Genomic Instability and DNA Damage Responses in Progeria Arising From Defective Maturation of Prelamin A.

Progeria syndromes have in common a premature aging phenotype and increased genome instability. The susceptibility to DNA damage arises from a compromised repair system, either in the repair proteins themselves or in the DNA damage response pathways. The most severe progerias stem from mutations affecting lamin A production, a filamentous protein of the nuclear lamina. Hutchinson-Gilford progeria syndrome (HGPS) patients are heterozygous for aLMNA gene mutation while Restrictive Dermopathy (RD) individuals have a homozygous deficiency in the processing protease Zmpste24. These mutations generate the mutant lamin A proteins progerin and FC-lamina A, respectively, which cause nuclear deformations and chromatin perturbations. Genome instability is observed even though genome maintenance and repair genes appear normal. The unresolved question is what features of the DNA damage response pathways are deficient in HGPS and RD cells. Here we review and discuss recent findings which resolve some mechanistic details of how the accumulation of progerin/FC-lamin A proteins may disrupt DNA damage response pathways in HGPS and RD cells. As the mutant lamin proteins accumulate they sequester replication and repair factors, leading to stalled replication forks which collapse into DNA double-strand beaks (DSBs). In a reaction unique to HGPS and RD cells these accessible DSB termini bind Xeroderma pigmentosum group A (XPA) protein which excludes normal binding by DNA DSB repair proteins. The bound XPA also signals activation of ATM and ATR, arresting cell cycle progression, leading to arrested growth. In addition, the effective sequestration of XPA at these DSB damage sites makes HGPS and RD cells more sensitive to ultraviolet light and other mutagens normally repaired by the nucleotide excision repair pathway of which XPA is a necessary and specific component

Phosphorylation-Dependent Pin1 Isomerization of ATR: Its Role in Regulating ATR’s Anti-Apoptotic Function at Mitochondria, and the Implications in Cancer

Peptidyl-prolyl isomerization is an important post-translational modification of protein because proline is the only amino acid that can stably exist as cis and trans, while other amino acids are in the trans conformation in protein backbones. This makes prolyl isomerization a unique mechanism for cells to control many cellular processes. Isomerization is a rate-limiting process that requires a peptidyl-prolyl cis/trans isomerase (PPIase) to overcome the energy barrier between cis and trans isomeric forms. Pin1, a key PPIase in the cell, recognizes a phosphorylated Ser/Thr-Pro motif to catalyze peptidyl-prolyl isomerization in proteins. The significance of the phosphorylation-dependent Pin1 activity was recently highlighted for isomerization of ATR (ataxia telangiectasia- and Rad3-related). ATR, a PIKK protein kinase, plays a crucial role in DNA damage responses (DDR) by phosphorylating hundreds of proteins. ATR can form cis or trans isomers in the cytoplasm depending on Pin1 which isomerizes cis-ATR to trans-ATR. Trans-ATR functions primarily in the nucleus. The cis-ATR, containing an exposed BH3 domain, is anti-apoptotic at mitochondria by binding to tBid, preventing activation of pro-apoptotic Bax. Given the roles of apoptosis in many human diseases, particularly cancer, we propose that cytoplasmic cis-ATR enables cells to evade apoptosis, thus addicting cancer cells to cis-ATR formation for survival. But in normal DDR, a predominance of trans-ATR in the nucleus coordinates with a minimal level of cytoplasmic cis-ATR to promote DNA repair while preventing cell death; however, cells can die when DNA repair fails. Therefore, a delicate balance/equilibrium of the levels of cis- and trans-ATR is required to ensure the cellular homeostasis. In this review, we make a case that this anti-apoptotic role of cis-ATR supports oncogenesis, while Pin1 that drives the formation of trans-ATR suppresses tumor growth. We offer a potential, novel target that can be specifically targeted in cancer cells, without killing normal cells, to significantly reduce the adverse effects usually seen in cancer treatment. We also raise important issues regarding the roles of phosphorylation-dependent Pin1 isomerization of ATR in diseases and propose areas of future studies that would shed more understanding on this important cellular mechanism

XPA-Mediated Regulation of Global Nucleotide Excision Repair by Atr Is p53-Dependent and Occurs Primarily in S-Phase

Cell cycle checkpoints play an important role in regulation of DNA repair pathways. However, how the regulation occurs throughout the cell cycle remains largely unknown. Here we demonstrate that nucleotide excision repair (NER) is regulated by the ATR/p53 checkpoint via modulation of XPA nuclear import and that this regulation occurs in a cell cycle-dependent manner. We show that depletion of p53 abrogated the UV-induced nuclear translocation of XPA, while silencing of Chk1 or MAPKAP Kinase-2 (MK2) had no effect. Inhibition of p53 transcriptional activities and silencing of p53-Ser15 phosphorylation also reduced the damage-induced XPA nuclear import. Furthermore, in G1-phase cells the majority of XPA remained in the cytoplasm even after UV treatment. By contrast, while most of the XPA in S-phase cells was initially located in the cytoplasm before DNA damage, UV irradiation stimulated bulk import of XPA into the nucleus. Interestingly, the majority of XPA molecules always were located in the nucleus in G2-phase cells no matter whether the DNA was damaged or not. Consistently, the UV-induced Ser15 phosphorylation of p53 occurred mainly in S-phase cells, and removal of cyclobutane pyrimidine dimers (CPDs) was much more efficient in S-phase cells than in G1-phase cells. Our results suggest that upon DNA damage in S phase, NER could be regulated by the ATR/p53-dependent checkpoint via modulation of the XPA nuclear import process. In contrast, the nuclear import of XPA in G 1 or G 2 phase appears to be largely independent of DNA damage and p53

Chimeric Antigen Receptor T-Cell Therapy in Glioblastoma: Charging the T Cells to Fight

Glioblastoma multiforme (GBM) is the most common malignant brain cancer that invades normal brain tissue and impedes surgical eradication, resulting in early local recurrence and high mortality. In addition, most therapeutic agents lack permeability across the blood brain barrier (BBB), further reducing the efficacy of chemotherapy. Thus, effective treatment against GBM requires tumor specific targets and efficient intracranial drug delivery. With the most recent advances in immunotherapy, genetically engineered T cells with chimeric antigen receptors (CARs) are becoming a promising approach for treating cancer. By transducing T lymphocytes with CAR constructs containing a tumor-associated antigen (TAA) recognition domain linked to the constant regions of a signaling T cell receptor, CAR T cells may recognize a predefined TAA with high specificity in a non-MHC restricted manner, and is independent of antigen processing. Active T cells can travel across the BBB, providing additional advantage for drug delivery and tumor targeting. Here we review the CAR design and technical innovations, the major targets that are in pre-clinical and clinical development with a focus on GBM, and multiple strategies developed to improve CAR T cell efficacy

PP2A Regulates Phosphorylation-Dependent Isomerization of Cytoplasmic and Mitochondrial-Associated ATR by Pin1 in DNA Damage Responses

Ataxia telangiectasia and Rad3-related protein (ATR) is a serine/threonine-protein kinase of the PI3K family and is well known for its key role in regulating DNA damage responses in the nucleus. In addition to its nuclear functions, ATR also was found to be a substrate of the prolyl isomerase Pin1 in the cytoplasm where Pin1 isomerizes cis ATR at the Ser428-Pro429 motif, leading to formation of trans ATR. Cis ATR is an antiapoptotic protein at mitochondria upon UV damage. Here we report that Pin1’s activity on cis ATR requires the phosphorylation of the S428 residue of ATR and describe the molecular mechanism by which Pin1-mediated ATR isomerization in the cytoplasm is regulated. We identified protein phosphatase 2A (PP2A) as the phosphatase that dephosphorylates Ser428 following DNA damage. The dephosphorylation led to an increased level of the antiapoptotic cis ATR (ATR-H) in the cytoplasm and, thus, its accumulation at mitochondria via binding with tBid. Inhibition or depletion of PP2A promoted the isomerization by Pin1, resulting in a reduction of cis ATR with an increased level of trans ATR. We conclude that PP2A plays an important role in regulating ATR’s anti-apoptotic activity at mitochondria in response to DNA damage. Our results also imply a potential strategy in enhancing cancer therapies via selective moderation of cis ATR levels

Dissociation Dynamics of XPC-RAD23B from Damaged DNA Is a Determining Factor of NER Efficiency

XPC-RAD23B (XPC) plays a critical role in human nucleotide excision repair (hNER) as this complex recognizes DNA adducts to initiate NER. To determine the mutagenic potential of structurally different bulky DNA damages, various studies have been conducted to define the correlation of XPC-DNA damage equilibrium binding affinity with NER efficiency. However, little is known about the effects of XPC-DNA damage recognition kinetics on hNER. Although association of XPC is important, our current work shows that the XPC-DNA dissociation rate also plays a pivotal role in achieving NER efficiency.We characterized for the first time the binding of XPC to mono- versus di-AAF-modified sequences by using the real time monitoring surface plasmon resonance technique. Strikingly, the half-life (t1/2 or the retention time of XPC in association with damaged DNA) shares an inverse relationship with NER efficiency. This is particularly true when XPC remained bound to clustered adducts for a much longer period of time as compared to mono-adducts. Our results suggest that XPC dissociation from the damage site could become a rate-limiting step in NER of certain types of DNA adducts, leading to repression of NER

Dissociation Dynamics of XPC-RAD23B From Damaged Dna Is a Determining Factor of NER Efficiency

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. XPC-RAD23B (XPC) plays a critical role in human nucleotide excision repair (hNER) as this complex recognizes DNA adducts to initiate NER. To determine the mutagenic potential of structurally different bulky DNA damages, various studies have been conducted to define the correlation of XPC-DNA damage equilibrium binding affinity with NER efficiency. However, little is known about the effects of XPC-DNA damage recognition kinetics on hNER. Although association of XPC is important, our current work shows that the XPC-DNA dissociation rate also plays a pivotal role in achieving NER efficiency. We characterized for the first time the binding of XPC to mono- versus di-AAF-modified sequences by using the real time monitoring surface plasmon resonance technique. Strikingly, the half-life (t1/2 or the retention time of XPC in association with damaged DNA) shares an inverse relationship with NER efficiency. This is particularly true when XPC remained bound to clustered adducts for a much longer period of time as compared to mono-adducts. Our results suggest that XPC dissociation from the damage site could become a rate-limiting step in NER of certain types of DNA adducts, leading to repression of NER

DSTYK Promotes Metastasis and Chemoresistance via EMT in Colorectal Cancer

Objective: Tumor metastasis and resistance to chemotherapy are two critical factors that contribute to the high death rate of colorectal cancer (CRC) patients. Metastasis is facilitated by the epithelial-mesenchymal transition (EMT) of tumor cells, which has emerged not only as a fundamental process during metastasis, but is also a key process leading to chemoresistance of cancer cells. However, the underlying mechanisms of EMT in CRC cell remain unknown. Here, we aim to assess the role of dual serine/threonine and tyrosine protein kinase (DSTYK) in CRC metastasis and chemoresistance. Methods: To study the role of DSTYK in TGF-β-induced EMT, we employed techniques including Crispr/Cas9 knockout (KO) to generate DSTYK KO cell lines, RT-PCR to detect the mRNA expression, immunofluorescence analyses, and western blots to detect protein levels of DSTYK in the following 4 cell lines: control LS411N-TβRII and LS411N-TβRII/DSTYK KO, control LS513 and LS513/DSTYK KO cells, treated with/without TGF-β. The effects of DSTYK on apoptosis were investigated by MTT assays, flow cytometry assays, and TUNEL assays. The expression of DSTYK in CRC patients and its correlation with EMT markers were determined by bioinformatics analysis. For in vivo analysis, both xenograft and orthotopic tumor mouse models were employed to investigate the function of DSTYK in chemoresistance and metastasis of tumors. Results: In this study, we demonstrate that the novel kinase DSTYK promotes both TGF-β-induced EMT and the subsequent chemoresistance in CRC cells. DSTYK KO significantly attenuates TGF-β–induced EMT and chemoresistance in CRC cells. According to the Gene Expression Omnibus (GEO) database, the expression of DSTYK is not only positively correlated to the expression of TGF-β, but proportional to the death rate of CRC patients as well. Evidently, the expression of DSTYK in the metastatic colorectal cancer samples from patients was significantly higher than that of primary colorectal cancer samples. Further, we demonstrate in mouse models that chemotherapeutic drug treatment suppresses the growth of DSTYK KO tumors more effectively than control tumors. Conclusion: Our findings identify DSTYK as a novel protein kinase in regulating TGF-β–mediated EMT and chemoresistance in CRC cells, which defines DSTYK as a potential therapeutic target for CRC therapy

Checkpoint Kinase ATR Promotes Nucleotide Excision Repair of UV-Induced DNA Damage via Physical Interaction Withxeroderma Pigmentosum Group A

In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helixturn-helix motif in the minimal DNA-binding domain of XPA where anATRphosphorylation site (serine 196) is located.XPAdeficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistentDNAdamage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation

ATR Prevents Ca\u3csup\u3e2+\u3c/sup\u3e Overload-Induced Necrotic Cell Death Through Phosphorylation-Mediated Inactivation of PARP1 Without DNA Damage Signaling

Hyperactivation of PARP1 is known to be a major cause of necrotic cell death by depleting NAD+/ATP pools during Ca2+ overload which is associated with many ischemic diseases. However, little is known about how PARP1 hyperactivity is regulated during calcium overload. In this study we show that ATR kinase, well known for its role in DNA damage responses, suppresses ionomycin, glutamate, or quinolinic acid-induced necrotic death of cells including SH-SY5Y neuronal cells. We found that the inhibition of necrosis requires the kinase activity of ATR. Specifically, ATR binds to and phosphorylates PARP1 at Ser179 after the ionophore treatments. This site-specific phosphorylation inactivates PARP1, inhibiting ionophore-induced necrosis. Strikingly, all of this occurs in the absence of detectable DNA damage and signaling up to 8 hours after ionophore treatment. Furthermore, little AIF was released from mitochondria/cytoplasm for nuclear import, supporting the necrotic type of cell death in the early period of the treatments. Our results reveal a novel ATR-mediated anti-necrotic mechanism in the cellular stress response to calcium influx without DNA damage signaling