Biology subjects

Frame, M. C.

Publications and source records attributed to Frame, M. C..

15 recordsLinked to original sources

bioRxiv2025

FAK inhibitor treatment systemically regulates the glioblastoma immune environment via blocking monocyte trafficking

Glioblastoma (GBM) is the most common primary malignant brain tumour in adults with dismal survival rates, and current therapies, including most immunotherapies, are not efficacious due to the highly immunosuppressive microenvironment. Studies in other solid cancers report that impairment of the integrin effector pathway involving focal adhesion kinase (FAK) can promote anti-tumour immune responses. Therefore, we set out to address whether, and if so how, suppressing FAK function may influence GBM by using both tumour cell-specific FAK gene deletion and systemic delivery of a clinically relevant FAK kinase inhibitor (FAKi) VS-4718 in an orthotopic murine stem cell model of GBM. We found that treatment with the FAKi, but not tumour cell-specific FAK gene deletion, resulted in GBM clearance and improved survival. This was dependent on adaptive immunity, and tumour-infiltrating T cells in FAKi-treated tumours displayed increased cytotoxic potential and reduced exhaustion. We also found a significant reduction in immuno-suppressive peripherally-derived macrophages and FAKi treatment caused sequestration of inflammatory monocytes within the bone marrow, resulting in impaired monocyte trafficking to tumours as judged by adoptive transfer. This is due to suppression of key adhesion and migration signalling through 4{beta}1 integrin and CX3CR1 in peripheral monocytes. Our work here describes a previously unidentified role for FAK in trafficking of peripheral suppressive macrophages to GBM tumours, reducing T cell exhaustion and promoting anti-tumour immunity. This highlights a new way in which systemic FAK inhibitors can be used to provide a beneficial immune modulatory strategy for the treatment of GBM.

immunology↗

bioRxiv2025

Tissue scarring provides a biomechanical framework to promote mammalian bile duct regeneration through the activation of integrin-SRC/FAK signalling.

Following chronic injury, the adult mammalian bile duct regenerates by forming new branches, essentially replumbing the ductular system to overcome blockages and breaks. To regenerate effectively, biliary epithelial cells (BECs) receive a range of pro-mitogenic signals from myofibroblasts, which concurrently deposit a collagen-rich scar around the duct as it regrows. Despite epithelial regeneration and scarring occurring side-by-side, whether the deposition of scar tissue regulates ductular regeneration per se remains unclear. By inducing ductular fibrosis and regeneration in vivo, we show that the formation of collagen-I-rich scars around regenerating ducts changes the local biomechanical properties of these tissues, promoting the growth of ducts. Critically, this changing structural landscape is perceived by a spatially restricted population of biliary epithelial cells which forms a "leading-tip" of integrin-2-high cells. This leading-tip undergoes partial-EMT-type reprogramming, allowing it to become migratory and coordinate ductular regeneration. We show that this process is directly driven through an integrin-2-SRC/FAK signalling axis; thereby connecting epithelial regeneration directly to the changing fibrotic environment in chronic ductular disease. HighlightsO_LIChronic liver disease results in the formation of stiff, collagen scars around ducts. C_LIO_LINew ducts acquire high levels of integrin-2 which is spatially localised to a "leading-tip", which loses epithelial features. C_LIO_LIIntegrin-2{beta}1-SRC/FAK signalling regulates ductular migration by linking changes in the bio-structural composition of the liver with ductular cells. C_LI

cell biology↗

bioRxiv2024

FAK modulates glioblastoma stem cell energetics via actomyosin contractility: regulation of glycolysis and mitochondrial function

Glycolysis and the TCA cycle are reprogrammed in cancer cells to meet bioenergetic and biosynthetic demands, including by engagement with the extracellular matrix (ECM). We show that focal adhesion kinase (FAK), a mediator of integrin-ECM signalling, is driving cellular energetics in a stem cell model of glioblastoma (GBM). FAK gene deletion inhibits both glycolysis and glutamine oxidation, with increased mitochondrial fragmentation and elevated phosphorylation of the mitochondrial protein MTFR1L at S235. Simultaneously, FAK loss causes a mesenchymal to epithelial transition, enhanced acto-myosin contractility as shown by phospho-myosin light chain (p-MLC S19) and impaired cell migration/invasiveness. Rho-kinase (ROCK) inhibitors suppress p-MLC (S19) and restore glutamine oxidation and elongation of mitochondria. Thus, FAK is a key regulator of both glycolysis and glutamine oxidation mediated by acto-myosin contractility that controls both cell and mitochondrial morphology. Moreover, FAK-dependent cellular energetics are coincident properties with GBM stem cell migration, invasiveness and tumour growth in vivo.

cancer biology↗

bioRxiv2024

A comprehensive pharmacological survey across heterogeneous patient-derived GBM stem cell models

Despite substantial drug discovery investments, the lack of any significant therapeutic advancement in the treatment of glioblastoma (GBM) over the past two decades calls for more innovation in the identification of effective treatments. The inter-and intra-patient heterogeneity of GBM presents significant obstacles to effective clinical progression of novel treatments by contributing to tumour plasticity and rapid drug resistance that confounds contemporary target directed drug discovery strategies. Phenotypic drug screening is ideally suited to heterogeneous diseases, where targeting specific oncogenic drivers have been broadly ineffective. Our hypothesis is that a modern phenotypic led approach using disease relevant patient-derived GBM stem cell systems will be the most productive approach to identifying new therapeutic targets, drug classes and future drug combinations that target the heterogeneity of GBM. In this study we incorporate a panel of patient-derived GBM stem cell lines into an automated and unbiased Cell Painting assay to quantify multiple GBM stem cell phenotypes. By screening several compound libraries at multiple concentrations across a panel of patient-derived GBM stem cells we provide the first comprehensive survey of distinct pharmacological classes and known druggable targets, including all clinically approved drug classes and oncology drug candidates upon multiple GBM stem cell phenotypes linked to cell proliferation, survival and differentiation. Our data set representing, 3866 compounds, 2.2million images and 64000 datapoints is the largest phenotypic screen carried out to date on a panel of patient-derived GBM stem cell models that we are aware of. We seek to identify agents and target classes which engender potent activity across heterogenous GBM genotypes and phenotypes, in this study we further characterize two validated target classes, histone deacetylase inhibitors and cyclin dependent kinases that exert broad and potent effects on the phenotypic and transcriptomic profiles of GBM stem cells. Here we present all validated hit compounds and their target assignments for the GBM community to explore.

cancer biology↗

bioRxiv2024

Drug combinations targeting FAK and MEK overcomes tumour heterogeneity in glioblastoma

Glioblastoma (GBM) is an aggressive brain tumour with limited treatment options and poor prognosis, largely due to its heterogeneity and the involvement of multiple intracellular signalling pathways that contribute to drug resistance. Standard therapies have not significantly improved patient outcomes over the past two decades. While recent advancements in targeted drug combination therapies, such as dabrafenib and trametinib, show promise for certain GBM subgroups, identifying drug combinations effective across the broader GBM population remains a challenge. Integrin-mediated signalling, particularly through Focal Adhesion Kinase (FAK), plays a pivotal role in GBM pathogenesis and invasion, making it a potential therapeutic target [1]. In our study, we utilized a chemogenomic screening approach to identify synergistic drug combinations that target FAK in glioblastoma. We initially employed a CRISPR-engineered GBM model to assess the effects of FAK depletion and discovered that combining FAK inhibitors with MEK inhibitors, particularly trametinib, demonstrated synergistic effects. This potent combination was validated through various 2D & 3D assays, including cell viability/apoptotic assessment, synergistic analysis, cellular imaging, and target engagement assays. The combination also effectively inhibited spheroid growth and invasion across a diverse panel of patient derived GBM stem cells. Molecular mechanisms underlying these effects included suppression of multiple kinase signalling pathways and enhanced apoptosis, elucidated using Reverse Phase Protein Array (RPPA) profiling and western blot validation. In vivo, the combination therapy significantly reduced tumour volume in orthotopic transplantation models. These findings suggest that combining FAK and MEK inhibitors represent a promising therapeutic strategy to overcome the challenges of GBM treatment.

cancer biology↗

bioRxiv2023

A novel ILK/STAT3 pathway controls plasticity in a neural stem cell model of glioblastoma.

Glioblastomas (GBM) are driven by malignant neural stem-like cells that display extensive heterogeneity and phenotypic plasticity, which drives tumour progression and therapeutic resistance. Here we show that the nodal extracellular matrix-cell adhesion protein integrin-linked kinase (ILK; a pseudokinase), is a key determinant of phenotypic plasticity and the mesenchymal-like, invasive cell state in mouse GBM stem cells. We found that a novel ILK-STAT3 signalling pathway is required for plasticity that enables the transition of GBM stem cells to an astrocyte-like state both in vitro and in vivo. GBM cells genetically depleted of ILK become predominantly stabilised in a transcriptionally-defined progenitor-like state that is characterised by lack of response to differentiation cues and constitutive proliferation. Loss of ILK or interference with STAT3 impairs differentiation potential, reducing phenotypic plasticity of tumour cell populations; additionally, ILK loss causes a mesenchymal- to epithelial-like morphological transition and suppression of malignancy-associated features. Our work defines ILK as a central regulator of multiple GBM phenotypes including phenotypic plasticity and mesenchymal state.

cancer biology↗

bioRxiv2022

Quantitative proteomics identifies tumour matrisome signatures in patients with non-small cell lung cancer

The composition and remodelling of the extracellular matrix (ECM) are important factors in the development and progression of cancers, and the ECM is implicated in promoting tumour growth and restricting anti-tumour therapies through multiple mechanisms. The characterisation of differences in ECM composition between normal and diseased tissues may aid in identifying novel diagnostic markers, prognostic indicators and therapeutic targets for drug development. Using tissue from non-small cell lung cancer (NSCLC) patients undergoing curative intent surgery, we characterised quantitative tumour-specific ECM proteome signatures by mass spectrometry, identifying 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue. We defined a collagen hydroxylation functional protein network that is enriched in the lung tumour microenvironment. We validated two novel putative extracellular markers of NSCLC, the collagen cross-linking enzyme peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), for discrimination of malignant and non-malignant lung tissue. These proteins were up-regulated in lung tumour samples, and high PXDN and ADAMTS16 gene expression was associated with shorter survival of lung adenocarcinoma and squamous cell carcinoma patients, respectively. These data reveal tumour matrisome signatures in human NSCLC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=19 SRC="FIGDIR/small/510064v1_ufig1.gif" ALT="Figure 1"> View larger version (8K): org.highwire.dtl.DTLVardef@b34045org.highwire.dtl.DTLVardef@40a5a9org.highwire.dtl.DTLVardef@3c150corg.highwire.dtl.DTLVardef@89c6f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Loss of the adhesion protein Kindlin-1 stimulates tumor clearance via modulation of Tregs

The adhesion protein Kindlin-1 is over-expressed in breast cancer where it has been shown to be associated with metastasis-free survival, however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in a mouse model of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells leads to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs and impairment of their immune-suppressive activities in Kindlin-1 depleted tumors. Similar changes in T cell populations were seen following depletion of Kindlin-1 in the polyomavirus middle T antigen (PyV MT)-driven mouse model of mammary tumorigenesis. Analysis of cytokines secreted from the Met-1 cells identified a significant increase in IL-6 secretion when Kindlin-1 was depleted. Conditioned media from Kindlin-1 depleted cells lead to a decrease in the ability of Tregs to suppress the proliferation of CD8+ T cells, which was dependent on IL-6 and depletion of CD25+ Tregs resulted in a reduction of Met-1 tumor growth in mice. Overall, these data identify a novel function for Kindlin-1 in the regulation of anti-tumor immunity through cytokine regulation of Treg number and function.

cancer biology↗

bioRxiv2021

Mena regulates the LINC complex to control actin-nuclear lamina associations, trans-nuclear membrane signalling and cancer gene expression

Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes (IACs), play key roles in cancer progression and metastasis. We investigated systems-level changes in the integrin adhesome during metastatic progression of a patient-derived cutaneous squamous cell carcinoma (cSCC), and found that the actin regulatory protein Mena is enriched in IACs in metastatic cSCC cells. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope of metastatic cells. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We have uncovered an unexpected novel role for Mena at the nuclear membrane, where it controls the LINC complex, nuclear architecture, chromatin repositioning and cancer gene expression. This is the first description of an adhesion protein regulating gene transcription via direct signalling across the nuclear envelope. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/458340v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@14e2251org.highwire.dtl.DTLVardef@af341corg.highwire.dtl.DTLVardef@8624dforg.highwire.dtl.DTLVardef@deeb50_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Characterisation of a nucleo-adhesome

In addition to central functions in cell adhesion signalling, integrin-associated proteins have wider roles at sites distal to adhesion receptors. In experimentally defined adhesomes, we noticed that there is clear enrichment of proteins that localise to the nucleus, and conversely, we now report that nuclear proteomes contain a class of adhesome components that localise to the nucleus. We here defined a nucleo-adhesome, providing experimental evidence for a remarkable scale of nuclear localisation of adhesion proteins, establishing a framework for interrogating nuclear adhesion protein functions. In adding to nuclear FAKs known roles in regulating transcription, we now show that nuclear FAK regulates expression of many adhesion-related proteins that localise to the nucleus and that nuclear FAK binds to the adhesome component and nuclear protein Hic-5. FAK and Hic-C work together in the nucleus, co-regulating a subset of genes transcriptionally. We describe the first nucleo-adhesome using a squamous cancer cell model, and demonstrate the new principle that there are nuclear adhesion protein subcomplexes that cooperate to control transcription. O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/458428v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1c841ceorg.highwire.dtl.DTLVardef@586a75org.highwire.dtl.DTLVardef@b54439org.highwire.dtl.DTLVardef@921537_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Pathway profiling of a novel SRC inhibitor, AZD0424, in combination with MEK inhibitors.

A more comprehensive understanding of how cells respond to drug intervention, the likely immediate signalling responses and how resistance may develop within different microenvironments allows us anticipate how cells adapt to targeted therapy enabling more informed prediction of rational drug combinations. The non-receptor tyrosine kinase SRC regulates many cellular signalling processes and pharmacological inhibition has long been a target of drug discovery projects for the treatment of cancer. Here we describe the in vitro and in vivo characterisation of the small molecule SRC inhibitor, AZD0424. We show that AZD0424 potently inhibits the phosphorylation of tyrosine-416 of SRC (IC50 [~] 100 nM) in many cancer cell lines; however inhibition of cell viability, via a G1 cell cycle arrest, was observed only in a sub-set of cancer cell lines in the low (on target) micromolar range. We profiled the changes in intracellular pathway signalling in cancer cells following exposure to AZD0424 and other targeted therapies using Reverse Phase Protein Array analysis. We demonstrate that SRC is activated in response to MEK inhibitor (trametinib or AZD6244)-treatment of KRAS mutant colorectal cell lines (HCT116 and DLD1) and that AZD0424 abrogates this. Cell lines treated with trametinib or AZD6244 in combination with AZD0424 revealed reduction of EGFR, FAK and SRC compensatory activation, and, synergistically inhibits cell viability in vitro. In vivo, trametinib-treatment of mice bearing HCT116 tumours increased phosphorylation of SRC on Tyr416, and when combined with AZD0424, inhibition of tumour growth is greater than trametinib alone. We also demonstrate that drug-induced resistance to trametinib is not re-sensitised by AZD0424 treatment in vitro, likely as a result of multiple compensatory signalling mechanisms; however inhibition of SRC remains an effective way to block invasion of trametinib resistant tumour cells. These data imply that inhibiting SRC may offer a useful addition to MEK inhibitor combination strategies.

cancer biology↗

bioRxiv2021

Toll-like receptor 2 orchestrates a potent anti-tumor response in non-small cell lung cancer

Targeting early-stage lung cancer is vital to improve overall survival. We previously identified Toll-like receptor 2 (TLR2) as a regulator of oncogene-induced senescence (OIS) and the senescence-associated secretory phenotype (SASP), both key for tumor suppression. Here, we demonstrate that TLR2 is widely expressed in human lung tumor epithelium where it correlates with improved survival and clinical regression. Using genetically engineered mouse models of lung cancer we have shown that Tlr2 is a tumor suppressor in lung cancer initiation via regulation of proliferation and the SASP. The SASP is integral in the regulation of immune surveillance of premalignant cells, and we observe impaired myeloid derived immune surveillance following Tlr2 loss. Lastly, we show that administration of a synthetic Tlr2 agonist significantly reduces preinvasive lung tumor growth. Our data highlight an unexpected tumor surveillance pathway in early-stage lung cancer with therapeutic potential. Statement of significanceLung cancer is a major cancer of unmet need. This study identifies a novel tumor suppressor mechanism in lung cancer. Not only does this highlight a potential therapeutic target for early-stage disease but also multiple secreted candidate biomarkers that could be exploited to augment lung cancer screening approaches.

cancer biology↗

bioRxiv2020

FAK regulates IL-33 expression via chromatin accessibility and c-Jun

Focal adhesion kinase (FAK) localizes to focal adhesions and is overexpressed in many cancers. FAK can also translocate to the nucleus, where it binds to, and regulates, several transcription factors, including MBD2, p53 and IL-33, to control gene expression by unknown mechanisms. We have used ATAC-seq to reveal that FAK controls chromatin accessibility at a subset of regulated genes. Integration of ATAC-seq and RNA-seq data showed that FAK-dependent chromatin accessibility is linked to differential gene expression, including of the FAK-regulated cytokine and transcriptional regulator interleukin-33 (Il33), which controls anti-tumor immunity. Analysis of the accessibility peaks on the Il33 gene promoter/enhancer regions revealed sequences for several transcription factors, including ETS and AP-1 motifs, and we show that c-Jun, a component of AP-1, regulates Il33 gene expression by binding to its enhancer in a FAK kinase-dependent manner. This work provides the first demonstration that FAK controls transcription via chromatin accessibility, identifying a novel mechanism by which nuclear FAK regulates biologically important gene expression.

genomics↗

bioRxiv2020

A novel transcriptional signalling pathway mediated by the trafficking protein Ambra1 via scaffolding Atf2 complexes

Previously, we reported that Ambra1 is a core component of a cytoplasmic trafficking network, acting as a spatial rheostat to control active Src and FAK levels in addition to its critical roles in autophagy during neurogenesis. Here we identify a novel nuclear scaffolding function for Ambra1 that controls gene expression. Ambra1 binds to nuclear pore proteins, to other adaptor proteins like FAK and Akap8 in the nucleus, as well as to chromatin modifiers and transcriptional regulators such as Brg1, Cdk9 and the cAMP-regulated transcription factor Atf2. Ambra1 contributes to their association with chromatin and we identified genes whose transcription is regulated by Ambra1 complexes, likely via histone modifications and phospho-Atf2-dependent transcription. Therefore, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signalling in the nucleus; in particular, it recruits chromatin modifiers and transcriptional regulators to control expression of genes such as angpt1, tgfb2, tgfb3, itga8 and itgb7 that likely contribute to the role of Ambra1 in cancer cell invasion.

cell biology↗

bioRxiv2019

A Synergistic Anti-Cancer FAK and HDAC Inhibitor Combination Discovered by a Novel Chemical-Genetic High Content Phenotypic Screen

We mutated the Focal Adhesion Kinase (FAK) catalytic domain to inhibit binding of the chaperone, Cdc37 and ATP, mimicking the actions of a FAK kinase inhibitor. We re-expressed mutant and wild-type FAK in squamous cell carcinoma (SCC) cells from which endogenous FAK had been deleted, genetically fixing one axis of a FAK inhibitor combination high-content phenotypic screen to discover drugs that may synergize with FAK inhibitors. HDAC inhibitors represented the major class of compounds that potently induced multi-parametric phenotypic changes when FAK was rendered kinase-defective, or inhibited pharmacologically in SCC cells. Indeed, combined FAK and HDAC inhibitors arrest proliferation and induce apoptosis in a sub-set of cancer cell lines in vitro and efficiently inhibits tumor growth in vivo. Mechanistically, HDAC inhibitors potentiate inhibitor-induced FAK inactivation and reverses FAK-associated nuclear YAP in sensitive cancer cell lines. Here we report the discovery of a new clinically actionable synergistic combination between FAK and HDAC inhibitors.\n\nSignificanceWe describe a chemical-genetic, high-content phenotypic screening approach to discover effective anti-cancer combinations of targeted therapeutics through which we identified a novel, synergistic and clinically actionable, combination of FAK and HDAC inhibitors.

cancer biology↗