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Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

Rashmi J. Kumar, Hui Xiao Chao, Victoria R. Roberts, Aurora R. Sullivan, Sonam J. Shah, Dennis A. Simpson, Wanjuan Feng, Anne-Sophie Wozny, Sunil Kumar, Jeremy E. Purvis, Gaorav P. Gupta

Preprint posted on 4 April 2020 https://www.biorxiv.org/content/10.1101/2020.04.01.021253v2.full

P53 deficiency upregulates Pol theta and promotes alternative end joining mechanisms to repair damage induced by radiomimetic therapies providing an insight into the mechanisms of resistance to DNA damaging therapies in P53 deficient cancers.

Selected by Matthew Burke

Overview

Deleterious TP53 mutations are found in many cancers and are associated with poor disease outcome. Previously, the loss of functional P53 protein has primarily been associated with reduced tumour suppression but converging lines of evidence now suggest those cancers with a lack of P53 are also resistant to DNA damaging therapies via an unknown mechanism(s). In this study, Kumar et al., shed light on this mechanism(s) and show via single cell tracking that P53 deficiency causes a DNA repair hyperactivity that prevents DNA damage accumulation, and this is partially dependent on DNA protein kinase and canonical NHEJ. However, in a showcasing of the inherent flexibility of NHEJ, Kumar et al., also demonstrate that Pol Theta Mediated End Joining (TMEJ) can readily resolve these treatment induced DNA breaks in a P53 deficient background. The authors show that Pol theta is massively upregulated by P53 deficiency and that dual inhibition of pol theta and DNA PK during treatment with a radiomimetic small molecule can significantly reduce cell viability. These novel findings may provide an exciting new avenue to the eventual prevention of treatment resistance in many cancers associated with the loss of P53.

Main findings

P53 deficient epithelial cells were radio-resistant and had a significant growth advantage over wild type cells at any dose level of ionizing radiation and during treatment with the radiomimetic drug Neocarzinostatin (NCS).

Single cell tracking on a live cell imaging platform revealed NCS-induced DNA damage foci were repaired significantly faster in P53 deficient cells and this difference was most pronounced during S phase. Importantly, the authors showed this hyper-activity could largely be normalized by inhibition of DNA protein kinase (DNA PKi), which is a key NHEJ component that is integral to the resolution of complex DNA breaks. These data indicate P53 deficiency allows cells to rapidly resolve most treatment induced DNA damage to remain viable and this is at least partially dependent on DNA PK.

Indeed, while wild type cells were arrested at the G1/S phase checkpoint following NCS treatment as DNA damage accumulated, P53 deficient cells continued to cycle as their damage was rapidly repaired. However, the number of P53 deficient cells arrested at G1/S phase following NCS treatment could be significantly increased by simultaneous DNA PKi, as this prolonged DNA damage foci. Notably, DNA PKi also prolonged G2/mitosis checkpoints in both wild type and P53 deficient cells following NCS treatment, clearly suggesting DNA PKi-induced damage accumulates primarily during S phase in a manner that is partially independent of P53 levels.

However, P53 deficient cells were more likely to progress from the G2 checkpoint and remain viable. Indeed, while over 90% of wild type cells exposed to NCS and DNA PKi in S phase remained arrested at G2 for the 72 hr duration of the imaging, nearly 50% of P53 deficient cells from the same treatment group were still viable and proliferative.

How then, are the P53 deficient cells still resolving their DNA breaks and re-entering the cell cycle even when canonical NHEJ is inhibited? Remarkably, in accordance with previous studies, P53 deficiency here led to a 10 to 20-fold increase in polymerase theta (Pol θ), a polymerase strongly associated with alternative end joining mechanisms. To assess the role of Pol θ in this context, the authors generated a break at a known chromosomal site (LBR locus) and analysed break points from the different groups via next generation sequencing. DNA PKi led to a reduction in typical NHEJ repair products and a significant increase in Pol θ -dependant end joining. While Pol θ knock out, together with DNA PKi, was associated with the greatest reduction in the overall detectability of the LBR locus as measured by digital PCR, suggesting the lowest level of overall DNA repair. Finally, dual inhibition of both Pol θ and DNA PK could significantly reduce cell viability upon NCS treatment to a level similar to wild type cells. This implicates a significant role for theta-mediated end joining in treatment resistance to DNA damaging agents.

Overall, this study elegantly demonstrates the flexibility of DNA End Joining and elucidates the role of this flexibility in resistance to DNA damaging therapies. Notably, given the inherent error prone nature of such alternative end joining mechanisms, it is likely that those cells that escape treatment-induced cell death may carry a greater load of somatic mutations. Future genomic analyses will be important in assessing the contribution of these treatments to cancer heterogeneity, and whether this is reduced by combination therapies that block both TMEJ and canonical NHEJ during exposure to DNA damaging treatments.

 

Questions for Authors

  1. Are there any pol theta inhibitors currently in clinical use? Could dual targeting of these pathways reduce the somatic mutation burden in some P53-deficient cancers to potentially improve disease outcome?
  2. In your different study conditions, did you analyse any cells for mutations in known cancer driver genes? Or is that a further study you intend to undertake?

 

Posted on: 24 June 2020

doi: https://doi.org/10.1242/prelights.22125

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