An ILK/STAT3 pathway controls glioblastoma stem cell plasticity.

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.

The intrinsic substrate specificity of the human tyrosine kinome.

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.

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

Introduction: 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. Methods: 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. Results: We identified 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue, and 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. Discussion: These data chart extensive remodelling of the lung extracellular niche and reveal tumour matrisome signatures in human NSCLC.

Kindlin-1 regulates IL-6 secretion and modulates the immune environment in breast cancer models.

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.

Mena regulates nesprin-2 to control actin-nuclear lamina associations, trans-nuclear membrane signalling and gene expression.

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.

Toll-like receptor 2 orchestrates a tumor suppressor response in non-small cell lung cancer.

Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target.

A fluorogenic probe for granzyme B enables in-biopsy evaluation and screening of response to anticancer immunotherapies.

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies.

Glioblastoma and the search for non-hypothesis driven combination therapeutics in academia.

Glioblastoma (GBM) remains a cancer of high unmet clinical need. Current standard of care for GBM, consisting of maximal surgical resection, followed by ionisation radiation (IR) plus concomitant and adjuvant temozolomide (TMZ), provides less than 15-month survival benefit. Efforts by conventional drug discovery to improve overall survival have failed to overcome challenges presented by inherent tumor heterogeneity, therapeutic resistance attributed to GBM stem cells, and tumor niches supporting self-renewal. In this review we describe the steps academic researchers are taking to address these limitations in high throughput screening programs to identify novel GBM combinatorial targets. We detail how they are implementing more physiologically relevant phenotypic assays which better recapitulate key areas of disease biology coupled with more focussed libraries of small compounds, such as drug repurposing, target discovery, pharmacologically active and novel, more comprehensive anti-cancer target-annotated compound libraries. Herein, we discuss the rationale for current GBM combination trials and the need for more systematic and transparent strategies for identification, validation and prioritisation of combinations that lead to clinical trials. Finally, we make specific recommendations to the preclinical, small compound screening paradigm that could increase the likelihood of identifying tractable, combinatorial, small molecule inhibitors and better drug targets specific to GBM.

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

A more comprehensive understanding of how cells respond to drug intervention, the likely immediate signalling responses and how resistance may develop within different microenvironments will help inform treatment regimes. The nonreceptor tyrosine kinase SRC regulates many cellular signalling processes, and pharmacological inhibition has long been a target of cancer drug discovery projects. 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-419 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 subset 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 (RPPA) analysis. We demonstrate that SRC is activated in response to treatment of KRAS-mutant colorectal cell lines with MEK inhibitors (trametinib or AZD6244) and that AZD0424 abrogates this. Cell lines treated with trametinib or AZD6244 in combination with AZD0424 had reduced EGFR, FAK and SRC compensatory activation, and cell viability was synergistically inhibited. In vivo, trametinib treatment of mice-bearing HCT116 tumours increased phosphorylation of SRC on Tyr419, and, when combined with AZD0424, inhibition of tumour growth was greater than with 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 SRC inhibition may offer a useful addition to MEK inhibitor combination strategies.

A Conformation Selective Mode of Inhibiting SRC Improves Drug Efficacy and Tolerability.

Despite the approval of several multikinase inhibitors that target SRC and the overwhelming evidence of the role of SRC in the progression and resistance mechanisms of many solid malignancies, inhibition of its kinase activity has thus far failed to improve patient outcomes. Here we report the small molecule eCF506 locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions that prevent phosphorylation and complex formation with its partner FAK. This mechanism of action resulted in highly potent and selective pathway inhibition in culture and in vivo. Treatment with eCF506 resulted in increased antitumor efficacy and tolerability in syngeneic murine cancer models, demonstrating significant therapeutic advantages over existing SRC/ABL inhibitors. Therefore, this mode of inhibiting SRC could lead to improved treatment of SRC-associated disorders. SIGNIFICANCE: Small molecule-mediated inhibition of SRC impairing both catalytic and scaffolding functions confers increased anticancer properties and tolerability compared with other SRC/ABL inhibitors.

Oncogenic BRAF, unrestrained by TGFß-receptor signalling, drives right-sided colonic tumorigenesis.

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFß signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFß-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFß-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.

Glioblastomas acquire myeloid-affiliated transcriptional programs via epigenetic immunoediting to elicit immune evasion.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.

Targeting FAK in anticancer combination therapies.

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.

FAK regulates IL-33 expression by controlling chromatin accessibility at c-Jun motifs.

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.

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer.

BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.

Structural basis of Focal Adhesion Kinase activation on lipid membranes.

Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.

The autophagy protein Ambra1 regulates gene expression by supporting novel transcriptional complexes.

Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.

Novel roles of PRK1 and PRK2 in cilia and cancer biology.

PRK1 and PRK2 are two closely related AGC-family serine/threonine protein kinases. Here we demonstrate novel roles for them at cilia and in cancer biology. In both instances serum withdrawal leads to increased activating PRK1 and PRK2 phosphorylation (pPRK1/pPRK2) and their depletion results in reduced spheroid growth. pPRK1/pPRK2 localise to the transition zone of cilia and their co-depletion results in reduced cilia size, impaired planer polarity and impaired cilia associated signalling. High PRK2 (but not PRK1) expression correlates with poor outcome in patients with basal-like/Triple Negative (TN) Breast Cancer (BC) where there is also higher expression relative to other BC tumour subtypes. In agreement, depletion of PRK1 and PRK2 in mouse TNBC cells, or CRISPR/Cas9 mediated deletion of PRK2 alone, significantly reduces cell proliferation and spheroid growth. Finally proteomic analysis to identify PRK2 binding partners in mouse TNBC cells revealed proteins that are important for both cilia and BC biology. Taken together these data demonstrate novel roles for PRK1 and PRK2 at cilia and in BC biology and in the case of PRK2 in particular, identifies it as a novel TNBC therapeutic target.

A Synergistic Anticancer 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 reexpressed 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. Histone deacetylase (HDAC) inhibitors represented the major class of compounds that potently induced multiparametric phenotypic changes when FAK was rendered kinase-defective or inhibited pharmacologically in SCC cells. Combined FAK and HDAC inhibitors arrest proliferation and induce apoptosis in a subset of cancer cell lines in vitro and efficiently inhibit their growth as tumors in vivo Mechanistically, HDAC inhibitors potentiate inhibitor-induced FAK inactivation and impair 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.