Phosphorylation at S2053 in Murine (S2056 in Human) DNA-PKcs Is Dispensable for Lymphocyte Development and Class Switch Recombination

Jiang, Wenxia; Estes, Verna M; Wang, Xiaobin S; Shao, Zongping; Lee, Brian J.; Lin, Xiaohui; Crowe, Jennifer; Zha, Shan

doi:10.4049/jimmunol.1801657

Cited by 30 publications

(43 citation statements)

References 53 publications

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“…While a single dose of 3.3 Gy radiation produced a relatively marginal inhibition of Calu-6 tumor growth, the concurrent administration of NU5455 enhanced the antitumor activity significantly to induce a cytostatic response for 6 days following treatment (mean tumor volume = 104 mm 3 at day 0 vs. 102 mm 3 at day 6) (Figure 3A). As antibodies against human phospho–DNA-PK (Ser2056) do not cross-react with the complementary phosphorylation site in murine DNA-PK (Ser2053) (28), further ex vivo comparisons between mouse skin and human tumor tissues were based on quantification of γH2AX, reflecting DNA-DSBs. A histological analysis of Calu-6 subcutaneous tumors indicated that the number of radiation-induced γH2AX foci remaining 5 hours after irradiation was 3.0-fold greater ( P = 0.01) with concomitant NU5455 treatment (Figure 3B and Supplemental Figure 11A).…”

Section: Resultsmentioning

confidence: 99%

Selective DNA-PKcs inhibition extends the therapeutic index of localized radiotherapy and chemotherapy

Willoughby¹,

Jiang²,

Thomas³

et al. 2019

Journal of Clinical Investigation

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Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.

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Section: Resultsmentioning

confidence: 99%

Selective DNA-PKcs inhibition extends the therapeutic index of localized radiotherapy and chemotherapy

Willoughby¹,

Jiang²,

Thomas³

et al. 2019

Journal of Clinical Investigation

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“…Impaired phosphorylation at either or both clusters increases IRsensitivity in CHO cells with ectopic expression of DNA-PKcs [70]. Yet, alanine substitution at the S2056 cluster (corresponding to S2053 in mouse) does not affect V(D) J recombination or CSR, and only causes moderate IR sensitivity in B cells [73]. Meanwhile, alanine substitution at the T2609 cluster (mouse T2605A/T2634A/T2643A, DNA-PKcs 3A ) caused lethal p53-dependent bone marrow failure [74] without abolishing chromosomal V(D)J recombination [75].…”

Section: Dna-dependent Protein Kinase Catalytic Subunit (Dna-pkcs)mentioning

confidence: 99%

“…Finally, despite the normal development of mice or horses in the absence of DNA-PKcs or KU, cultured human cells, including cancer cells, cannot tolerate the loss of DNA-PKcs or KU [76,77]. This essential function of DNA-PKcs and KU in human cells seems to be independent of cNHEJ, since (1) the protein levels of KU and DNA-PKcs increase 50 fold in human cells independently from all the other NHEJ factors [73], (2) the loss of LIG4 or XRCC4 can be well tolerated in cultured human cells [78]. Correspondingly, DNA-PKcs protein expression is preserved in the two patients with DNA-PKcs deficiency identified thus far-one patient carries the L3062R mutation in the FAT domain, with preserved kinase activity and isolated SCID [79], and the other one has reduced kinase activity with SCID and severe microcephaly [80], similar to patients with hypomorphic mutations in LIG4 or XRCC4 [81][82][83].…”

Section: Dna-dependent Protein Kinase Catalytic Subunit (Dna-pkcs)mentioning

confidence: 99%

ATM, ATR and DNA-PKcs kinases—the lessons from the mouse models: inhibition ≠ deletion

2020

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DNA damage, especially DNA double strand breaks (DSBs) and replication stress, activates a complex post-translational network termed DNA damage response (DDR). Our review focuses on three PI3-kinase related protein kinases-ATM, ATR and DNA-PKcs, which situate at the apex of the mammalian DDR. They are recruited to and activated at the DNA damage sites by their respective sensor protein complexes-MRE11/RAD50/NBS1 for ATM, RPA/ ATRIP for ATR and KU70-KU80/86 (XRCC6/XRCC5) for DNA-PKcs. Upon activation, ATM, ATR and DNA-PKcs phosphorylate a large number of partially overlapping substrates to promote efficient and accurate DNA repair and to coordinate DNA repair with other DNA metabolic events (e.g., transcription, replication and mitosis). At the organism level, robust DDR is critical for normal development, aging, stem cell maintenance and regeneration, and physiological genomic rearrangements in lymphocytes and germ cells. In addition to endogenous damage, oncogene-induced replication stresses and genotoxic chemotherapies also activate DDR. On one hand, DDR factors suppress genomic instability to prevent malignant transformation. On the other hand, targeting DDR enhances the therapeutic effects of anti-cancer chemotherapy, which led to the development of specific kinase inhibitors for ATM, ATR and DNA-PKcs. Using mouse models expressing kinase dead ATM, ATR and DNA-PKcs, an unexpected structural function of these kinases was revealed, where the expression of catalytically inactive kinases causes more genomic instability than the loss of the proteins themselves. The spectrum of genomic instabilities and physiological consequences are unique for each kinase and depends on their activating complexes, suggesting a model in which the catalysis is coupled with DNA/chromatin release and catalytic inhibition leads to the persistence of the kinases at the DNA lesion, which in turn affects repair pathway choice and outcomes. Here we discuss the experimental evidences supporting this mode of action and their implications in the design and use of specific kinase inhibitors for ATM, ATR and DNA-PKcs for cancer therapy.

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“…The increased use of MHs and end-resection in DNA-PKcs 5A/5A cells are also much less prominent than those from DNA-PKcs KD/KD cells (16, 40). Together with the normal CSR in DNA-PKcs PQR/PQR B cells with alanine substitutions at the S2056 cluster(32), these results suggest that lack of DNA-PKcs phosphorylation alone is not sufficient to explain the strong cNHEJ-defects in DNA-PKcs KD/KD cells. The possibilities include other phosphorylation sites (23) or phosphorylation-independent effects of catalysis itself on the structure or organization of DNA-PKcs protein.…”

Section: Discussionmentioning

confidence: 92%

“…Human DNA-PKcs with alanine substitutions at the S2056 cluster and/or the T2609 cluster, cannot restore radiation resistance in DNA-PKcs-deficient Chinese hamster ovary (CHO) cells (22, 28–31), suggesting a role of DNA-PKcs phosphorylation in DSB repair. The mouse model carrying alanine substitutions in the S2056 cluster ( DNA-PKcs PQR/PQR ) has no defects in chromosomal V(D)J Recombination or CSR, despite moderate radiation sensitivity (32). Mouse models with alanine substitutions of three or all five threonines at the T2609 cluster ( DNA-PKcs 3A/3A or DNA-PKcs 5A/5A ) developed neonatal bone marrow failure that abolishes both myeloid and lymphoid progenitors (33–35).…”

Section: Introductionmentioning

confidence: 99%

DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

Crowe

Wang

Shao

et al. 2020

Preprint

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18The DNA-dependent protein kinase (DNA-PK), composed of the KU heterodimer and the 19 large catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) 20 factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with 21 different isotypes by joining two DNA double-strand breaks at different switching regions via 22 the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair 23 phase of CSR. To initiate cNHEJ, KU binds to DNA ends, and recruits and activates DNA-PK. 24 DNA-PKcs is the best-characterized substrate of DNA-PK, which phosphorylates DNA-PKcs at 25 both the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation 26 sensitivity, suggesting a role of T2609 phosphorylation in DNA repair. Using the DNA-PKcs 5A 27 mouse model carrying an alanine substitution at the T2609 cluster, here we show that loss of 28 T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR 29 junctions recovered from DNA-PKcs 5A/5A B cells reveal increased chromosomal translocation, 30 excess end-resection, and preferential usage of micro-homologyall signs of the alternative 31 end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation 32 in promoting cNHEJ repair pathway choice during CSR. 33 34 Key points 35 Loss of T2069 cluster phosphorylation of DNA-PKcs promotes Alt-EJ-mediated CSR.36 37 58 recombination. Correspondingly, DNA-PKcs -/-(null) or Artemis -/mice are born of normal size at 59 the expected ratio but develop severe combined immunodeficiency (SCID) (4-6). Patients with 60 4hypomorphic mutations in DNA-PKcs or Artemis also develop SCID (12, 13). Mature B cells 61 undergo CSR, which replace the initially expressed IgM constant region with another 62 downstream constant region encoding a different isotype, to generate antibodies with different 63 effector functions. The cNHEJ pathway plays an important role in CSR. But in cells lacking a 64 core cNHEJ factor (e.g., Lig4 or Xrcc4), up to 25-50% of CSR can be achieved by the alternative 65 end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. 66 Consistent with DNA-PKcs and Artemis being dispensable for direct end-ligation, DNA-PKcs -/-67 B cells only have moderate defects in CSR (14, 15). Nevertheless, in recent high-throughput 68 sequence analyses, we found that CSR junctions recovered from DNA-PKcs -/-B cells contained 69 increased chromosomal translocations and extensive end-resection, and preferentially used MHs 70 (16), suggesting that the seemingly robust CSR achieved in DNA-PKcs -/cells is primarily 71 mediated by the Alt-EJ pathway like those in Lig4 -/or Xrcc4 -/-B cells (17, 18). Accordingly, 72 human patients with spontaneous mutations in DNA-PKcs show severe defects in both V(D)J 73 recombination and CSR and increased MH in the residual CSR junctions (12, 19). 74Active DNA-PK phosphorylates many substrates, among which, DNA-PKcs is the best-75 characterized...

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Phosphorylation at S2053 in Murine (S2056 in Human) DNA-PKcs Is Dispensable for Lymphocyte Development and Class Switch Recombination

Cited by 30 publications

References 53 publications

Selective DNA-PKcs inhibition extends the therapeutic index of localized radiotherapy and chemotherapy

Selective DNA-PKcs inhibition extends the therapeutic index of localized radiotherapy and chemotherapy

ATM, ATR and DNA-PKcs kinases—the lessons from the mouse models: inhibition ≠ deletion

DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

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