Background/Objectives: Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options and poor prognosis, largely owing to its heterogeneity and the involvement of multiple intracellular signaling pathways that contribute to drug resistance. While recent advancements in targeted drug combination therapies, such as dabrafenib and trametinib, show promise for certain GBM subgroups, identifying effective drug combinations across the broader GBM population remains a challenge. Integrin-mediated signaling, particularly through Focal Adhesion Kinase (FAK), plays a pivotal role in GBM pathogenesis and invasion, making it a potential therapeutic target and component of future drug combination strategies. Methods: In this 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 subsequently discovered that combining FAK inhibitors such as VS4718 with MEK inhibitors, particularly trametinib, demonstrated synergistic effects. This potent combination was validated using various 2D and 3D assays, including cell viability/apoptosis assessment, synergistic analysis, cellular imaging, and target engagement assays. This combination also effectively inhibited spheroid growth and invasion across a diverse panel of patient-derived GBM stem cells. Molecular mechanisms underlying these effects include suppression of multiple kinase signaling pathways and enhanced apoptosis, elucidated using Reverse-Phase Protein Array (RPPA) profiling and Western blot validation. Result: In vivo, combination therapy significantly reduced the tumor volume in orthotopic transplantation models. Conclusions: These findings suggest that the combination of FAK and MEK inhibitors represents a promising therapeutic strategy to overcome the challenges of GBM treatment.

Glioblastoma (GBM) is a heterogeneous and aggressive brain tumour that is invariably fatal despite maximal treatment. Genetic or transcriptomic 'biomarkers' could be used to stratify patients for treatments, however, pairing biomarkers with appropriate therapeutic 'targets' is challenging. Consequently, therapeutics have not yet been optimised for specific GBM patient subsets. Here we integrate genome-wide CRISPR/Cas9 knockout screening and genetic-annotation data for 60 distinct patient-derived, IDHwildtype, adult GBM cell lines, quantifying the essentiality of 15,145 genes. We describe a novel method using Targeted Learning, to estimate the effect size of GBM-relevant biomarkers on context-dependent gene essentiality (GBM-CoDE). We derive multiple target-biomarker pair hypotheses, which we release in an accessible platform to accelerate translation to biomarker-stratified clinical trials. Two of these (WWTR1 with EGFR mutation/amplification, and VRK1 with VRK2 expression suppression) have been validated in GBM, implying that our additional novel findings may be valid. Our method is readily translatable to other cancers of unmet need. ### Competing Interest Statement MTF, AE, MF, PMB, AK, SVB, CS: None. NC: is a scientific advisory board member and shareholder in Amplia Therapeutics Ltd (Melbourne, Australia); is and founder, shareholder, and management consultant of PhenoTherapeutics Ltd (Edinburgh, UK); is Director of Ther-IP Ltd (Edinburgh); and is Director of Edinburgh Innovations Ltd.

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-α2-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-α2-SRC/FAK signalling axis; thereby connecting epithelial regeneration directly to the changing fibrotic environment in chronic ductular disease. Highlights ### Competing Interest Statement The authors have declared no competing interest. European Commission, , EP/Y028546/1 Dutch Cancer Foundation, , COCOON KWF-14364 Chief Scientist Office, , EPD/22/12 Carnegie Trust for the Universities of Scotland, , RIG012508 Cancer Research UK, https://ror.org/054225q67, C52499/A27948 Medical Research Council, https://ror.org/03x94j517, MR/Z506199/1

Glycolysis and the tricarboxylic acid cycle (TCA) cycle are reprogrammed in cancer cells to meet bioenergetic and biosynthetic demands, including by engagement with the extracellular matrix (ECM). However, the mechanisms by which the ECM engagement reprograms core energy metabolism is still unknown. We showed that the canonical cell-ECM adhesion protein focal adhesion kinase (FAK, also known as PTK2) and, specifically, its kinase activity, is driving cellular energetics. Using a mouse stem cell model of glioblastoma, we showed that deletion of the FAK gene simultaneously inhibits glycolysis and glutamine oxidation, increases mitochondrial fragmentation, elevates phosphorylation of the mitochondrial protein MTFR1L at serine residue 235 (S235) and triggers a mesenchymal-to-epithelial transition. These metabolic and structural changes arise through altered contractility of actomyosin, as shown by myosin light chain type II (MYL2, also known as MLC2) phosphorylated (p) at S19. This process can be reversed by Rho-kinase (ROCK) inhibitors revealing mechanotransduction pathway control of both mitochondrial dynamics and glutamine oxidation. FAK-dependent metabolic programming is associated with regulation of cell migration, invasive capacity and tumour growth in vivo. Our work describes a previously unrecognised FAK-ROCK axis that couples mechanical cues to the rewiring of energy metabolism, linking cell shape, mitochondrial function and malignant behaviour.

The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis: Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high, risk of developing colorectal cancer.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

Glioblastoma (GBM) is driven by malignant neural stem-like cells that display extensive heterogeneity and phenotypic plasticity, which drive tumor progression and therapeutic resistance. Here, we show that the extracellular matrix-cell adhesion protein integrin-linked kinase (ILK) stimulates phenotypic plasticity and mesenchymal-like, invasive behavior in a murine GBM stem cell model. ILK is required for the interconversion of GBM stem cells between malignancy-associated glial-like states, and its loss produces cells that are unresponsive to multiple cell state transition cues. We further show that an ILK/STAT3 signaling pathway controls the plasticity that enables transition of GBM stem cells to an astrocyte-like state in vitro and in vivo. Finally, we find that ILK expression correlates with expression of STAT3-regulated proteins and protein signatures describing astrocyte-like and mesenchymal states in patient tumors. This work identifies ILK as a pivotal regulator of multiple malignancy-associated GBM phenotypes, including phenotypic plasticity and mesenchymal state.

Abstract BACKGROUND Owing to the lack of therapeutic advancement in the treatment of glioblastoma (GBM) over the past two decades, Cancer Research UK has designated malignant brain tumours a ‘strategic priority⋯ as a cancer of unmet need’. The inter- and intra-molecular heterogeneity of glioblastoma presents significant obstacles to effective clinical progression of novel treatments. Phenotypic drug screening is ideally suited to heterogeneous diseases, where targeting specific oncogenic drivers (such as EGFR) are broadly ineffective. Phenotypic hits can then be used to systematically identify synergistic combinations which overcome variable therapeutic response observed across multi-cell panels. Synergistic drug combinations between approved drugs (or drug candidates in early clinical trials) provides a repurposing context that could accelerate the bench to bedside model. Our preliminary objective was to incorporate a panel of patient derived GBM stem cell lines into an unbiased ‘cell painting’ assay to quantify multiple GBM stem cell phenotypes and create an updated pharmacological survey by screening these cell lines across several drug libraries containing commercially available, FDA approved and/or Phase I/II/III compounds. MATERIAL AND METHODS We profiled patient derived GBM stem cells against several thousand annotated compounds covering multiple doses, using our established image-based high throughput screening protocols. Multiplexed ‘cell painting’ dyes were used to quantify in detail the cellular response to each drug via CellProfiler image analysis software. Data processing, QC, dimensionality reduction and hit thresholding was carried out primarily using Stratominer-HCA platform. Putative hits based on phenotypic distance and potency were selected for validation and further characterisation. RESULTS Predominant drug sensitivities include the highly potent but expected pan-cytotoxic effects of compounds that target microtubules, the proteasome, topoisomerases, DNA/RNA synthesis and histone deacetylases. Beyond these, approximately 50 compounds have been selected for further studies including mechanism-of-action and pathway profiling, dose-ratio matrix combination testing in 2D and 3D GBM models, neuronal cytotoxicity and in vivo proof-of-concept studies via intracranial implantation. CONCLUSION Our preliminary results serve as a foundation for a systematic approach to identify novel drug combinations including combinations that could be used concurrently with standard treatment protocols for GBM.

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 off. We seek to identify agents and targets 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. ### Competing Interest Statement S.P. is a co-founder, shareholder and Chief Scientific Officer of Trogenix Ltd. N.O.C. is a co-founder, shareholder and management consultant for PhenoTherapeutics Ltd. and N.O.C and M.C.F have held advisory positions and are shareholders in Amplia Therapeutics Ltd. N.O.C. had patents pertaining to discovery of the SRC/YES1 inhibitor, eCF506/NXP900 ( EP3298015B1, JP6684831B2, US10294227B2, CN107849050B, and CA3021550A1) licensed to Nuvectis Pharma.

Supplementary video for Fig. 2A (Src527F panels) from Identification of Src-Specific Phosphorylation Site on Focal Adhesion Kinase: Dissection of the Role of Src SH2 and Catalytic Functions and Their Consequences for Tumor Cell Behavior

Supplementary Figure S4 - PDF file 1182K, Measuring the organization and ultrastructure of distinct ECM manipulations in complex 3D-organotpyic matrices

FAK kinase-dependent dysregulation of specific FAK-binding proteins.

Supplementary Figure and Movie Legends- PDF file 97K, Legend to Supplemental Movies 1-5 and Supplemental Figures S1-S5

Supplementary Movie 4 - WMV file 1818K, Representative maximum projection SHG image of fibrillar collagen I used for GLCM analysis, control

FAK combination phenotypic screen data.

Supplementary Figures 1-6 from Quantitative In vivo Imaging of the Effects of Inhibiting Integrin Signaling via Src and FAK on Cancer Cell Movement: Effects on E-cadherin Dynamics

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.

Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes, play key roles in fundamental cellular processes, including the sensing and transduction of mechanical cues. Here, we investigate systems-level changes in the integrin adhesome in patient-derived cutaneous squamous cell carcinoma cells and identify the actin regulatory protein Mena as a key node in the adhesion complex network. 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. 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 uncover an unexpected role for Mena at the nuclear membrane, where it controls nuclear architecture, chromatin repositioning and gene expression. Our findings identify an adhesion protein that regulates gene transcription via direct signalling across the nuclear envelope.

Proteomic analysis of FAK protein complexes and turnover.

Analysis of HDAC and FAK inhibition across a panel of 35 human cell lines.