-cell cycle. The CDT1 licensing factor is particularly relevant during licensing due to its critical function in recruiting an inactive form of the replicative MCM2-7 helicase on DNA origins. In order to prevent illegitimate origin firing, licensing is restricted upon origin initiation and helicase unwinding of DNA through tight regulation of each licensing component. It has been demonstrated that CDT1 undergoes multiple control mechanisms in mammalians, underlining its critical relevance when promoting DNA licensing. Accordingly, overexpression of CDT1 is induces genomic instability and malignant behavior in vitro and in vivo and is a genomic trait of several tumor types. One of the mechanisms controlling CDT1 activity involves Geminin, a cell-cycle regulated protein that operates after helicase unwinding of DNA at the onset of S phase until G2, to bind and inhibit CDT1. Certain studies proposed that Geminin could function as a backup pathway to limit CDT1 activity in highly proliferative cells where CDT1 activity is found upregulated. The potential dependency of highly proliferative cells to Geminin could be a genomic trait of therapeutic significance towards the identification of new therapeutic approaches in cancer treatment. However, the molecular mechanisms leading to malignant transformation of cells presenting licensing aberrations was still far from being elucidated. During the initial steps of this study, we validated that CDT1 over-expression leads to re-replication and DNA damage in human cells, and that it is a genomic hallmark of multiple tumor types associated with poor survival. We then focused on the licensing inhibitor Geminin, and we demonstrated its activity is essential to prevent origin re-licensing and re-replication selectively in cancer cells. Cancer cells depleted of Geminin exhibited more than two copies of DNA, evident from cell cycle analysis and increased DNA damage when compared to normal cells. Moreover, we showed that silencing of Geminin triggers replication stress even from the first round of replication, impairing fork progression and
1
replication completion at late-replicating loci. Single-strand DNA gaps were observed to accumulate after progressive rounds of re-replication in Geminin-deficient cancer cells, suggesting that Geminin inactivation triggers deregulated origin initiation. Consistent with this, activation of the DNA damage G2/M checkpoint was required for re- replication, whereby inhibiting checkpoint signaling led to mitotic catastrophe and increased genomic instability during the next cell cycle.
Given that deregulated licensing is a genomic signature of cancer, we then sought to identify targetable molecular pathways signaling and responding to this phenomenon by performing a synthetic-lethal high content screening in cell models with aberrant licensing by examining proliferation and DNA damage. After validation of primary hits, we demonstrated the central effector of the Fanconi anemia repair pathway FANCD2 as critical during protection of cells with induced re-replication. Geminin and FANCD2 double-depletes exhibited reduced survival and increased DNA damage and these results were also confirmed in patient-derived FANCD2-KO cells depleted of Geminin. Remarkably, we observed that Geminin absence induces FA signaling early during the first round of replication, as demonstrated by immunostaining and chromatin fractionation assays. FANCD2 foci were partially distinct from DSBs, indicating sites of impaired fork progression followed by DNA breakage. Subsequent analyses revealed that cells undergoing re-replication require both canonical and non-canonical FANCD2 activity to restrict extensive genomic instability; Loss of FANCD2 in Geminin-depleted cells induced deregulated origin initiation and increased replisome progression, triggering in turn replication through single-strand DNA templates, fork collapse and chromosome fragility. The results in this study provide a novel synthetic lethal interaction with therapeutic relevance in targeted-studies, by saturating the DNA repair capacity of cancer cells presenting licensing aberrations for selective cancer-cell killing.
|
|---|
