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1.
F1000Res ; 12: 954, 2023.
Article in English | MEDLINE | ID: mdl-37799492

ABSTRACT

With diminishing returns and high clinical failure rates from traditional preclinical and animal-based drug discovery strategies, more emphasis is being placed on alternative drug discovery platforms. Ex vivo approaches represent a departure from both more traditional preclinical animal-based models and clinical-based strategies and aim to address intra-tumoural and inter-patient variability at an earlier stage of drug discovery. Additionally, these approaches could also offer precise treatment stratification for patients within a week of tumour resection in order to direct tailored therapy. One tumour group that could significantly benefit from such ex vivo approaches are high-grade gliomas, which exhibit extensive heterogeneity, cellular plasticity and therapy-resistant glioma stem cell (GSC) niches. Historic use of murine-based preclinical models for these tumours has largely failed to generate new therapies, resulting in relatively stagnant and unacceptable survival rates of around 12-15 months post-diagnosis over the last 50 years. The near universal use of DNA damaging chemoradiotherapy after surgical resection within standard-of-care (SoC) therapy regimens provides an opportunity to improve current treatments if we can identify efficient drug combinations in preclinical models that better reflect the complex inter-/intra-tumour heterogeneity, GSC plasticity and inherent DNA damage resistance mechanisms. We have therefore developed and optimised a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. As a proof-of-concept for GliExP, we have optimised SoC therapy responses and screened 30+ small molecule therapeutics and preclinical compounds against tumours from 18 different patients, including multi-region spatial heterogeneity sampling from several individual tumours. Our data therefore provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with SoC therapies as an important preclinical alternative to murine models (reduction and replacement) to triage experimental therapeutics for clinical translation and deliver rapid identification of effective treatment strategies for individual gliomas.


Subject(s)
Brain Neoplasms , Glioblastoma , Glioma , Humans , Animals , Mice , Glioblastoma/drug therapy , Drug Evaluation, Preclinical , Avatar , Brain Neoplasms/drug therapy , Early Detection of Cancer , Neoplastic Stem Cells
2.
Br J Cancer ; 129(11): 1829-1840, 2023 11.
Article in English | MEDLINE | ID: mdl-37777579

ABSTRACT

BACKGROUND: High-grade gliomas are primary brain cancers with unacceptably low and persistent survival rates of 10-16 months for WHO grade 4 gliomas over the last 40 years, despite surgical resection and DNA-damaging chemo-radiotherapy. More recently, tumour-treating fields therapy (TTFields) has demonstrated modest survival benefit and been clinically approved in several countries. TTFields is thought to mediate anti-cancer activity by primarily disrupting mitosis. However, recent data suggest that TTFields may also attenuate DNA damage repair and replication fork dynamics, providing a potential platform for therapeutic combinations incorporating standard-of-care treatments and targeted DNA damage response inhibitors (DDRi). METHODS: We have used patient-derived, typically resistant, glioma stem-like cells (GSCs) in combination with the previously validated preclinical Inovitro™ TTFields system together with a number of therapeutic DDRi. RESULTS: We show that TTFields robustly activates PARP- and ATR-mediated DNA repair (including PARylation and CHK1 phosphorylation, respectively), whilst combining TTFields with PARP1 or ATR inhibitor treatment leads to significantly reduced clonogenic survival. The potency of each of these strategies is further enhanced by radiation treatment, leading to increased amounts of DNA damage with profound delay in DNA damage resolution. CONCLUSION: To our knowledge, our findings represent the first report of TTFields applied with clinically approved or in-trial DDRi in GSC models and provides a basis for translational studies toward multimodal DDRi/TTFields-based therapeutic strategies for patients with these currently incurable tumours.


Subject(s)
Glioma , Humans , Glioma/pathology , DNA Repair , DNA Damage , Mitosis
3.
Cancers (Basel) ; 13(5)2021 Feb 24.
Article in English | MEDLINE | ID: mdl-33668183

ABSTRACT

Brain tumours kill more children and adults under 40 than any other cancer, with approximately half of primary brain tumours being diagnosed as high-grade malignancies known as glioblastomas. Despite de-bulking surgery combined with chemo-/radiotherapy regimens, the mean survival for these patients is only around 15 months, with less than 10% surviving over 5 years. This dismal prognosis highlights the urgent need to develop novel agents to improve the treatment of these tumours. To address this need, we carried out a human kinome siRNA screen to identify potential drug targets that augment the effectiveness of temozolomide (TMZ)-the standard-of-care chemotherapeutic agent used to treat glioblastoma. From this we identified ERK5/MAPK7, which we subsequently validated using a range of siRNA and small molecule inhibitors within a panel of glioma cells. Mechanistically, we find that ERK5 promotes efficient repair of TMZ-induced DNA lesions to confer cell survival and clonogenic capacity. Finally, using several glioblastoma patient cohorts we provide target validation data for ERK5 as a novel drug target, revealing that heightened ERK5 expression at both the mRNA and protein level is associated with increased tumour grade and poorer patient survival. Collectively, these findings provide a foundation to develop clinically effective ERK5 targeting strategies in glioblastomas and establish much-needed enhancement of the therapeutic repertoire used to treat this currently incurable disease.

4.
Oncotarget ; 9(50): 29508-29524, 2018 Jun 29.
Article in English | MEDLINE | ID: mdl-30034634

ABSTRACT

BACKGROUND: Cyclin-Dependent Kinases (CDKs) are established anti-cancer drug targets and a new generation of CDK inhibitors are providing clinical benefits to a sub-set of breast cancer patients. We have recently shown that human CDK18 promotes efficient cellular responses to replication stress. In the current study, we have investigated the clinicopathological and functional significance of CDK18 expression levels in breast cancers. RESULTS: High CDK18 protein expression was associated with a triple negative and basal-like phenotype (p = 0.021 and 0.027 respectively) as well as improved patient survival, which was particularly significant in ER negative breast cancers (n = 594, Log Rank 6.724, p = 0.01) and those treated with chemotherapy (n = 270, Log Rank 4.575, p = 0.03). In agreement with these clinical findings, breast cancer cells genetically manipulated using a dCRISPR approach to express high levels of endogenous CDK18 exhibited an increased sensitivity to replication stress-inducing chemotherapeutic agents, as a consequence to defective replication stress signalling at the molecular level. CONCLUSIONS: These data reveal that CDK18 protein levels may predict breast cancer disease progression and response to chemotherapy, and provide further rationale for potential targeting of CDK18 as part of novel anti-cancer strategies for human cancers. MATERIALS AND METHODS: CDK18 protein expression was evaluated in 1650 breast cancers and correlated to clinicopathological parameters and survival outcomes. Similar analyses were carried out for genetic and transcriptomic changes in CDK18 within several publically available breast cancer cohorts. Additionally, we used a deactivated CRISPR/Cas9 approach (dCRISPR) to elucidate the molecular consequences of heightened endogenous CDK18 expression within breast cancer cells.

5.
Sci Rep ; 6: 35548, 2016 10 14.
Article in English | MEDLINE | ID: mdl-27739501

ABSTRACT

It was recently discovered that vertebrate genomes contain multiple endogenised nucleotide sequences derived from the non-retroviral RNA bornavirus. Strikingly, some of these elements have been evolutionary maintained as open reading frames in host genomes for over 40 million years, suggesting that some endogenised bornavirus-derived elements (EBL) might encode functional proteins. EBLN1 is one such element established through endogenisation of the bornavirus N gene (BDV N). Here, we functionally characterise human EBLN1 as a novel regulator of genome stability. Cells depleted of human EBLN1 accumulate DNA damage both under non-stressed conditions and following exogenously induced DNA damage. EBLN1-depleted cells also exhibit cell cycle abnormalities and defects in microtubule organisation as well as premature centrosome splitting, which we attribute in part, to improper localisation of the nuclear envelope protein TPR. Our data therefore reveal that human EBLN1 possesses important cellular functions within human cells, and suggest that other EBLs present within vertebrate genomes may also possess important cellular functions.


Subject(s)
Bornaviridae/metabolism , Cell Cycle/genetics , Genomic Instability , Microtubules/metabolism , Nucleoproteins/metabolism , CDC2 Protein Kinase , Cell Line , Centrosome/metabolism , Cyclin B1/metabolism , DNA Damage , Humans , Nuclear Pore Complex Proteins/metabolism , Nucleoproteins/deficiency , Protein Binding , Proteins/metabolism , Proto-Oncogene Proteins/metabolism
6.
Cell Rep ; 16(10): 2565-2575, 2016 09 06.
Article in English | MEDLINE | ID: mdl-27568553

ABSTRACT

Through an RNAi-based screen for previously uncharacterized regulators of genome stability, we have identified the human protein C5orf45 as an important factor in preventing the accumulation of DNA damage in human cells. Here, we functionally characterize C5orf45 as a binding partner of the MRE11-RAD50-NBS1 (MRN) damage-sensing complex. Hence, we rename C5orf45 as MRNIP for MRN-interacting protein (MRNIP). We find that MRNIP is rapidly recruited to sites of DNA damage. Cells depleted of MRNIP display impaired chromatin loading of the MRN complex, resulting in reduced DNA end resection and defective ATM-mediated DNA damage signaling, a reduced ability to repair DNA breaks, and radiation sensitivity. Finally, we show that MRNIP phosphorylation on serine 115 leads to its nuclear localization, and this modification is required for MRNIP's role in promoting genome stability. Collectively, these data reveal that MRNIP is an important component of the human DNA damage response.


Subject(s)
Carrier Proteins/metabolism , DNA Damage , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Checkpoint Kinase 2/metabolism , Chromatin/metabolism , DNA Breaks, Double-Stranded/radiation effects , DNA Repair/radiation effects , Endodeoxyribonucleases , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Protein Binding/radiation effects , Radiation Tolerance/radiation effects , Radiation, Ionizing , Sequence Homology, Amino Acid , Signal Transduction/radiation effects
7.
Nucleic Acids Res ; 44(18): 8772-8785, 2016 Oct 14.
Article in English | MEDLINE | ID: mdl-27382066

ABSTRACT

Cyclin-dependent kinases (CDKs) coordinate cell cycle checkpoints with DNA repair mechanisms that together maintain genome stability. However, the myriad mechanisms that can give rise to genome instability are still to be fully elucidated. Here, we identify CDK18 (PCTAIRE 3) as a novel regulator of genome stability, and show that depletion of CDK18 causes an increase in endogenous DNA damage and chromosomal abnormalities. CDK18-depleted cells accumulate in early S-phase, exhibiting retarded replication fork kinetics and reduced ATR kinase signaling in response to replication stress. Mechanistically, CDK18 interacts with RAD9, RAD17 and TOPBP1, and CDK18-deficiency results in a decrease in both RAD17 and RAD9 chromatin retention in response to replication stress. Importantly, we demonstrate that these phenotypes are rescued by exogenous CDK18 in a kinase-dependent manner. Collectively, these data reveal a rate-limiting role for CDK18 in replication stress signalling and establish it as a novel regulator of genome integrity.


Subject(s)
Cyclin-Dependent Kinases/metabolism , DNA Replication , Genomic Instability , Signal Transduction , Stress, Physiological , Cell Cycle Checkpoints/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Chromatin/genetics , Chromatin/metabolism , Chromosome Aberrations , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/genetics , DNA Damage , Humans , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , RNA Interference , RNA, Small Interfering/genetics
8.
Cell Cycle ; 13(21): 3450-9, 2014.
Article in English | MEDLINE | ID: mdl-25485589

ABSTRACT

We previously identified and characterized TELO2 as a human protein that facilitates efficient DNA damage response (DDR) signaling. A subsequent yeast 2-hybrid screen identified LARG; Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (also known as Arhgef12), as a potential novel TELO2 interactor. LARG was previously shown to interact with Pericentrin (PCNT), which, like TELO2, is required for efficient replication stress signaling. Here we confirm interactions between LARG, TELO2 and PCNT and show that a sub-set of LARG co-localizes with PCNT at the centrosome. LARG-deficient cells exhibit replication stress signaling defects as evidenced by; supernumerary centrosomes, reduced replication stress-induced γH2AX and RPA nuclear foci formation, and reduced activation of the replication stress signaling effector kinase Chk1 in response to hydroxyurea. As such, LARG-deficient cells are sensitive to replication stress-inducing agents such as hydroxyurea and mitomycin C. Conversely we also show that depletion of TELO2 and the replication stress signaling kinase ATR leads to RhoA signaling defects. These data therefore reveal a level of crosstalk between the RhoA and DDR signaling pathways. Given that mutations in both ATR and PCNT can give rise to the related primordial dwarfism disorders of Seckel Syndrome and Microcephalic osteodysplastic primordial dwarfism type II (MOPDII) respectively, which both exhibit defects in ATR-dependent checkpoint signaling, these data also raise the possibility that mutations in LARG or disruption to RhoA signaling may be contributory factors to the etiology of a sub-set of primordial dwarfism disorders.


Subject(s)
Rho Guanine Nucleotide Exchange Factors/metabolism , Signal Transduction , Antigens/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Survival/drug effects , Centrosome/metabolism , Checkpoint Kinase 1 , DNA Repair , DNA Replication/drug effects , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Hydroxyurea/pharmacology , Mitomycin/pharmacology , Phosphorylation/drug effects , Protein Kinases/metabolism , RNA, Small Interfering/metabolism , Rho Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Rho Guanine Nucleotide Exchange Factors/genetics , Telomere-Binding Proteins/antagonists & inhibitors , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , rhoA GTP-Binding Protein/metabolism
9.
Oncotarget ; 5(15): 6414-24, 2014 Aug 15.
Article in English | MEDLINE | ID: mdl-25071006

ABSTRACT

Brain tumours kill more children and adults under 40 than any other cancer. Around half of primary brain tumours are glioblastoma multiforme (GBMs) where treatment remains a significant challenge, where survival rates have improved little over the last 40 years, thus highlighting an unmet need for the identification/development of novel therapeutic targets and agents to improve GBM treatment. Using archived and fresh glioma tissue, we show that in contrast to normal brain or benign schwannomas GBMs exhibit re-expression of FANCD2, a key protein of the Fanconi Anaemia (FA) DNA repair pathway, and possess an active FA pathway. Importantly, FANCD2 expression levels are strongly associated with tumour grade, revealing a potential exploitable therapeutic window to allow inhibition of the FA pathway in tumour cells, whilst sparing normal brain tissue. Using several small molecule inhibitors of the FA pathway in combination with isogenic FA-proficient/deficient glioma cell lines as well as primary GBM cultures, we demonstrate that inhibition of the FA pathway sensitises gliomas to the chemotherapeutic agents Temozolomide and Carmustine. Our findings therefore provide a strong rationale for the development of novel and potent inhibitors of the FA pathway to improve the treatment of GBMs, which may ultimately impact on patient outcome.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Fanconi Anemia Complementation Group D2 Protein/biosynthesis , Fanconi Anemia/metabolism , Glioma/drug therapy , Glioma/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Carmustine/pharmacology , Cell Line, Tumor , Curcumin/metabolism , Dacarbazine/analogs & derivatives , Dacarbazine/pharmacology , Fanconi Anemia/genetics , Fanconi Anemia/pathology , Glioma/genetics , Glioma/pathology , Humans , Neoplasm Grading , Temozolomide
10.
J Cell Sci ; 127(Pt 13): 2910-9, 2014 Jul 01.
Article in English | MEDLINE | ID: mdl-24816561

ABSTRACT

Here, we identify coiled-coil domain-containing protein 13 (Ccdc13) in a genome-wide RNA interference screen for regulators of genome stability. We establish that Ccdc13 is a newly identified centriolar satellite protein that interacts with PCM1, Cep290 and pericentrin and prevents the accumulation of DNA damage during mitotic transit. Depletion of Ccdc13 results in the loss of microtubule organisation in a manner similar to PCM1 and Cep290 depletion, although Ccdc13 is not required for satellite integrity. We show that microtubule regrowth is enhanced in Ccdc13-depleted cells, but slowed in cells that overexpress Ccdc13. Furthermore, in serum-starved cells, Ccdc13 localises to the basal body, is required for primary cilia formation and promotes the localisation of the ciliopathy protein BBS4 to both centriolar satellites and cilia. These data highlight the emerging link between DNA damage response factors, centriolar and peri-centriolar satellites and cilia-associated proteins and implicate Ccdc13 as a centriolar satellite protein that functions to promote both genome stability and cilia formation.


Subject(s)
Cell Cycle Proteins/physiology , Centrioles/metabolism , Cilia/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Genomic Instability , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Transfection
11.
J Cell Sci ; 125(Pt 20): 4770-9, 2012 Oct 15.
Article in English | MEDLINE | ID: mdl-22797915

ABSTRACT

The centrosome acts as a centre for microtubule organisation and plays crucial roles in cell polarity, migration, growth and division. Cep131 has recently been described as a basal body component essential for cilium formation, but its function in non-ciliogenic cells is unknown. We identified human Cep131 (also known as AZI1) in a screen for regulators of genome stability. We show that centrosomal localisation of Cep131 is cell-cycle-regulated and requires both an intact microtubule network and a functional dynein-dynactin transport system. Cep131 is recruited to centriolar satellites by PCM1, and localised to the centriolar core region by both pericentrin and Cep290. Depletion of Cep131 results in a reduction in proliferation rate, centriole amplification, an increased frequency of multipolar mitosis, chromosomal instability and an increase in post-mitotic DNA damage. These data therefore highlight the importance of human Cep131 for maintaining genomic integrity.


Subject(s)
Cell Cycle Proteins , Centrioles , Centrosome , Genomic Instability , Microtubule Proteins , Antigens, Neoplasm/metabolism , Autoantigens/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Centrioles/genetics , Centrioles/ultrastructure , Centrosome/metabolism , Centrosome/ultrastructure , Chromosomal Instability , Cytoskeletal Proteins , Dynactin Complex , Dyneins/metabolism , Humans , Microtubule Proteins/genetics , Microtubule Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Mitosis/genetics , Neoplasm Proteins/metabolism
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