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Focal adhesion kinase (FAK) is both a non-receptor tyrosine kinase and an adaptor protein that primarily regulates adhesion signalling and cell migration, but FAK can also promote cell survival in response to stress. FAK is commonly overexpressed in cancer and is considered a high-value druggable target, with multiple FAK inhibitors currently in development. Evidence suggests that in the clinical setting, FAK targeting will be most effective in combination with other agents so as to reverse failure of chemotherapies or targeted therapies and enhance efficacy of immune-based treatments of solid tumours. Here, we discuss the recent preclinical evidence that implicates FAK in anticancer therapeutic resistance, leading to the view that FAK inhibitors will have their greatest utility as combination therapies in selected patient populations.Focal adhesion kinase (FAK) is overexpressed in many cancers and is involved in a multitude of oncogenic processes and resistance mechanisms. This Review discusses the rationale and preclinical evidence for FAK-based combination therapies and strategies for future development.

Key Points Focal adhesion kinase (FAK) is a scaffold and kinase protein that binds to itself and cellular partners through its four-point-one, ezrin, radixin, moesin (FERM) domain. Recent data on FAK structure and the sequence of activation have been deduced, largely from crystallographic and biosensor studies. These data have revealed that the activation sequence involves the release of FERM–kinase interactions, subsequent conformational changes, protein binding and key phosphorylation events at specific subcellular locations. The FERM domain of FAK binds to cellular protein and lipid partners, and these are variously involved in catalytic activation and/or recruitment of FAK into macromolecular complexes in cells — predominantly at cortical adhesion structures, but also in the nucleus. Among cytoplasmic FAK FERM domain binding partners recognized thus far are: phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ; generated by the action of phosphatidylinositol-4-phosphate 5-kinase type-1γ (PtdIns(4)P5KIγ)), which binds to the FERM domain and may trigger the activation cascade, perhaps along with molecular clustering, actin-related protein 3 (ARP3), receptor for activated kinase C 1 (RACK1), epidermal growth factor receptor (EGFR), c-Met, ezrin, epithelial and endothelial tyrosine kinase (ETK; also known as BMX), possibly insulin receptor substrate 1 (IRS1), JNK/SAPK-associated protein 1 (JSAP1; also known as JIP3) , and the β subunits of integrin cell–extracellular matrix receptors. FAK is required for optimal signalling from the tyrosine kinase receptors EGFR and c-Met, and modulates cortical actin and nascent adhesions by binding to the ARP2/3 complex and the molecular scaffold RACK1. The FERM domain is implicated in these important FAK functions. The FERM domains of FAK and PYK2 also bind to the tumour suppressor protein p53 and its regulator MDM2 in the nucleus on receipt of stress signals such as cell detachment. p53 degradation and survival can be promoted through these FERM-mediated interactions. The findings above imply that the FAK FERM domain may control and coordinate cellular responses at the cell cortex and in the nucleus, potentially linking regulation of cell migration with the life and death choices that cells have to make. We speculate that the FERM domain may act as a 'sensor' and 'shuttle' of information and events occurring in and between distinct subcellular locations. From data in the literature on other FERM domain-containing proteins (FDCPs) and from sequence gazing, we speculate that a 'cortex to nuclear' information shuttle may be a more general function of FERM domains in biology, which could be exploited for therapy. Focal adhesion kinase (FAK) is a scaffold and tyrosine kinase protein that binds to itself and cellular partners through its four-point-one, ezrin, radixin, moesin (FERM) domain. Recent structural work reveals how regulatory proteins activate FAK by binding to its FERM domain, enabling it to coordinate diverse cellular responses. Focal adhesion kinase (FAK) is a scaffold and tyrosine kinase protein that binds to itself and cellular partners through its four-point-one, ezrin, radixin, moesin (FERM) domain. Recent structural work reveals that regulatory protein partners convert auto-inhibited FAK into its active state by binding to its FERM domain. Further, the identity of FAK FERM domain-interacting proteins yields clues as to how FAK coordinates diverse cellular responses, including cell adhesion, polarization, migration, survival and death, and suggests that FERM domains might mediate information transfer between the cell cortex and nucleus. Importantly, the FAK FERM domain might act as a paradigm for the actions of other FERM domain-containing proteins.

TP53 mutation occurs in 50-75% of human pancreatic ductal adenocarcinomas (PDAC) following an initiating activating mutation in the KRAS gene. These p53 mutations frequently result in expression of a stable protein, p53R¹⁷⁵H, rather than complete loss of protein expression. In this study we elucidate the functions of mutant p53 (Trp53R¹⁷²H), compared to knockout p53 (Trp53fl), in a mouse model of PDAC. First we find that although KrasG¹²D is one of the major oncogenic drivers of PDAC, most KrasG¹²D-expressing pancreatic cells are selectively lost from the tissue, and those that remain form premalignant lesions. Loss, or mutation, of Trp53 allows retention of the KrasG¹²D-expressing cells and drives rapid progression of these premalignant lesions to PDAC. This progression is consistent with failed growth arrest and/or senescence of premalignant lesions, since a mutant of p53, p53R¹⁷²P, which can still induce p21 and cell cycle arrest, is resistant to PDAC formation. Second, we find that despite similar kinetics of primary tumor formation, mutant p53R¹⁷²H, as compared with genetic loss of p53, specifically promotes metastasis. Moreover, only mutant p53R¹⁷²H-expressing tumor cells exhibit invasive activity in an in vitro assay. Importantly, in human PDAC, p53 accumulation significantly correlates with lymph node metastasis. In summary, by using 'knock-in' mutations of Trp53 we have identified two critical acquired functions of a stably expressed mutant form of p53 that drive PDAC; first, an escape from KrasG¹²D-induced senescence/growth arrest and second, the promotion of metastasis.

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. Cells transmit mechanical force to the nucleus via the cytoskeleton. Here, the authors reveal a role for the actin regulator Mena in force transmission at the nuclear envelope, where it regulates nuclear architecture, chromatin organization and gene expression.

Key Points Focal-adhesion kinase (FAK) is a non-receptor tyrosine kinase that provides signalling and scaffolding functions at sites of integrin adhesion. It is involved in the regulation of turnover of these adhesion sites, a process that is crucial in the control of cell migration. FAK is linked to the protection of cells from anoikis (suspension-induced cell death). This anti-apoptotic function is potentially linked to the ability of FAK to sequester receptor-interacting protein (RIP) from the death-receptor machinery. Substantial circumstantial evidence has accumulated linking overexpression of FAK to a wide range of human epithelial cancers. Levels of FAK expression correlate with the invasive potential of tumours. Using a mouse model of skin carcinogenesis, a direct requirement for FAK has now been shown during tumour progression in vivo . These observations are probably linked to the ability of FAK to protect cells from apoptosis. Inhibition of FAK function might provide an attractive anticancer target, however it is not yet clear what the most effective strategy would be. Potential intervention routes are inhibition of the kinase activity of FAK or disruption of crucial protein–protein interactions. Focal-adhesion kinase (FAK) is an important mediator of growth-factor signalling, cell proliferation, cell survival and cell migration. Given that the development of malignancy is often associated with perturbations in these processes, it is not surprising that FAK activity is altered in cancer cells. Mouse models have shown that FAK is involved in tumour formation and progression, and other studies showing that FAK expression is increased in human tumours make FAK a potentially important new therapeutic target.

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 define 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. Adding to nuclear FAK’s 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-5 work together in the nucleus, co-regulating a subset of genes transcriptionally. We demonstrate the principle that there are subcomplexes of nuclear adhesion proteins that cooperate to control transcription. Cell adhesion proteins have been described at sites away from the cell surface, including in the nucleus. Here, the authors report the scale of nuclear localisation of adhesion proteins, establishing a nucleo-adhesome and showing that nuclear adhesion proteins can cooperate to control transcription.

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.

The adhesion protein Kindlin-1 is over-expressed in breast cancer where it is associated with metastasis-free survival; however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in mouse models of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells led to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs. 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 spontaneous mammary tumorigenesis. There was a significant increase in IL-6 secretion from Met-1 cells when Kindlin-1 was depleted and conditioned media from Kindlin-1-depleted cells led to a decrease in the ability of Tregs to suppress the proliferation of CD8 + T cells, which was dependent on IL-6. In addition, deletion of tumor-derived IL-6 in the Kindlin-1-depleted tumors reversed the reduction of tumor-infiltrating Tregs. Overall, these data identify a novel function for Kindlin-1 in regulation of anti-tumor immunity, and that Kindlin-1 dependent cytokine secretion can impact the tumor immune environment.

Elevated expression and activation of the focal adhesion kinase (FAK) occurs in a large proportion of human breast cancers. Although several studies have implicated FAK as an important signaling molecule in cell culture systems, evidence supporting a role for FAK in mammary tumor progression is lacking. To directly assess the role of FAK in this process, we have used the Cre/loxP recombination system to disrupt FAK function in the mammary epithelium of a transgenic model of breast cancer. Using this approach, we demonstrate that FAK expression is required for the transition of premalignant hyperplasias to carcinomas and their subsequent metastases. This dramatic block in tumor progression was further correlated with impaired mammary epithelial proliferation. These observations provide direct evidence that FAK plays a critical role in mammary tumor progression.

The MacBlue transgenic mouse uses the Csf1r promoter and first intron to drive expression of gal4-VP16, which in turn drives a cointegrated gal4-responsive UAS-ECFP cassette. The Csf1r promoter region used contains a deletion of a 150 bp conserved region covering trophoblast and osteoclast-specific transcription start sites. In this study, we examined expression of the transgene in embryos and adult mice. In embryos, ECFP was expressed in the large majority of macrophages derived from the yolk sac, and as the liver became a major site of monocytopoiesis. In adults, ECFP was detected at high levels in both Ly6C+ and Ly6C- monocytes and distinguished them from Ly6C+, F4/80+, CSF1R+ immature myeloid cells in peripheral blood. ECFP was also detected in the large majority of microglia and Langerhans cells. However, expression was lost from the majority of tissue macrophages, including Kupffer cells in the liver and F4/80+ macrophages of the lung, kidney, spleen and intestine. The small numbers of positive cells isolated from the liver resembled blood monocytes. In the gut, ECFP+ cells were identified primarily as classical dendritic cells or blood monocytes in disaggregated cell preparations. Immunohistochemistry showed large numbers of ECFP+ cells in the Peyer's patch and isolated lymphoid follicles. The MacBlue transgene was used to investigate the effect of treatment with CSF1-Fc, a form of the growth factor with longer half-life and efficacy. CSF1-Fc massively expanded both the immature myeloid cell (ECFP-) and Ly6C+ monocyte populations, but had a smaller effect on Ly6C- monocytes. There were proportional increases in ECFP+ cells detected in lung and liver, consistent with monocyte infiltration, but no generation of ECFP+ Kupffer cells. In the gut, there was selective infiltration of large numbers of cells into the lamina propria and Peyer's patches. We discuss the use of the MacBlue transgene as a marker of monocyte/macrophage/dendritic cell differentiation.