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Dual inhibition of DNA-PK and DNA polymerase theta overcomes radiation resistance induced by p53 deficiency.

ABSTRACT: TP53 deficiency in cancer is associated with poor patient outcomes and resistance to DNA damaging therapies. However, the mechanisms underlying treatment resistance in p53-deficient cells remain poorly characterized. Using live cell imaging of DNA double-strand breaks (DSBs) and cell cycle state transitions, we show that p53-deficient cells exhibit accelerated repair of radiomimetic-induced DSBs arising in S phase. Low-dose DNA-dependent protein kinase (DNA-PK) inhibition increases the S-phase DSB burden in p53-deficient cells, resulting in elevated rates of mitotic catastrophe. However, a subset of p53-deficient cells exhibits intrinsic resistance to radiomimetic-induced DSBs despite DNA-PK inhibition. We show that p53-deficient cells under DNA-PK inhibition utilize DNA polymerase theta (Pol θ)-mediated end joining repair to promote their viability in response to therapy-induced DSBs. Pol θ inhibition selectively increases S-phase DSB burden after radiomimetic therapy and promotes prolonged G2 arrest. Dual inhibition of DNA-PK and Pol θ restores radiation sensitivity in p53-deficient cells as well as in p53-mutant breast cancer cell lines. Thus, combination targeting of DNA-PK- and Pol θ-dependent end joining repair represents a promising strategy for overcoming resistance to DNA damaging therapies in p53-deficient cancers.

SUBMITTER: Kumar RJ

PROVIDER: S-EPMC7751686 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Modeling amplified p53 responses under DNA-PK inhibition in DNA damage response.

Project description:During DNA double strand breaks (DSBs) repair, coordinated activation of phosphatidylinositol 3-kinase (PI3K)-like kinases can activate p53 signaling pathway. Recent findings have identified novel interplays among these kinases demonstrating amplified first p53 pulses under DNA-PK inhibition. However, no theoretical model has been developed to characterize such dynamics. In current work, we modeled the prolonged p53 pulses with DNA-PK inhibitor. We could identify a dose-dependent increase in the first pulse amplitude and width. Meanwhile, weakened DNA-PK mediated ATM inhibition was insufficient to reproduce such dynamic behavior. Moreover, the information flow was shifted predominantly to the first pulse under DNA-PK inhibition. Furthermore, the amplified p53 responses were relatively robust. Taken together, our model can faithfully replicate amplified p53 responses under DNA-PK inhibition and provide insights into cell fate decision by manipulating p53 dynamics.

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Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK.

Project description:CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.

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Combined ATR and DNA-PK Inhibition Radiosensitizes Tumor Cells Independently of Their p53 Status.

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DNA polymerase theta suppresses mitotic crossing over.

Project description:Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3' single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we show that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.

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Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila.

Project description:DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or "alternative" end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s) are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta), encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair.

| S-EPMC2895639 | biostudies-literature

DNA-PK suppresses a p53-independent apoptotic response to DNA damage.

Project description:p53 is required for DNA damage-induced apoptosis, which is central to its function as a tumour suppressor. Here, we show that the apoptotic defect of p53-deficient cells is nearly completely rescued by inactivation of any of the three subunits of the DNA repair holoenzyme DNA-dependent protein kinase (DNA-PK). Intestinal crypt cells from p53 nullizygous mice were resistant to radiation-induced apoptosis, whereas apoptosis in DNA-PK(cs)/p53, Ku80/p53 and Ku70/p53 double-null mice was quantitatively equivalent to that seen in wild-type mice. This p53-independent apoptotic response was specific to the loss of DNA-PK, as it was not seen in ligase IV (Lig4)/p53 or ataxia telangiectasia mutated (Atm)/p53 double-null mice. Furthermore, it was associated with an increase in phospho-checkpoint kinase 2 (CHK2), and cleaved caspases 3 and 9, the latter indicating engagement of the intrinsic apoptotic pathway. This shows that there are two separate, but equally effective, apoptotic responses to DNA damage: one is p53 dependent and the other, engaged in the absence of DNA-PK, does not require p53.

| S-EPMC2613202 | biostudies-literature

Selective DNA-PK inhibition enhances chemotherapy and ionizing radiation activity in soft-tissue sarcomas

Project description:Purpose: Patients with advanced soft-tissue sarcomas (STSs) exhibit a poor prognosis and have few therapeutic options. DNA-dependent protein kinase catalytic subunit (DNA-PK) is a multifunctional serine—threonine protein kinase that plays a crucial role in DNA double-strand damage repair via nonhomologous end joining (NHEJ). Experimental design: To investigate the therapeutic potential of DNA-PK targeting in STS, we first evaluated the prognostic value of DNA-PK expression in two large cohorts of patients with STS. We then used the potent and selective DNA-PK inhibitor AZD7648 compound to investigate the antitumor effect of the pharmacological inhibition of DNA-PK in vitro via MTT, apoptosis, cell cycle, and proliferation assays. In vivo studies were performed with patient-derived xenograft models to evaluate the effects of AZD7648 in combination with chemotherapy or ionizing radiation on tumor growth. The mechanisms of sensitivity and resistance to DNA-PK inhibition were investigated by using a genome-wide CRISPR-Cas9 positive screen. Results: DNA-PK overexpression is significantly associated with poor prognosis in patients with sarcomas. Selective pharmacological inhibition of DNA-PK strongly synergizes with radiation- and doxorubicin-based regimen in sarcoma models. By using a genome-wide CRISPR-Cas9 positive screen, we identified genes involved in sensitivity to DNA-PK inhibition. Conclusion: DNA-PK inhibition deserves clinical investigation to improve response to current therapies in patients with sarcoma

2023-09-25 | GSE243758 | GEO

DNA-PK deficiency potentiates cGAS-mediated antiviral innate immunity.

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Impact of the DNA polymerase Theta on the DNA replication program.

Project description:The physiological function of the human DNA polymerase θ (pol θ) is still unclear despite its in vitro translesion synthesis capacity during DNA damage repair process. However this DNA polymerase is always present along the cell cycle in the absence of replication stress and DNA damage. Is there a different molecular function? We present the genomic data of replication timing in depleted pol θ cells (GSE49693) and in cells overexpressing pol θ (GSE53070) indicating that Pol θ holds a novel role in the absence of external stress as a critical determinant of the replication timing program in human cells.

| S-EPMC4535461 | biostudies-literature

Genetic determinants of cellular addiction to DNA polymerase theta.

Project description:Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.

| S-EPMC6753077 | biostudies-literature

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