Increased Non-Homologous End Joining Makes DNA-PK a Promising Target for Therapeutic Intervention in Uveal Melanoma

Abstract

Uveal melanoma (UM) is the most common primary intraocular tumour in adults, with a mean survival of six months following metastasis. The survival rates have not improved in over 30 years. This study has shown that sister chromatid exchange (SCE) is low in UM which is likely due to a reduced expression of FANCD2. As FANCD2 can function to suppress non-homologous end joining (NHEJ), this study therefore investigated NHEJ in UM. The activation of the catalytic subunit of the NHEJ pathway protein DNA-dependent protein kinase (DNA-PK) was measured by analysing the foci formation and the ligation efficiency by NHEJ determined using a plasmid-based end-joining assay. Using small-interfering RNA (siRNA) knock-down, and chemical inhibitors of DNA-PK, the survival of primary UM cultures and two cell lines were determined. To assess the homologous recombination capacity in response to the inhibition of DNA-PK, a SCE analysis was performed. In addition, to support the findings, the messenger RNA (mRNA) expression of genes associated with NHEJ was analysed using the Cancer Genome Atlas (TCGA)-UM RNAseq data (n = 79). The NHEJ activity and DNA-PKcs activation was upregulated in UM and the inhibition of DNA-PK selectively induced apoptosis and sensitized to ionising radiation and inter-strand cross-linking agents. The inhibition of the NHEJ protein DNA-PK is lethal to UM, indicating a potentially effective therapeutic option, either alone or as a sensitizer for other treatments.

Inhibition of ATM Increases the Radiosensitivity of Uveal Melanoma Cells to Photons and Protons

“…Interestingly, in a recent study, greater gene expression of DNA-Pk was observed in metastatic UM, and was significantly associated with an adverse clinical outcome [ 13 ]. A second study also confirmed greater DNA-Pk expression in UM, although, similar to our study, no changes in ATM expression were observed [ 14 ]. This compares to recent data identifying loss of ATM and ATR in UM with a high risk of metastasis [ 15 , 16 ], although it should be noted that the goal of our study was not to determine whether specific DNA repair proteins are biomarkers of metastatic risk.…”

“…We are aware that more potent and selective inhibitors targeting ATM, particularly AZD1390, are now available, and this will be the focus of our future studies, in combination with both photons and protons in enhancing the radiosensitivity of UM cell models. Interestingly, there are recent studies suggesting that the inhibition of another kinase involved in DSB repair, DNA-Pk, as a monotherapy can reduce the survival of UM cell lines [ 13 , 14 ]. There are also very limited data suggesting that DNA-Pk inhibition can work effectively in combination with X-rays in preventing UM cell survival [ 14 ].…”

“…Interestingly, there are recent studies suggesting that the inhibition of another kinase involved in DSB repair, DNA-Pk, as a monotherapy can reduce the survival of UM cell lines [ 13 , 14 ]. There are also very limited data suggesting that DNA-Pk inhibition can work effectively in combination with X-rays in preventing UM cell survival [ 14 ]. Despite this, we didn’t observe any dramatic changes in DNA-Pk protein levels in the UM cell lines that showed variations in radiosensitivity utilized in this study.…”

“…Interestingly, in UM, there is recent evidence demonstrating that the DNA-Pk inhibitor, NU7026, can decrease the survival of UM cell lines (OMM1, OMM2.5, Mel270 and MM28) as a monotherapy [ 13 ]. Similarly, the survival of UM cells (SOM 157D and SOM 196B) was also significantly reduced using inhibitors of DNA-Pk (NU7026 and NU7441) alone, and there was limited data presented that SOM 196B displayed increased radiosensitivity in the presence of NU7026 [ 14 ]. Interestingly, recent data suggest that loss of ATM and ATR has been observed in UM, and has been associated with metastasis [ 15 , 16 ].…”

Uveal Melanoma

“…New targets for therapy are addressed by Rezzola et al who describe the fibroblast growth factors (FGFs) and their receptors as potential therapy targets in uveal melanoma and show the efficacy of FGF traps [22]. Doherty et al introduce the DNA-PK as a therapy target since its inhibition leads to increased non-homologous end joining and apoptosis [23]. Vivet-Noguer and colleagues review our knowledge of the molecular biology of uveal melanoma and how this might lead to the identification of new therapies [50].…”