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The loss of DNA polymerase epsilon accessory subunits POLE3-POLE4 leads to BRCA1-independent PARP inhibitor sensitivity

Hasan Mamar, Roberta Fajka-Boja, Mónika Mórocz, Eva Pinto Jurado, Siham Zentout, Alexandra Mihuț, Anna Georgina Kopasz, Mihály Mérey, Rebecca Smith, Lajos Haracska, Sébastien Huet, Gyula Timinszky

Posted on: 14 November 2023

Preprint posted on 22 September 2023

POLE4: how to make cancer cells more sensitive to treatment

Selected by Audrey Noireterre

Categories: cancer biology, cell biology, molecular biology

Background

Genome integrity is constantly challenged by DNA lesions. To counteract these assaults, cells have evolved a complex DNA Damage Response (DDR) to mediate DNA repair. In this network, poly(ADP-ribose) polymerase 1 and 2 (PARP1/2) enzymes are crucial in recognizing DNA lesions and modulating DDR. Notably, PARP1 is recruiting repair factors during the early stages of DDR.

PARP inhibitors (PARPi) are widely used compounds in the treatment of cancers. It is particularly effective in patients who present BRCA1/2-mutations, as their cancer cells are deficient in the repair of DNA through homologous recombination (HR). PARPi induce trapping of PARP enzymes on chromatin, increasing the reliance on HR-mediated repair (Rudolph, Jung, and Luger 2022). This ultimately results in increased cell death in BRCA-deficient cancers. However, resistance to PARPi is emerging, and cancer cells are able to reactivate HR via alternative pathways (Gogola, Rottenberg, and Jonkers 2019).

Genetic screens have revealed that disruption of DNA polymerase epsilon (POLε) – specifically the loss of its accessory components POLE3 and POLE4 – had a synergistic effect with PARPi in cancer treatment. Therefore, the authors of this preprint decided to investigate the mechanism underlying the interplay between POLε and PARPi, aiming to provide mechanistic insights that can be used to design alternative approaches to combat cancer.

Their findings demonstrate that the loss of the POLE4 component of POLε contributes to PARPi sensitivity, regardless of BRCA status.

Key findings

The authors initiated their study by confirming the sensitivity of POLE3 knockout (KO) and POLE4 KO cells to Olaparib, a commonly used PARPi. These cells exhibited sensitivity not only to Olaparib but also to other PARPi. Interestingly, the KO of either subunit affected the protein expression of the other, emphasizing the essential interaction between POLE3 and POLE4. Given that POLE3 is also part of the CHRAC15 complex, the authors concentrated their efforts on POLE4 to avoid any confounding factors. It is worth noting that PARPi are effective only in the presence of PARP enzymes. To investigate whether the observed phenotype in POLE4 KO was more linked to PARP1, PARP2, or both, they performed siRNA depletion targeting these enzymes in POLE4 KO cells treated with Olaparib. The results showed that only PARP1 depletion significantly aided the survival of POLE4 KO cells treated with Olaparib, emphasizing the requirement of PARP1 in this context.

After showing that POLE4 KO is not responsible for DNA lesions activating PARP1, the authors asked whether POLE4 regulates PARP1 mobilization at DNA damage sites. Employing laser micro-irradiation and a GFP-PARP1 nanobody, they followed PARP1 dynamics at irradiated locations. Interestingly, both WT and POLE4 KO cells exhibited similar PARP1 recruitment and dissociation times from DNA, regardless of whether they were treated with Olaparib. This result suggests that POLE4 does not influence PARP1’s access to or release from DNA.

As PARPi are known to generate single-strand DNA (ssDNA) gaps, the authors wondered if Olaparib in POLE4 KO cells induces accumulation of ssDNA. They used BrdU immunostaining to specifically recognize BrdU opposite of ssDNA, indicating the level of ssDNA gaps. This experiment showed a substantial increase in BrdU signal in POLE4 KO cells upon Olaparib treatment. Since the accumulation of ssDNA gaps often leads to replication stress, the authors hypothesized that cell cycle progression of POLE4 KO cells might be affected. Indeed, while a slight cell cycle perturbation was observed in WT cells following Olaparib treatment, POLE4 KO cells arrested in the G2/M phases.

The authors then questioned whether the cell cycle arrest in Olaparib-treated POLE4 KO cells was mitotic. By following a mitotic marker (pH3S10), they observed that these cells did not enter mitosis, probably due to the high load of ssDNA gaps. RPA, a protein that is known to bind to ssDNA and become phosphorylated (pRPA). Accordingly, the authors saw an increase in pRPA levels in POLE4 KO upon Olaparib exposure, in line with the increase in ssDNA gaps. These findings further confirm the idea that POLE4 counteracts the formation of ssDNA gaps caused by PARPi, and concurrently prevents replication stress.

At this stage, the authors wondered whether the Phosphatidylinositol 3-kinase-related kinase (PIKK) family is part of the signaling pathways in POLE4 KO cells. They focused on ATR, ATM and DNA-PK, known for protecting replication forks and regulating cell cycle progression. In absence of POLE4, they observed an interesting interplay between PIKK members, where inhibition of ATR (ATRi) led to the activation of DNA-PK and ATM. ATRi alone also caused G2/M arrest, while DNA-PK inhibition reduced the number of arrested cells. The combination of ATRi and Olaparib in POLE4 KO cells resulted in nearly complete cell death. Using ATRi forces cells to enter into mitosis, which causes replication problems and cell death in absence of POLE4.

Finally, the authors addressed the similarities between BRCA1 and POLE4, both of which lead to PARPi sensitivity when absent. Depleting BRCA1 by siRNA increased RPA activation, mirroring the observations in POLE4 KO cells. Interestingly, codepletion of BRCA1 and POLE4 significantly enhanced RPA activation, revealing independent roles for BRCA1 and POLE4. Assessing HR activity using Rad51 as a marker, a factor binding ssDNA, they showed that BRCA1 depletion reduced Rad51 foci formation, whereas Olaparid-treated POLE4 KO cells exhibited an increase in Rad51 foci, probably due to the recognition of the ssDNA. As BRCA1 and POLE4 are not redundant, the authors exploited this fact to reveal a strong synthetic lethality in POLE4 KO siBRCA1 cells treated with Olaparib, opening up promising possibilities for future cancer therapies.

Figure 1 – Suggested model for the POLE4-KO mediated sensitivity to PARPi. Figure taken from Mamar, Fajka-Boja et al., 2023. BioRxiv published under the CC-BY-NC-ND 4.0 International license. BRCA1-deficient cells are sensitive to PARPi, but loss of 53BP1 causes resistance (left panel). In absence of POLE4, PARPi are effective even in BRCA1-proficient cells. Absence of both BRCA1 and POLE4 leads to sensitivity, even with 53BP1 loss (right panel).

Why I chose this preprint

I really enjoyed reading this preprint, because it provides a concise yet clear investigation into the role of POLE4. I think it may improve the identification of patients who could potentially benefit from PARPi therapies, irrespective of their BRCA1 status. The issue of resistance to PARP inhibitor therapies in clinics is a pressing concern, and the study responds to the need of exploring predispositions and strategies to overcome this resistance.

Questions for the authors

  1. My first question may be naïve, but do all PARP inhibitors generate ssDNA gaps? You mainly used Olaparib in your study, although you showed that POLE4 KO cells are sensitive to several PARPi. Did you conduct additional experiments with Talazoparib and Rucaparib, and if not, do you anticipate similar results?

  2. POL4E KO cells downregulated for BRCA1 are extremely sensitive to Olaparib. Did you observe sensitivity of POLE4 loss with BRCA1 deficiency even in absence of PARPi?

  3. You propose that POLE4 loss could serve as a marker for cancers that could be treated with PARPi. Is there some available small compounds capable of targeting POLE4? And, do you think it is also worth to explore the benefits of combining inhibitors targeting sensitizer proteins (like POLE4) with PARPi?

References

Gogola, E., S. Rottenberg, and J. Jonkers. 2019. ‘Resistance to PARP Inhibitors: Lessons from Preclinical Models of BRCA-Associated Cancer’, Annual Review of Cancer Biology, 3: 235-54.

Rudolph, J., K. Jung, and K. Luger. 2022. ‘Inhibitors of PARP: Number crunching and structure gazing’, Proc Natl Acad Sci U S A, 119: e2121979119.

Tags: cell culture, dna repair, parpi

doi: https://doi.org/10.1242/prelights.35949

Read preprint (No Ratings Yet)

Author's response

Hasan Mamar, Roberta Fajka-Boja and Gyula Timinszky shared

We are delighted that you showcase our study in such a neat and informative summary for the wide scientific community.

  1. My first question may be naïve, but do all PARP inhibitors generate ssDNA gaps? You mainly used Olaparib in your study, although you showed that POLE4 KO cells are sensitive to several PARPi. Did you conduct additional experiments with Talazoparib and Rucaparib, and if not, do you anticipate similar results?

The mechanism of action of all clinically approved PARP inhibitors is the same, therefore, PARP trapping through their use is expected generate ssDNA gaps. Yes, in all assays we tested other PARP inhibitors, they had the same effect on POLE4 knockouts as Olaparib only differing in their effective concentrations.

  1. POL4E KO cells downregulated for BRCA1 are extremely sensitive to Olaparib. Did you observe sensitivity of POLE4 loss with BRCA1 deficiency even in absence of PARPi?

Indeed, BRCA1 depletion mildly reduces the proliferation of POLE4 deficient cells, which suggests that these two proteins have safeguarding roles beyond dealing with PARP inhibitor-induced PARP trapping.

  1. You propose that POLE4 loss could serve as a marker for cancers that could be treated with PARPi. Is there some available small compounds capable of targeting POLE4? And, do you think it is also worth to explore the benefits of combining inhibitors targeting sensitizer proteins (like POLE4) with PARPi?

We are not aware of any POLE4 inhibitor so far. POLE4 is not an enzyme with a catalytic pocket, and according to online tools it may not be easily druggable. Yet, POLE3 and POLE4 being obligate heterodimers, one could envision generating POLE3 mimics, inhibitors that would in interfere with POLE3-POLE4 dimerization. And the answer to your last question is a definitive ‘yes’. Generally combining inhibitors is expected to achieve their beneficial effects at lower concentrations as compared to their single use, which is expected to have less side effects and be better tolerated by patients. In fact, we and other labs described that the oncogene ALC1 (Amplified in Live Cancer 1) induces sensitivity to PARP inhibitors upon its deletion, and within two years of publishing those results the first inhibitors of ALC1 were published. Undeniably, the field is moving rapidly down this road.

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