Inhibition of CDK9 activity compromises global splicing in prostate cancer cells

Cyclin-dependent kinase 9 (CDK9) phosphorylates RNA polymerase II to promote productive transcription elongation. Here we show that short-term CDK9 inhibition affects the splicing of thousands of mRNAs. CDK9 inhibition impairs global splicing and there is no evidence for a coordinated response between the alternative splicing and the overall transcriptome. Alternative splicing is a feature of aggressive prostate cancer (CRPC) and enables the generation of the anti-androgen resistant version of the ligand-independent androgen receptor, AR-v7. We show that CDK9 inhibition results in the loss of AR and AR-v7 expression due to the defects in splicing, which sensitizes CRPC cells to androgen deprivation. Finally, we demonstrate that CDK9 expression increases as PC cells develop CRPC-phenotype both in vitro and also in patient samples. To conclude, here we show that CDK9 inhibition compromises splicing in PC cells, which can be capitalized on by targeting the PC-specific addiction androgen receptor.Peer reviewe

Endoplasmic Reticulum Stress signalling - from basic mechanisms to clinical applications

The endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one-third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling-centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes

Spatially resolved clonal copy number alterations in benign and malignant tissue

Publisher Copyright: © 2022, The Author(s).Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.Peer reviewe

Spatially resolved clonal copy number alterations in benign and malignant tissue

Publisher Copyright: © 2022, The Author(s).Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.Peer reviewe

Ciblage d'IRE1 dans le glioblastome multiforme

Le réticulum endoplasmique (RE) est un organite membranaire intracellulaire et le premier compartiment de la voie de sécrétion. En tant que tel, le RE contribue à la production et au repliement d’environ un tiers des protéines cellulaires et est donc lié au maintien de l’homéostasie cellulaire. L’UPR est un processus biochimique intégré et adaptatif activé en réponse au stress du RE qui contrôle l'homéostasie cellulaire et maintient une function physiologique normale. L'accumulation de protéines mal conformées dans le RE entraîne un stress qui peut pousser l'UPR à la signalisation apoptotique. L'UPR et l'un de ses principaux capteurs IRE1 contribuent ainsi au début, au maintien et à l'exacerbation d'une multitude d'états pathologiques, y compris le glioblastome multiforme (GBM), ce qui en fait une cible thérapeutique novatrice. La GBM est la tumeur primitive du système nerveux central la plus fréquente avec une incidence de 3 sur 100 000. Le pronostic est sombre avec des patients qui succombent à la tumeur entre 15 et 18 mois après le diagnostic, avec une survie médiane à 5 ans inférieure à 6%. Dans cette thèse, des approches in silico, in vitro et in vivo sont utilisées pour déterminer si IRE1 est un médiateur physiopathologique majeur et une cible pharmacologique valide dans le GBM et si sa modulation peut fournir de nouvelles options thérapeutiques comme traitement adjuvant modifiant la maladie. Il est montré ici que IRE1 peut jouer un role différentiel dans la physiopathologie des GBM par le biais d'angiogenèse et de croissance, ainsi que dans l'adaptation à la chimiothérapie et le maintien du phénotype différencié des cellules GBM par le biais de XBP1 et de la signalisation RIDD. La signalisation XBP1 favorise l'infiltration des macrophages dans la tumeur, l'angiogenèse, l'invasion et le maintien d'un phénotype agressif différencié, tandis que le RIDD peut atténuer les propriétés angiogénétiques et invasives, ainsi que contrôler la ré-différenciation cellulaire et l'environnement. IRE1 est évalué en tant que cible thérapeutique en générant des modèles cellulaires traductionnels de GBM portant des variants génétiques modulés par IRE1 et en testant leur sensibilisation au témozolomide en presence d'inhibiteurs de la kinase IRE1 ciblés, en établissant un nouveau pipeline de découverte de médicaments et en produisant six médicaments non encore impactés. Activité, inhibiteurs. L’ensemble de ces travaux montre que IRE1 fait partie intégrante de la pathogenèse et de la progression de la GBM. Son ciblage peut s'avérer bénéfique dans des sous-ensembles spécifiques de patients atteints de GBM.The endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one-third of cellular proteins, and is thus linked to the maintenance of cellular homeostasis and the fine balance between health and disease. The unfolded protein response (UPR) is an integrated, adaptive biochemical process that controls cell homeostasis and maintains normal physiological function. Accumulation of improperly folded proteins in the ER leads to stress, which may push the UPR past beneficial functions such as reduced protein production and increased folding and clearance, to apoptotic signalling. The UPR and one of its major sensors IRE1 are thus contributory to the commencement, maintenance, and exacerbation of a multitude of disease states, including Glioblastoma multiforme (GBM) making it an attractive global target to tackle conditions sorely in need of novel therapeutic intervention. GBM is the commonest primary CNS tumour with an incidence of 3 per 100000. The disease has a dismal prognosis with patients succumbing to the tumour between 15 and 18 months post diagnosis, with a median 5 year survival at less than 6%. In this thesis, in silico, in vitro and in vivo approaches are utilised to assess whether IRE1 is a major pathophysiological mediator and valid pharmacological target in GBM and whether its modulation may provide novel therapeutic options as an adjuvant disease modifying treatment. It is here shown that IRE1 may play a differential role in GBM pathophysiology through angiogenesis and growth as well as adaptation to chemotherapy and maintenance of differentiated GBM cell phenotype through XBP1s and RIDD signalling. XBP1s signalling promotes macrophage infiltration to the tumour, angiogenesis, invasion and maintenance of a differentiated aggressive phenotype, whilst RIDD may attenuate angiogenetic and invasive properties as well control miRNA environment and cell redifferentiation. IRE1 is assessed as a therapeutic target, by generating translational cellular models of GBM carrying IRE1 modulated genetic variants and testing their sensitisation to Temozolomide in the presence of targeted IRE1 kinase inhibitors by establishing a novel drug discovery pipeline and producing six as yet unknown to impact IRE1 activity, inhibitors. This body of work shows that IRE1 is an integral part of GBM pathogenesis and progression and targeting it may prove beneficial in specific subsets of GBM patients

The role of the androgen receptor as a driver and mitigator of cellular stress

Prostate cancer is a high-incidence male cancer, which is dependent on the activity of a nuclear hormone receptor, the androgen receptor (AR). Since the AR is required for both normal prostate gland development and for prostate cancer progression, it is possible that prostate cancer evolves from perturbations in AR-dependent biological processes that sustain specialist glandular functions. The archetypal example of course is the use of PSA, an organ-type specific component of the normal prostate secretome, as a biomarker of prostate cancer. Furthermore, localised prostate cancer is characterised by a low proliferative index and a heterogenous array of somatic mutations aligned to a multifocal disease pattern. We and others have identified a number of biological processes that are AR-dependent and represent aberrations in significant glandular processes. Glands are characterised by high-rates of metabolic activity including protein synthesis supported by co-dependent processes such as glycosylation, organelle biogenesis and vesicle trafficking. Impairment in anabolic metabolism, protein folding and processing will inevitably impose proteotoxic and oxidative stress on glandular cells, and in particular, luminal epithelial cells for which secretion is their primary function. As cancer develops there is also significant metabolic dysregulation including impaired negative feedback effects on glycolytic and anabolic activity under conditions of hypoxia and heightened protein synthesis due to dysregulated PI 3-kinase/mTOR activity. In this review we will focus on the components of the AR regulome that support cancer development as well as glandular functions focussing on the unfolded protein response and on regulators of mTOR activity

Derivation and Application of Molecular Signatures to Prostate Cancer: Opportunities and Challenges

Prostate cancer is a high-incidence cancer that requires improved patient stratification to ensure accurate predictions of risk and treatment response. Due to the significant contributions of transcription factors and epigenetic regulators to prostate cancer progression, there has been considerable progress made in developing gene signatures that may achieve this. Some of these are aligned to activities of key drivers such as the androgen receptor, whilst others are more agnostic. In this review, we present an overview of these signatures, the strategies for their derivation, and future perspectives on their continued development and evolution

Control of the Unfolded Protein Response in Health and Disease

International audienceThe unfolded protein response (UPR) is an integrated, adaptive biochemical process that is inextricably linked with cell homeostasis and paramount to maintenance of normal physiological function. Prolonged accumulation of improperly folded proteins in the endoplasmic reticulum (ER) leads to stress. This is the driving stimulus behind the UPR. As such, prolonged ER stress can push the UPR past beneficial functions such as reduced protein production and increased folding and clearance to apoptotic signaling. The UPR is thus contributory to the commencement, maintenance, and exacerbation of a multitude of disease states, making it an attractive global target to tackle conditions sorely in need of novel therapeutic intervention. The accumulation of information of screening tools, readily available therapies, and potential pathways to drug development is the cornerstone of informed clinical research and clinical trial design. Here, we review the UPR's involvement in health and disease and, beyond providing an in-depth description of the molecules found to target the three UPR arms, we compile all the tools available to screen for and develop novel therapeutic agents that modulate the UPR with the scope of future disease intervention

Pharmacological Targeting of IRE1 in Cancer

International audienceIRE1α (inositol requiring enzyme 1 alpha) is one of the main transducers of the unfolded protein response (UPR). IRE1α plays instrumental protumoral roles in several cancers, and high IRE1α activity has been associated with poorer prognoses. In this context, IRE1α has been identified as a potentially relevant therapeutic target. Pharmacological inhibition of IRE1α activity can be achieved by targeting either the kinase domain or the RNase domain. Herein, the recent advances in IRE1α pharmacological targeting is summarized. We describe the identification and optimization of IRE1α inhibitors as well as their mode of action and limitations as anticancer drugs. The potential pitfalls and challenges that could be faced in the clinic, and the opportunities that IRE1α modulating strategies may present are discussed

Peptidomimetic-based identification of FDA approved compounds inhibiting IRE1 activity

International audienceInositol Requiring Enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease that is a major mediator of the Unfolded Protein Response (UPR) during endoplasmic reticulum (ER) stress. Tumour cells experience ER stress due to adverse environmental cues such as hypoxia or nutrient shortage and high metabolic/protein folding demand. To cope with those stresses, cancer cells utilise IRE1 signalling as an adaptive mechanism. Here we report the discovery of the FDA approved compounds methotrexate, cefoperazone, folinic acid and fludarabine phosphate as IRE1 inhibitors. These were identified through a structural exploration of the IRE1 kinase domain using IRE1 peptide fragment docking and further optimization and pharmacophore development. The inhibitors were verified to have an impact on IRE1 activity in vitro and were tested for their ability to sensitise human cell models of glioblastoma multiforme (GBM) to chemotherapy. We show that all molecules identified sensitise glioblastoma cells to the standard of care chemotherapy temozolomide (TMZ)