Development and Optimisation of Tumour Treating Fields (TTFields) Delivery within 3D Primary Glioma Stem Cell-like Models of Spatial Heterogeneity.

Glioblastoma is an aggressive, incurable brain cancer with poor five-year survival rates of around 13% despite multimodal treatment with surgery, DNA-damaging chemoradiotherapy and the recent addition of Tumour Treating Fields (TTFields). As such, there is an urgent need to improve our current understanding of cellular responses to TTFields using more clinically and surgically relevant models, which reflect the profound spatial heterogeneity within glioblastoma, and leverage these biological insights to inform the rational design of more effective therapeutic strategies incorporating TTFields. We have recently reported the use of preclinical TTFields using the inovitroTM system within 2D glioma stem-like cell (GSC) models and demonstrated significant cytotoxicity enhancement when co-applied with a range of therapeutically approved and preclinical DNA damage response inhibitors (DDRi) and chemoradiotherapy. Here we report the development and optimisation of preclinical TTFields delivery within more clinically relevant 3D scaffold-based primary GSC models of spatial heterogeneity, and highlight some initial enhancement of TTFields potency with temozolomide and clinically approved PARP inhibitors (PARPi). These studies, therefore, represent an important platform for further preclinical assessment of TTFields-based therapeutic strategies within clinically relevant 3D GSC models, aimed towards accelerating clinical trial implementation and the ultimate goal of improving the persistently dire survival rates for these patients.

Inhibition of ATR opposes glioblastoma invasion through disruption of cytoskeletal networks and integrin internalization via macropinocytosis.

BACKGROUND: Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist that counter glioblastoma invasion. Here, we report that inhibition of ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking. METHODS: Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR overexpression using time-lapse microscopy, two orthotopic glioblastoma models, and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high-throughput, super-resolution and intravital imaging. RESULTS: High ATR expression was associated with significantly poorer survival in clinical datasets while histological, protein expression, and spatial transcriptomics using glioblastoma tumor specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed the invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalization of integrins at growth-cone-like structures, resulting in a tumor microtube retraction defect. The biological relevance and translational potential of these findings were confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging. CONCLUSIONS: We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far-reaching clinical benefits beyond radiosensitization.

Ex-vivo drug screening of surgically resected glioma stem cells to replace murine avatars and provide personalise cancer therapy for glioblastoma patients.

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.

DNA damage response inhibitors enhance tumour treating fields (TTFields) potency in glioma stem-like cells.

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.

Development of a Personalised Device for Systemic Magnetic Drug Targeting to Brain Tumours.

Delivering therapies to deeply seated brain tumours (BT) is a major clinical challenge. Magnetic drug targeting (MDT) could overcome this by rapidly transporting magnetised drugs directly into BT. We have developed a magnetic device for application in murine BT models using an array of neodymium magnets with a combined strength of 0.7T. In a closed fluidic system, the magnetic device trapped magnetic nanoparticles (MNP) up to distances of 0.8cm. In mice, the magnetic device guided intravenously administered MNP (<50nm) from the circulation into the brain where they localised within mouse BT. Furthermore, MDT of magnetised Temozolomide (TMZmag+) significantly reduced tumour growth and extended mouse survival to 48 days compared to the other treatment groups. Using the same principles, we built a proof of principle scalable magnetic device for human use with a strength of 1.1T. This magnetic device demonstrated trapping of MNP undergoing flow at distances up to 5cm. MDT using our magnetic device provides an opportunity for targeted delivery of magnetised drugs to human BT.

Precision oncology using ex vivo technology: a step towards individualised cancer care?

Despite advances in cancer genomics and the increased use of genomic medicine, metastatic cancer is still mostly an incurable and fatal disease. With diminishing returns from traditional drug discovery strategies, and high clinical failure rates, more emphasis is being placed on alternative drug discovery platforms, such as ex vivo approaches. Ex vivo approaches aim to embed biological relevance and inter-patient variability at an earlier stage of drug discovery, and to offer more precise treatment stratification for patients. However, these techniques also have a high potential to offer personalised therapies to patients, complementing and enhancing genomic medicine. Although an array of approaches are available to researchers, only a minority of techniques have made it through to direct patient treatment within robust clinical trials. Within this review, we discuss the current challenges to ex vivo approaches within clinical practice and summarise the contemporary literature which has directed patient treatment. Finally, we map out how ex vivo approaches could transition from a small-scale, predominantly research based technology to a robust and validated predictive tool. In future, these pre-clinical approaches may be integrated into clinical cancer pathways to assist in the personalisation of therapy choices and to hopefully improve patient experiences and outcomes.

DDRugging glioblastoma: understanding and targeting the DNA damage response to improve future therapies.

Glioblastoma is the most frequently diagnosed type of primary brain tumour in adults. These aggressive tumours are characterised by inherent treatment resistance and disease progression, contributing to ~ 190 000 brain tumour-related deaths globally each year. Current therapeutic interventions consist of surgical resection followed by radiotherapy and temozolomide chemotherapy, but average survival is typically around 1 year, with < 10% of patients surviving more than 5 years. Recently, a fourth treatment modality of intermediate-frequency low-intensity electric fields [called tumour-treating fields (TTFields)] was clinically approved for glioblastoma in some countries after it was found to increase median overall survival rates by ~ 5 months in a phase III randomised clinical trial. However, beyond these treatments, attempts to establish more effective therapies have yielded little improvement in survival for patients over the last 50 years. This is in contrast to many other types of cancer and highlights glioblastoma as a recognised tumour of unmet clinical need. Previous work has revealed that glioblastomas contain stem cell-like subpopulations that exhibit heightened expression of DNA damage response (DDR) factors, contributing to therapy resistance and disease relapse. Given that radiotherapy, chemotherapy and TTFields-based therapies all impact DDR mechanisms, this Review will focus on our current knowledge of the role of the DDR in glioblastoma biology and treatment. We also discuss the potential of effective multimodal targeting of the DDR combined with standard-of-care therapies, as well as emerging therapeutic targets, in providing much-needed improvements in survival rates for patients.

Identification and Validation of ERK5 as a DNA Damage Modulating Drug Target in Glioblastoma.

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.

EML4-ALK V3 oncogenic fusion proteins promote microtubule stabilization and accelerated migration through NEK9 and NEK7.

EML4-ALK is an oncogenic fusion present in ∼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4-ALK with different patient outcomes. Here, we show that, in cell models, EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4-ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer.

The 'Ins and Outs' of Early Preclinical Models for Brain Tumor Research: Are They Valuable and Have We Been Doing It Wrong?

In this perspective, we congratulate the international efforts to highlight critical challenges in brain tumor research through a recent Consensus Statement. We also illustrate the importance of developing more accurate and clinically relevant early translational in vitro brain tumor models-a perspective given limited emphasis in the Consensus Statement, despite in vitro models being widely used to prioritize candidate therapeutic strategies prior to in vivo studies and subsequent clinical trials. We argue that successful translation of effective novel treatments into the clinic will require investment into the development of more predictive early pre-clinical models. It is in the interest of researchers, clinicians, and ultimately, patients that the most promising therapeutic candidates are identified and translated toward use in the clinic. Highlighting the value of early pre-clinical brain tumor models and debating how such models can be improved is of the utmost importance to the neuro-oncology research community and cancer research more broadly.

The relationship of CDK18 expression in breast cancer to clinicopathological parameters and therapeutic response.

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.

The bornavirus-derived human protein EBLN1 promotes efficient cell cycle transit, microtubule organisation and genome stability.

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.

MRNIP/C5orf45 Interacts with the MRN Complex and Contributes to the DNA Damage Response.

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.

Human CDK18 promotes replication stress signaling and genome stability.

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.

Ciliogenesis and the DNA damage response: a stressful relationship.

Both inherited and sporadic mutations can give rise to a plethora of human diseases. Through myriad diverse cellular processes, sporadic mutations can arise through a failure to accurately replicate the genetic code or by inaccurate separation of duplicated chromosomes into daughter cells. The human genome has therefore evolved to encode a large number of proteins that work together with regulators of the cell cycle to ensure that it remains error-free. This is collectively known as the DNA damage response (DDR), and genome stability mechanisms involve a complex network of signalling and processing factors that ensure redundancy and adaptability of these systems. The importance of genome stability mechanisms is best illustrated by the dramatic increased risk of cancer in individuals with underlying disruption to genome maintenance mechanisms. Cilia are microtubule-based sensory organelles present on most vertebrate cells, where they facilitate transduction of external signals into the cell. When not embedded within the specialised ciliary membrane, components of the primary cilium's basal body help form the microtubule organising centre that controls cellular trafficking and the mitotic segregation of chromosomes. Ciliopathies are a collection of diseases associated with functional disruption to cilia function through a variety of different mechanisms. Ciliopathy phenotypes can vary widely, and although some cellular overgrowth phenotypes are prevalent in a subset of ciliopathies, an increased risk of cancer is not noted as a clinical feature. However, recent studies have identified surprising genetic and functional links between cilia-associated proteins and genome maintenance factors. The purpose of this mini-review is to therefore highlight some of these discoveries and discuss their implications with regards to functional crosstalk between the DDR and ciliogenesis pathways, and how this may impact on the development of human disease.

The leukemia-associated Rho guanine nucleotide exchange factor LARG is required for efficient replication stress signaling.

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.

FANCD2 re-expression is associated with glioma grade and chemical inhibition of the Fanconi Anaemia pathway sensitises gliomas to chemotherapeutic agents.

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.

Ccdc13 is a novel human centriolar satellite protein required for ciliogenesis and genome stability.

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.

The centriolar satellite protein Cep131 is important for genome stability.

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.

CK2 phospho-dependent binding of R2TP complex to TEL2 is essential for mTOR and SMG1 stability.

TEL2 interacts with and is essential for the stability of all phosphatidylinositol 3-kinase-related kinases (PIKKs), but its mechanism of action remains unclear. Here, we show that TEL2 is constitutively phosphorylated on conserved serines 487 and 491 by casein kinase 2 (CK2). Proteomic analyses establish that the CK2 phosphosite of TEL2 confers binding to the R2TP/prefoldin-like complex, which possesses chaperon/prefoldin activities required during protein complex assembly. The PIH1D1 subunit of the R2TP complex binds directly to the CK2 phosphosite of TEL2 in vitro and is required for the TEL2-R2TP/prefoldin-like complex interaction in vivo. Although the CK2 phosphosite mutant of TEL2 retains association with the PIKKs and HSP90 in cells, failure to interact with the R2TP/prefoldin-like complex results in instability of the PIKKs, principally mTOR and SMG1. We propose that TEL2 acts as a scaffold to coordinate the activities of R2TP/prefoldin-like and HSP90 chaperone complexes during the assembly of the PIKKs.