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DNA-PKcs

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a large serine/threonine protein kinase belonging to the phosphatidylinositol 3-kinase-related kinase (PIKK) family, encoded by the PRKDC gene (also known as XRCC7) on human chromosome 8q11.^[1] With a molecular mass of approximately 469 kDa and consisting of 4,129 amino acids, it forms the catalytic core of the DNA-PK holoenzyme, which plays a central role in repairing DNA double-strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway.^[1] This pathway is critical for maintaining genomic stability, as DSBs represent one of the most severe forms of DNA damage induced by ionizing radiation, chemotherapeutic agents, or replication stress.^[2] The DNA-PK activity was first observed in 1985, when double-stranded DNA was shown to stimulate the phosphorylation of several HeLa cell nuclear proteins, including poly(ADP-ribose) polymerase, revealing a novel DNA-activated kinase system. In 1990, the catalytic subunit was purified and identified from HeLa cell nuclei, confirming DNA-PKcs as a DNA-end-activated protein kinase distinct from other known kinases. Structurally, DNA-PKcs features an N-terminal region rich in HEAT repeats that form a flexible circular cradle for protein interactions, a central head unit containing the kinase domain, and C-terminal FAT (FRAP-ATM-TRRAP) and FATC domains that regulate kinase activity.^[1] Upon DNA damage, the Ku70/Ku80 heterodimer binds to DSB ends, recruits DNA-PKcs to form the holoenzyme, and activates its autophosphorylation and substrate phosphorylation capabilities, targeting over 700 proteins involved in repair.^[1] Beyond DSB repair, DNA-PKcs contributes to V(D)J recombination during T- and B-cell development, telomere end protection, and cell cycle checkpoints at G1/S and G2/M phases.^[1] It also participates in responses to replication stress, autophagy induction, and cellular senescence, with nuclear and cytoplasmic localization enabling diverse regulatory roles.^[1] Mutations in PRKDC lead to severe combined immunodeficiency in animal models and human inflammatory diseases characterized by granulomas, autoimmunity, and recurrent infections.^[3] In cancer, DNA-PKcs overexpression promotes tumor survival and resistance to radiotherapy and chemotherapy, positioning it as a promising therapeutic target for inhibitors like NU7441 and M3814.^[4]

Discovery and Structure

Historical Discovery

The discovery of DNA-dependent protein kinase (DNA-PK) activity occurred serendipitously in 1985 during experiments on protein phosphorylation in HeLa cell extracts. Researchers observed that double-stranded DNA stimulated the phosphorylation of several nuclear proteins, including a prominent 350-400 kDa polypeptide, revealing a novel DNA-activated serine/threonine kinase involved in the cellular response to DNA damage. In 1990, independent studies identified the catalytic subunit of this kinase, later termed DNA-PKcs, through biochemical purification from human cell extracts. Carter et al. isolated a ~350 kDa protein that exhibited DNA-dependent autophosphorylation and phosphorylated substrates such as the heat shock protein Hsp90, confirming its role as the enzymatic core. Concurrently, Lees-Miller et al. demonstrated that this subunit phosphorylated the tumor suppressor p53 at specific serine residues, linking it to DNA damage signaling pathways via assays with purified nuclear fractions. These findings established DNA-PKcs as a distinct entity separate from the DNA-binding component later identified as Ku. The cloning of the PRKDC gene encoding DNA-PKcs was achieved in 1995 by Hartley et al., who sequenced the full-length cDNA from human cells, revealing a massive 4,128-amino-acid protein of approximately 469 kDa with homology to phosphatidylinositol 3-kinase family members. This molecular characterization enabled targeted studies, including the pivotal demonstration that mutations in the murine Prkdc gene cause severe combined immunodeficiency (SCID) in mice. Kirchgessner et al. showed that SCID fibroblasts and lymphocytes lacked DNA-PK activity due to defects in the catalytic subunit, directly tying DNA-PKcs to V(D)J recombination and double-strand break repair. Contributions from the laboratories of Graeme C.M. Smith and Stephen P. Jackson were instrumental in defining the DNA-PK holoenzyme composition, integrating DNA-PKcs with the Ku70/Ku80 heterodimer through immunoprecipitation and reconstitution assays.^[5]^[6]

Molecular Structure

DNA-PKcs, the catalytic subunit of the DNA-dependent protein kinase complex, is encoded by the PRKDC gene located on human chromosome 8q11 and consists of 4,128 amino acids, yielding a molecular mass of approximately 469 kDa.^[7]^[8]^[9] The domain architecture of DNA-PKcs features an extensive N-terminal region of HEAT repeats (residues 1–2801) that folds into an open, ring-like scaffold facilitating regulatory interactions and conformational flexibility.^[10]^[11] This is followed by the FAT domain (residues 2802–3564), which stabilizes the adjacent catalytic regions. The kinase domain (residues 3565–4100) encompasses conserved motifs, including the ATP-binding site and catalytic loop, essential for substrate phosphorylation. The short C-terminal FATC domain (residues 4101–4128) maintains autoinhibition in the apo state by interacting with the kinase domain, a constraint relieved upon complex activation.^[12]^[13]30907-2) High-resolution cryo-EM structures from 2022 and 2023 have elucidated the conformational transitions of DNA-PKcs, revealing a compact, closed (autoinhibited) form in isolation and an extended, open (activated) state upon engagement with DNA-bound Ku.^[14]^[15] These structures demonstrate how DNA-induced stretching and twisting of the HEAT repeats propagate to the head domain, repositioning the kinase active site for catalysis.30907-2) Within the DNA-PK holoenzyme, DNA-PKcs integrates with the Ku70/Ku80 heterodimer, where double-strand DNA ends serve as a bridge to stabilize the assembly and align the components for non-homologous end joining.^[16]30907-2)

Mechanism of Action

Activation and Kinase Activity

The activation of DNA-PKcs, the catalytic subunit of the DNA-dependent protein kinase complex, is tightly regulated and occurs primarily at DNA double-strand break (DSB) ends in a process dependent on the Ku70/Ku80 heterodimer. The Ku heterodimer initially binds to the DSB ends with high affinity, forming a ring structure that slides along the DNA and recruits DNA-PKcs through interactions with its N-terminal HEAT repeats and circular cradle domain. This recruitment positions DNA-PKcs such that the DNA end docks beneath its head domain, inducing a conformational change that transitions the enzyme from an autoinhibited, closed state to an open, active configuration, thereby releasing the autoinhibitory interaction between the FAT domain and the kinase domain.^[15] This synapsis-dependent activation is further stabilized by ATP binding, which promotes dissociation of inhibitory elements and enables subsequent autophosphorylation.^[17] Central to this activation is the autophosphorylation of DNA-PKcs at specific sites within clustered motifs, which modulates its accessibility to DNA ends and facilitates downstream repair processes. Key autophosphorylation occurs in the ABCDE cluster (residues 2609–2647), including sites such as Thr2609, Ser2612, Thr2620, Ser2624, Thr2638, and Thr2647; phosphorylation here, particularly at Thr2609 and Thr2620, induces structural rearrangements that open the DNA-binding cleft, allowing end processing.^[10] These events are cis-autophosphorylation within the monomer but can involve trans-phosphorylation in dimeric complexes for other clusters like PQR (residues 2023–2056). Recent cryo-EM structures from 2022 reveal that in the active state, the DNA-end-binding (DEB) helix (residues 2735–2768) interacts directly with the DNA terminus and the ABCDE cluster, constraining DNA movement and fine-tuning activation to prevent premature dissociation.^[15] As a serine/threonine protein kinase, DNA-PKcs catalyzes the transfer of the γ-phosphate from ATP to target substrates, exhibiting a preference for the consensus motif S/T-Q (serine or threonine followed by glutamine), often extended as S/T-Q-X-E or involving prior phospho-serine/threonine. This motif is evident in its autophosphorylation sites and substrates like XRCC4 and Artemis, enabling coordinated phosphorylation events during repair. Kinase activity is typically assayed in vitro using histone H1 as a model substrate, where phosphorylation is quantified by incorporation of radiolabeled ATP or luminescent detection of ADP production. Kinetic studies indicate a Km for ATP of approximately 18 μM under optimized conditions with Mg²⁺, reflecting its efficiency at physiological nucleotide levels.^[18]^[19]

Role in DNA Repair Pathways

DNA-PKcs functions as a core component of the non-homologous end joining (NHEJ) pathway, the primary mechanism for repairing DNA double-strand breaks (DSBs) in mammalian cells, particularly during the G1 phase of the cell cycle. Upon DSB induction, the Ku70/Ku80 heterodimer initially binds to the free DNA ends, serving as a scaffold that recruits DNA-PKcs to form the active DNA-PK holoenzyme. This assembly promotes DNA end synapsis by tethering the broken ends in close proximity, which facilitates the subsequent recruitment of the XRCC4-DNA ligase IV (LIG4) complex responsible for end ligation, often with limited nucleotide loss or addition to minimize genomic alterations.^[20]^[15] This process ensures rapid, albeit error-prone, repair to maintain genome stability in non-replicating cells. In DSB sensing and initial response, DNA-PKcs enhances the stability of the Ku complex at break sites, thereby shielding DNA ends from nucleases and preventing excessive end resection that would otherwise favor homologous recombination (HR). This protective role inhibits the action of resection-initiating factors such as the MRN complex (MRE11-RAD50-NBS1) and CtIP, preserving blunt or minimally processed ends suitable for direct NHEJ ligation.^[21]^[22] By doing so, DNA-PKcs enforces pathway fidelity, prioritizing NHEJ over HR in contexts where sister chromatids are absent. Recent 2024 phosphoproteomic analyses have further uncovered non-canonical kinase signaling by DNA-PKcs, including novel S/T-ψ-D/E phosphorylation motifs.^[23] Beyond its canonical NHEJ function, DNA-PKcs exerts regulatory influence on alternative repair pathways, acting as a suppressor of HR by blocking end resection and thereby serving as a backup to channel unrepaired DSBs away from error-free HR templating.^[24] In situations of NHEJ compromise or high cellular stress, such as ionizing radiation exposure, DNA-PKcs can contribute to microhomology-mediated end joining (MMEJ) in certain contexts, while primarily suppressing excessive MMEJ to curb illegitimate chromosomal rearrangements.^[25] This pathway hierarchy underscores NHEJ's dominance in G1, where DNA-PKcs activity predominates, contrasted by competitive dynamics with HR in S/G2 phases, where cyclin-dependent kinase-mediated modifications can tip the balance toward resection and homology-directed repair.^[26]

Biological Functions

Non-Homologous End Joining

Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells, particularly during the G0/G1 phases of the cell cycle, where it operates in a template-independent manner to rejoin broken DNA ends with minimal sequence homology. DNA-PKcs, the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex, plays a central and multifaceted role in this classical NHEJ (cNHEJ) process by facilitating end recognition, protection, processing, and ligation, thereby maintaining genomic stability. Defects in DNA-PKcs impair NHEJ efficiency, leading to persistent DSBs and chromosomal instability.^[27] The NHEJ pathway initiates with the rapid binding of the Ku70/Ku80 heterodimer (Ku) to the free DNA ends at a DSB, forming a ring-like structure that slides inward along the DNA to protect the termini from degradation and nucleate further repair complex assembly. Ku then recruits DNA-PKcs through direct protein-protein interactions, assembling the DNA-PK holoenzyme, which is essential for stabilizing the break and promoting the alignment of DNA ends in a process known as synapsis. This tethering function of DNA-PKcs prevents diffusion of the broken ends and sets the stage for subsequent repair steps, with the holoenzyme's kinase activity becoming activated upon DNA binding.^[28]^[27] Once formed, the DNA-PK holoenzyme undergoes autophosphorylation at multiple sites on DNA-PKcs, which induces conformational changes that enable end processing for non-ligatable termini. This phosphorylation activates the endonuclease Artemis, which associates with DNA-PKcs to resolve complex structures such as hairpins, 3' overhangs, or damaged nucleotides through limited nucleolytic resection, typically removing fewer than 20 nucleotides to generate compatible ends without extensive loss of genetic information. The processed ends are then bridged by DNA-PKcs-mediated tethering to align them for ligation.^[28]^[29] The final stage involves recruitment of the ligation machinery, including the XRCC4/LIG4 DNA ligase complex and the accessory factor XLF (also known as XRCC4-like factor), which form a multi-subunit scaffold to seal the nick between the processed DNA ends. DNA-PKcs facilitates this recruitment and, upon successful ligation, undergoes further phosphorylation and dissociation from the DNA, allowing completion of repair and release of the core components. In mammalian cells, NHEJ mediated by DNA-PKcs repairs approximately 80% of DSBs, underscoring its dominance over alternative pathways, though inefficiencies or defects in this process result in chromosomal aberrations and heightened radiosensitivity.^[27]^[30] Recent investigations as of 2024 have elucidated an additional protective role for DNA-PKcs in NHEJ, where the holoenzyme shields "clean" blunt or minimally overhang-ended DSBs from resection by exonucleases such as Exo1 or Mre11, thereby favoring rapid and faithful rejoining over error-prone homologous recombination or alternative end-joining pathways. This end-blocking function, mediated by structural elements like the DNA end-blocking helix in DNA-PKcs, enhances repair fidelity for undamaged breaks, with implications for therapeutic targeting in genome editing.^[31]^[32]

V(D)J Recombination

V(D)J recombination is a site-specific process in developing B and T lymphocytes that assembles variable (V), diversity (D), and joining (J) gene segments to generate diverse antigen receptors essential for adaptive immunity.^[33] The recombination-activating genes RAG1 and RAG2 form a complex that recognizes recombination signal sequences (RSS) flanking the V, D, and J segments, adhering to the 12/23 rule where a 12-base pair spacer RSS pairs only with a 23-base pair spacer RSS.^[33] RAG1/2 introduces double-strand breaks (DSBs) at the RSS-coding segment borders through nicking and transesterification, yielding blunt signal ends (SEs) attached to RSS and covalently sealed hairpin coding ends (CEs).^[33] DNA-PKcs, as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) holoenzyme, is recruited to these DSBs by the Ku70/Ku80 heterodimer, which initially binds the DNA ends to form a scaffold for non-homologous end joining (NHEJ).^[33] In the joining phase, DNA-PKcs facilitates the formation of signal joints (SJs) and coding joints (CJs). Signal ends, being blunt, are ligated directly by the NHEJ ligase complex (XRCC4-LIG4-XLF) with minimal processing, often forming non-replicative extrachromosomal circles.^[33] Coding ends require more extensive processing due to their hairpin structure; DNA-PKcs phosphorylates and activates the Artemis endonuclease, enabling it to open the hairpins asymmetrically and generate 3' overhangs that undergo nucleotide additions by terminal deoxynucleotidyl transferase (TdT) or exonucleolytic nibbling.^[33] This processing, coordinated by DNA-PKcs, ensures precise joining of coding segments while introducing variability at junctions. Genetic studies underscore the essential role of DNA-PKcs in V(D)J recombination. Mutations in the Prkdc gene encoding DNA-PKcs, as observed in severe combined immunodeficient (scid) mice, abolish V(D)J recombination by preventing hairpin opening and joint formation, resulting in a complete block of B and T cell development.^[34] Similar defects occur in human patients with DNA-PKcs deficiencies, such as radiosensitive T-B- SCID, confirming its indispensability.^[35] The fidelity of V(D)J recombination, supported by DNA-PKcs, generates junctional diversity through random nucleotide deletions and additions at CE junctions, contributing to an estimated 10^{11} to 10^{18} unique antigen receptor specificities theoretically in humans, with the realized repertoire around 10^9.^[36] This diversity is crucial for recognizing a vast array of pathogens while maintaining recombination accuracy to avoid genomic instability.^[33] DNA-PKcs also plays an essential role in class switch recombination (CSR) in mature B cells, where activation-induced cytidine deaminase (AID) generates DSBs in switch regions, and DNA-PKcs facilitates their repair via NHEJ to enable antibody isotype switching. Defects in DNA-PKcs impair CSR, leading to immunodeficiencies.^[33]

Regulation

Protein Interactions

DNA-PKcs forms the core of the DNA-dependent protein kinase (DNA-PK) complex through direct binding to the Ku70/Ku80 heterodimer, which recognizes and binds DNA double-strand breaks to recruit DNA-PKcs via interactions with its N-terminal HEAT repeats.^[37] This stable association positions DNA-PKcs at DNA ends, enabling its kinase activation and initiation of non-homologous end joining (NHEJ) repair.^[38] Following autophosphorylation, DNA-PKcs facilitates the recruitment of XRCC4 and DNA ligase IV (LIG4), which bind at DNA termini to promote ligation; this post-phosphorylation step enhances ligase activity and complex stability.^[39] Phosphorylation effects on these interactions further modulate downstream assembly.^[39] Beyond the core NHEJ machinery, DNA-PKcs engages regulatory partners that influence its stability and signaling. Heat shock protein 90 (HSP90) interacts with DNA-PKcs to chaperone its folding and maintain protein stability, as HSP90 inhibition reduces DNA-PKcs levels and impairs DNA repair.^[40] Similarly, DNA-PKcs binds p53, phosphorylating it to regulate apoptosis and cell cycle responses, establishing crosstalk between DNA repair and tumor suppression pathways.^[41] Recent mass spectrometry-based studies have mapped non-canonical interactors, revealing associations with metabolic enzymes such as those in glycolysis, which modulate enzymatic activity and energy production under DNA damage conditions.^[42] These interactions exhibit distinct dynamics: the Ku70/Ku80 binding is relatively stable, stabilizing DNA-PKcs at DNA ends for several minutes to allow repair initiation, whereas associations like those with XRCC4/LIG4 are more transient, occurring post-activation to facilitate end processing and dissociation.^[43] Such temporal regulation prevents prolonged occupancy that could block repair progression.^[44] Yeast two-hybrid screening and co-immunoprecipitation (co-IP) assays have been instrumental in identifying over 50 protein interactors of DNA-PKcs, mapping binding domains and validating functional complexes in vitro and in cells.^[45]^[46]

Post-Translational Modifications

DNA-PKcs undergoes extensive post-translational modifications, predominantly phosphorylation, which dynamically regulate its conformation, activity, and interactions during DNA damage response. These modifications are critical for controlling access to DNA ends and facilitating the progression of non-homologous end joining (NHEJ). Phosphorylation occurs at multiple sites across the protein, with over 40 identified, including key clusters that respond to double-strand breaks induced by ionizing radiation or other genotoxic stresses.^[47]^[48] Autophosphorylation is a primary regulatory mechanism for DNA-PKcs, enabling conformational changes that promote DNA end accessibility and complex disassembly. The ABCDE cluster, encompassing residues Thr2609 to Thr2647 (including sites Thr2609, Ser2612, Thr2620, Ser2624, Thr2638, and Thr2647), is autophosphorylated upon DNA-PK activation, facilitating the opening of the DNA-PK holoenzyme to allow access for downstream repair factors like Artemis and ligase IV.^[10] In contrast, autophosphorylation at the PQR cluster (Ser2023 to Ser2053, including Ser2056) promotes the disassembly of the DNA-PK complex post-ligation, preventing prolonged retention at repair sites and ensuring repair fidelity.^[49]^[19] These clustered phosphorylations are interdependent, with ABCDE modifications often preceding PQR events to coordinate the repair cycle.^[50] Hetero-phosphorylation by related kinases further fine-tunes DNA-PKcs function, particularly in checkpoint activation. ATM phosphorylates DNA-PKcs at Ser2056 and the Thr2609 cluster, while ATR phosphorylates DNA-PKcs under conditions of replication stress, enhancing intra-S-phase checkpoints and coordinating with homologous recombination pathways.^[10]^[51] For instance, ATM-mediated phosphorylation at Thr2609 stabilizes DNA-PKcs at damage sites, amplifying signaling for cell cycle arrest.^[47] Recent phosphoproteomic analyses have expanded the understanding of DNA-PKcs modifications, identifying dozens of additional sites and linking them to non-canonical signaling beyond classical NHEJ. A 2024 study mapped over 1,200 conserved phosphorylation events in response to ionizing radiation, revealing DNA-PKcs-dependent motifs (e.g., S/T-ψ-D/E) that extend its substrate specificity to transcription and replication factors, thereby influencing broader cellular responses like ribosome biogenesis.^[23] Functional impacts of these modifications are evident in mutagenesis studies; for example, non-phosphorylatable mutants at the ABCDE cluster (e.g., 6A variant) impair end processing and increase radiosensitivity, while phosphorylation promotes efficient repair but requires precise dephosphorylation for resolution.^[52]

Physiological Roles

Immunity and Development

DNA-PKcs plays a critical role in lymphocyte development by facilitating V(D)J recombination, which is essential for the maturation of B and T cells in the adaptive immune system. Defects in DNA-PKcs lead to impaired V(D)J recombination, resulting in severe combined immunodeficiency (SCID) phenotypes characterized by a profound block in B and T cell development, as observed in DNA-PKcs-deficient mouse models.^[53]^[54] In these models, the absence of functional DNA-PKcs causes accumulation of unrepaired DNA double-strand breaks during recombination, preventing the generation of diverse antigen receptors and halting lymphocyte differentiation.^[55] Beyond V(D)J recombination, DNA-PKcs supports class switch recombination (CSR) in mature B cells, enabling the switching of immunoglobulin heavy chain constant regions from IgM to other isotypes like IgG, IgA, or IgE through non-homologous end joining (NHEJ) repair of activation-induced cytidine deaminase-generated double-strand breaks. Loss of DNA-PKcs significantly reduces CSR efficiency to most isotypes, except IgG1 in some contexts, leading to altered antibody responses and impaired humoral immunity.^[56]^[57] This NHEJ-dependent function underscores DNA-PKcs's necessity for B cell plasticity in responding to diverse pathogens.^[58] In non-immune developmental processes, DNA-PKcs contributes to neural development by promoting the survival of newly generated neurons in the embryonic brain. DNA-PKcs-deficient mice exhibit elevated DNA double-strand breaks and increased apoptosis in the central nervous system, particularly in developing neurons, leading to subtle cerebellar defects and impaired neurogenesis without causing embryonic lethality.^[59]^[60] These findings highlight DNA-PKcs's broader role in maintaining genomic stability during tissue development outside the immune system.^[61] Recent studies as of 2025 have revealed DNA-PKcs's involvement in innate immunity through modulation of cytokine signaling pathways, including the cGAS-STING axis, where it influences interferon and inflammatory cytokine production in response to cytosolic DNA.^[62]^[63] This function links DNA-PKcs to antiviral innate responses and broader inflammatory regulation during development and homeostasis.^[64]

Telomere Maintenance

DNA-PKcs plays a crucial role in telomere protection by being recruited to chromosome ends through interactions with the shelterin complex components TRF2 and RAP1. This recruitment forms a TRF2/RAP1-DNA-PK complex that represses classical non-homologous end joining (c-NHEJ) at telomeres, thereby preventing deleterious end-to-end fusions that could lead to genomic instability.^[65] The complex specifically inhibits the end-joining function of DNA-PK by blocking the recruitment of XRCC4-LIG4 to Ku-bound DNA ends, ensuring that telomeric DNA remains protected from inappropriate repair.^[66] This mechanism complements other shelterin-mediated protections, such as t-loop formation, to maintain telomere integrity during the cell cycle.^[66] A key 2025 discovery revealed the structural basis of this repression, showing that RAP1 directly interacts with the Ku heterodimer within the DNA-PK holoenzyme, occluding the LIG4 binding site and thereby inhibiting end ligation at telomeres. Cryo-electron microscopy structures at 3.58 Å resolution confirmed that the Myb and BRCT domains of RAP1 engage specific domains on Ku70, stabilizing the complex while preventing downstream NHEJ progression. This RAP1-mediated inhibition maintains telomere capping, particularly at blunt leading-strand ends or shortened telomeres where repair pathways might otherwise be activated.^[66] Without this regulation, telomeres become vulnerable to fusion events, as evidenced by increased chromosomal end-to-end joins in cells lacking RAP1 or TRF2.^[67] In addition to repressing c-NHEJ, DNA-PKcs suppresses microhomology-mediated end joining (MMEJ, also known as alternative NHEJ) at short or deprotected telomeres, limiting error-prone repairs that exacerbate telomere attrition. This suppression helps avert the activation of persistent DNA damage responses that drive replicative senescence, as MMEJ-dependent fusions at critically short ends can propagate genomic rearrangements.^[68] Studies indicate that DNA-PKcs promotes end stability to favor protective capping over mutagenic MMEJ, particularly in contexts of telomere shortening during prolonged cell division.^[69] Evidence from DNA-PKcs-deficient models underscores its essential function in telomere maintenance. In knockout mouse embryonic fibroblasts, loss of DNA-PKcs results in telomere uncapping, manifested as elevated rates of end-to-end fusions and chromosomal fragments, leading to aneuploidy through breakage-fusion-bridge cycles.^[70] Conditional ablation approaches further demonstrate that tissue-specific DNA-PKcs deficiency induces telomere dysfunction and associated chromosomal instability, highlighting its non-redundant role in preventing aneuploidy in proliferative cells.^[71] These findings establish DNA-PKcs as a key guardian of telomere ends against repair-mediated damage.

Pathological Implications

Cancer

Dysregulation of DNA-PKcs plays a significant role in cancer development and progression, primarily through its involvement in DNA double-strand break repair via non-homologous end joining (NHEJ), which allows tumor cells to survive genotoxic stress. Overexpression of DNA-PKcs is frequently observed in various malignancies, including breast, prostate, non-small cell lung, gastric, ovarian, pancreatic, and hepatocellular cancers, where it enhances cell survival and resistance to DNA-damaging therapies by promoting efficient NHEJ-mediated repair.^[19] Genetic alterations in the PRKDC gene encoding DNA-PKcs occur in more than 10% of numerous cancer types and can contribute to genomic instability by impairing DNA repair fidelity, thereby facilitating tumor evolution and heterogeneity.^[72] Beyond its canonical repair functions, DNA-PKcs exerts non-canonical effects that drive tumorigenesis, as highlighted in a 2024 review. Its kinase activity, independent of DNA repair, promotes metabolic reprogramming by phosphorylating and activating glycolytic enzymes such as aldolase A (ALDOA) and pyruvate kinase M2 (PKM2), thereby enhancing glycolysis and supporting tumor growth in castration-resistant prostate cancer. Additionally, DNA-PKcs facilitates cancer cell migration and invasion through phosphorylation of transcription factors like Snail1, which increases motility and genomic instability, and by upregulating vascular endothelial growth factor (VEGF) expression to promote angiogenesis and metastasis. These kinase-dependent mechanisms contribute to protumorigenic signaling in multiple cancer contexts.^[73] High DNA-PKcs expression in tumors sensitizes them to radiosensitization strategies, as inhibiting its activity impairs NHEJ and exacerbates DNA damage from ionizing radiation, particularly in glioblastomas. In tumors with BRCA1/2 deficiencies, where homologous recombination is compromised, DNA-PKcs becomes critical for NHEJ-dependent survival, making it a key mediator of resistance to therapies like PARP inhibitors; its upregulation in such contexts further promotes tumor persistence. Clinically, elevated DNA-PKcs levels correlate with poor prognosis in glioblastoma patients, associating with reduced survival following standard treatments including radiation.^[74]^[75]

Immunodeficiency and Disease

Mutations in the PRKDC gene, which encodes DNA-PKcs, cause rare forms of severe combined immunodeficiency (SCID), characterized by profound defects in T- and B-cell development due to impaired V(D)J recombination and non-homologous end joining (NHEJ).^[76] These mutations lead to radiosensitive SCID (RS-SCID), with affected individuals exhibiting hypersensitivity to ionizing radiation, recurrent infections, and failure to thrive.^[77] For instance, the recurrent p.L3062R missense mutation has been identified in multiple patients, resulting in atypical SCID phenotypes including low but detectable T-cell counts and neurological abnormalities such as microcephaly, seizures, and developmental delay.^[78] Growth retardation is a common feature, often accompanied by dysmorphic features and increased susceptibility to granulomatous disease.^[79] The prevalence of SCID overall is estimated at 1 in 50,000 to 100,000 live births, but PRKDC-related cases are exceptionally rare, with fewer than 20 human patients reported to date.^[80] Mouse models, including the classical scid strain with a truncating Prkdc mutation and complete DNA-PKcs knockout (DNA-PKcs^{-/-}) lines, closely recapitulate these human traits, displaying absent adaptive immunity, radiosensitivity, and growth defects, though without the severe neurological involvement seen in some human cases.^[77] These models have been instrumental in elucidating the role of DNA-PKcs in immune development and DNA repair.^[81] Beyond SCID, DNA-PKcs dysfunction has been linked to other immunodeficiencies and autoimmune disorders, including associations with rheumatoid arthritis through defects in immunoglobulin class-switch recombination (CSR).^[31] Impaired CSR in DNA-PKcs-deficient B cells leads to reduced production of switched antibody isotypes (IgG, IgA, IgE), contributing to humoral immune defects and potentially exacerbating autoimmune inflammation in conditions like rheumatoid arthritis. Recent 2025 studies in DNA-PKcs knockout mice have further revealed neurodegeneration phenotypes, including impaired synaptic transmission, deficits in long-term potentiation, and accumulation of DNA damage in neurons, suggesting a broader role in central nervous system maintenance.^[82] In the context of aging, accumulated double-strand breaks (DSBs) over time contribute to DNA-PKcs exhaustion, where chronic activation of the DNA damage response (DDR) leads to diminished repair capacity and cellular senescence.^[83] DNA-PKcs plays a protective role at telomeres, and its deficiency promotes telomere uncapping, triggering persistent DDR signaling and senescence-associated secretory phenotype (SASP), which accelerates tissue aging and age-related immune decline.^[84] This exhaustion mechanism underscores DNA-PKcs as a key mediator linking genomic instability to immunosenescence.^[85]

Therapeutic Targeting

DNA-PKcs Inhibitors

Small-molecule inhibitors of DNA-PKcs primarily target its kinase domain to disrupt non-homologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). These inhibitors are classified mainly as ATP-competitive agents that bind the ATP-binding pocket, preventing phosphorylation events essential for DNA repair. Early examples include NU7441, a chromen-4-one derivative with a biochemical IC₅₀ of 14 nM against DNA-PKcs, which demonstrates modest selectivity over related PI3K family kinases (IC₅₀ of 5 μM for PI3K).^[86]^[87] More advanced inhibitors, such as M3814 (peposertib), also operate via ATP competition, achieving a biochemical IC₅₀ of 0.6 nM at low ATP concentrations and showing greater than 100-fold selectivity over PI3K isoforms (e.g., IC₅₀ of 95 nM for PI3Kδ).^[88] Although some structural analyses suggest potential allosteric modulation in related inhibitors, M3814 and NU7441 are confirmed as ATP-competitive based on ATP-dependent potency shifts.^[88]^[86] The primary mechanism of these inhibitors involves blocking the kinase activity of DNA-PKcs, which halts autophosphorylation at key sites like Ser2056 and impairs downstream end-processing and ligation in NHEJ. This leads to accumulation of unrepaired DSBs, inducing genomic instability and apoptosis in response to DNA-damaging agents. In vitro, DNA-PKcs inhibitors enhance ionizing radiation (IR) sensitivity by 2- to 5-fold, as measured by dose enhancement factors in clonogenic survival assays across cancer cell lines such as A549 and HeLa, without significantly affecting normal cell repair at low doses.^[89]^[88] For instance, NU7441 prevents IR-induced autophosphorylation and increases persistent γH2AX foci, potentiating DSB persistence.^[86] AZD7648, a 7,9-dihydro-8H-purin-8-one with a biochemical IC₅₀ of 0.6 nM and over 1,000-fold selectivity against PI3Kγ (IC₅₀ >1 μM), was investigated in phase I/II trials but discontinued in 2025 due to greater-than-expected toxicity and limited antitumor activity in advanced solid tumors.^[90]^[91]^[92] It blocks autophosphorylation and induces genomic instability, showing radiosensitization in preclinical models. Off-target effects remain a consideration; NU7441 exhibits mild PI3K inhibition at higher doses, potentially contributing to cytotoxicity, while M3814 and AZD7648 minimize this through optimized scaffolds.^[86]^[88] Preclinical absorption, distribution, metabolism, and excretion (ADME) profiles support oral bioavailability for clinical translation. In phase I trials, M3814 demonstrates rapid absorption (t_max 1.1–2.5 hours), a half-life of approximately 5.5 hours, and dose-proportional exposure with minimal accumulation upon repeated dosing, enabling once- or twice-daily regimens. Similar favorable pharmacokinetics are reported for AZD7648 in xenograft models, with good tumor penetration and low systemic toxicity.^[93]^[89] These properties underpin their potential in enhancing IR and chemotherapy without exacerbating normal tissue damage in preclinical settings.

Clinical Applications

DNA-PKcs modulation has emerged as a promising strategy in cancer therapy, particularly through inhibitors that enhance the efficacy of DNA-damaging treatments. Peposertib (M3814), a selective DNA-PKcs inhibitor, is under investigation in a phase I trial (NCT04555577) combining it with radiotherapy followed by temozolomide for newly diagnosed MGMT-unmethylated glioblastoma. As of November 2025, data from 21 patients show favorable safety, with reduced DNA-PK phosphorylation in tumors observed.^[94]^[95]^[96] Early data indicate potential radiosensitization without excessive toxicity, though objective response rates remain under evaluation. Additionally, combinations of DNA-PKcs inhibitors with PARP inhibitors are being explored in phase I trials for homologous recombination-deficient (HRD) tumors, such as peposertib with pegylated liposomal doxorubicin (NCT04092270), which is active but not recruiting as of September 2025, aiming to exploit synthetic lethality by impairing alternative DNA repair pathways.^[97]^[98]^[99] In gene editing applications, DNA-PKcs inhibition facilitates homology-directed repair (HDR) over non-homologous end joining (NHEJ), improving precision in therapeutic CRISPR-based corrections. The inhibitor AZD7648 has been shown to boost HDR efficiency to approximately 60% at the CD40LG locus in mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs), enabling effective correction of X-linked hyper-IgM syndrome, a form of severe combined immunodeficiency (SCID).^[100] This approach reduces NHEJ-mediated insertions/deletions (indels) by up to 13-fold while enhancing long-term engraftment in xenotransplantation models. However, despite its preclinical promise, concerns about large-scale genomic alterations like megabase deletions have been noted, and a December 2024 study highlighted dangerous side effects of AZD7648 in gene editing, including increased genomic instability risks across cell types.^[101]^[32]^[102] Beyond oncology and gene therapy, DNA-PKcs inhibitors show potential in enhancing immunotherapy by promoting immunogenic cell death and antitumor immune responses. For instance, AZD7648 combined with radiotherapy induces apoptosis in tumor cells, activating the STING pathway and increasing T-cell infiltration, which synergizes with PD-1 checkpoint inhibitors to improve outcomes in preclinical models.^[103]^[104] Emerging explorations include telomere-targeted strategies leveraging DNA-PKcs's role in end protection, with preliminary links to Hutchinson-Gilford progeria syndrome where progerin disrupts DNA-PKcs function and accelerates telomere attrition, suggesting modulation could mitigate cellular senescence. Clinical translation faces challenges, including potential toxicity such as myelosuppression observed in early trials of DNA-PKcs inhibitors combined with DNA-damaging agents, though single-agent peposertib has shown minimal hematologic adverse events.^[92]^[105] Biomarker development, particularly PRKDC expression levels and mutation status, is critical for patient selection, as high PRKDC correlates with resistance to radiotherapy and immunotherapy response in various cancers.^[106] Ongoing trials emphasize the need for refined dosing to balance efficacy and safety.

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DNA-PK inhibition enhances gene editing efficiency in HSPCs for ...
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