Focal Adhesion Kinase Inhibitor Research Articles

Construction of crizotinib resistant models with CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutations to explore resistance mechanism and treatment strategy

Targeted therapy is an essential treatment for non-small cell lung cancer (NSCLC) that is always associated with the drug resistance. c-ros oncogene 1 (ROS1) gene point mutation is one of the leading factors causing drug resistance. However, the point mutation cell models of crizotinib are challenging to obtain, causing few reports on the drug resistance mechanism and the treatment strategy. We constructed CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant plasmids by fusion PCR technology and transfected them into A549 cells. Western blot and MTT assay proved that the drug-resistant cell lines were successfully transfected. The transwell assay confirmed that the mutant cells' motor abilities were significantly increased compared with the wild-type group. In addition, focal adhesion kinase (FAK) was significantly increased in mutant cells. Moreover, crizotinib resistance occurred in the mutant cells through the activation of FAK / phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) pathway. Next, crizotinib was combined with defactinib, a FAK inhibitor, to further explore its therapeutic effect. The results showed that the combination could significantly inhibit the proliferation, invasion and migration of mutant cells. In conclusion, we proved that CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant NSCLC cells were resistant to crizotinib through the activation of FAK/PI3K/AKT signaling pathway, and inhibiting FAK/PI3K/AKT signaling pathway activation by defactinib could overcome drug resistance in mutant cells.

Identification of defactinib derivatives targeting focal adhesion kinase using ensemble docking, molecular dynamics simulations and binding free energy calculations

Focal adhesion kinase (FAK) belongs to the nonreceptor tyrosine kinases, which selectively phosphorylate tyrosine residues on substrate proteins. FAK is associated with bladder, esophageal, gastric, neck, breast, ovarian and lung cancers. Thus, FAK has been considered as a potential target for tumor treatment. Currently, there are six adenosine triphosphate (ATP)-competitive FAK inhibitors tested in clinical trials but no approved inhibitors targeting FAK. Defactinib (VS-6063) is a second-generation FAK inhibitor with an IC50 of 0.6 nM. The binding model of VS-6063 with FAK may provide a reference model for developing new antitumor FAK-targeting drugs. In this study, the VS-6063/FAK binding model was constructed using ensemble docking and molecular dynamics simulations. Furthermore, the molecular mechanics/generalized Born (GB) surface area (MM/GBSA) method was employed to estimate the binding free energy between VS-6063 and FAK. The key residues involved in VS-6063/FAK binding were also determined using per-residue energy decomposition analysis. Based on the binding model, VS-6063 could be separated into seven regions to enhance its binding affinity with FAK. Meanwhile, 60 novel defactinib-based compounds were designed and verified using ensemble docking. Overall, the present study improves our understanding of the binding mechanism of human FAK with VS-6063 and provides new insights into future drug designs targeting FAK. Communicated by Ramaswamy H. Sarma

The actin cytoskeleton-MRTF/SRF cascade transduces cellular physical niche cues to entrain the circadian clock.

The circadian clock is entrained to daily environmental cues. Integrin-linked signaling via actin cytoskeleton dynamics transduces physical niche cues from the extracellular matrix to myocardin-related transcription factor (MRTF)/serum response factor (SRF)-mediated transcription. The actin cytoskeleton organization and SRF-MRTF activity display diurnal oscillations. By interrogating disparate upstream events in the actin cytoskeleton-MRTF-A/SRF signaling cascade, we show that this pathway transduces extracellular niche cues to modulate circadian clock function. Pharmacological inhibition of MRTF-A/SRF by disrupting actin polymerization or blocking the ROCK kinase induced period lengthening with augmented clock amplitude, and genetic loss of function of Srf or Mrtfa mimicked the effects of treatment with actin-depolymerizing agents. In contrast, actin polymerization shortened circadian clock period and attenuated clock amplitude. Moreover, interfering with the cell-matrix interaction through blockade of integrin, inhibition of focal adhesion kinase (FAK, encoded by Ptk2) or attenuating matrix rigidity reduced the period length while enhancing amplitude. Mechanistically, we identified that the core clock repressors Per2, Nr1d1 and Nfil3 are direct transcriptional targets of MRTF-A/SRF in mediating actin dynamics-induced clock response. Collectively, our findings defined an integrin-actin cytoskeleton-MRTF/SRF pathway in linking clock entrainment with extracellular cues that might facilitate cellular adaptation to the physical niche environment.

FOCAL ADHESION KINASE INHIBITION DECREASES CELL VIABILITY AND INDUCES APOPTOSIS IN SET-2 CELLS

Myeloproliferative Neoplasms (MPN) are diseases characterized by accumulation of myeloid cells. Polycythemia vera (PV), Essential Thrombocythemia (TE) and Primary Myelofibrosis (MF) are MPN BCR-ABL negative and mutations in JAK2, MPL and CALR genes are involved in their pathophysiology. For some PV and PMF patients, Ruxolitinib, a JAK inhibitor, has been used, leading to an improvement of constitutional symptoms. However, the allele burden is not completely reduced, and bone marrow transplantation is still the only curative option. Therefore, more studies are necessary to elucidate the mechanisms involved in these diseases. The Focal Adhesion Kinase (FAK) is a tyrosine kinase which participates in adhesion and migration and also has been described as participating in proliferation, cell survival and apoptosis process mainly by activating SRC kinase. FAK has been described as overexpressed in many types of cancer, including acute myeloid leukemia. Moreover, FAK seems to participate in inflammatory process. The aims of this study was to verify the participation of FAK protein in cell viability and apoptosis of SET-2 cells. With this purpose, SET-2 cells, a JAK2 V617F positive cell line, were treated with the FAK inhibitor PF-562,271 at 5 μM and 10 μM for 72h and cell viability and apoptosis were evaluated. The concentration that inhibits 50% of cell viability (IC50) was also determined. DMSO was used as negative control and Doxorrubicin 10 μM (DOXO) as positive control. SET-2 cells were also treated with Ruxolitinib at 300 nM for 72h. The cell viability and IC50 value for PF-562,271 were evaluated using MTT assay and cleaved PARP and cleaved Caspase 3 expression were detected by Western Blotting to verify apoptosis. P-SRC and SRC detection was performed to verify whether FAK inhibition inhibited SRC phosphorylation. The IC50 determined was 3,215μM. PF-562,271 reduced SET-2 cells viability by 70,2% and 85,4% at 5μM and 10μM, respectively. Ruxolitinib reduced cell viability by 88,4% at 300nM, compared to DMSO. It was possible to observe that cleaved PARP and cleaved CASP3 expression was increased in SET-2 cells treated with PF-562,271 at 5μM and 10μM compared to DMSO and also compared to Ruxolitinib 300nM. Therefore, it was possible to verify that FAK participate in cell viability and apoptosis of SET-2 cells. Considering that many studies have demonstrated that in MPN occurs modifications in bone marrow microenvironment, involving inflammation, resistance to apoptosis and excess of proliferation, FAK may participate in the deregulation of this process, justifying more studies regarding signaling pathways involving FAK in MPN. FAPEMIG (APQ02185/16); CT_INFRA2013/FINEP; ACMMV receives master's scholarship from FAPEMIG.

52 (PB042) - The RAF/MEK clamp VS-6766 shows strong anti-tumor activity across multiple MAPK pathway alterations, with a preferential effect on KRAS G12V

Background: KRAS is the most frequently mutated RAS/RAF family member (mutated in 15% of cancers), followed by BRAF (5%) and NRAS (3%). Several BRAF and MEK inhibitors (MEKi) are approved for BRAF V600E cancers, and the G12C inhibitor sotorasib is approved for KRAS G12C non-small cell lung cancer (NSCLC). However, there is still a need for agents targeting other RAS/RAF mutations (mt), including KRAS G12 V which is mutated in ∼7%, ∼9% and ∼19% of NSCLC, colorectal and pancreatic cancers, respectively. VS-6766 is a unique RAF/MEK clamp that potently inhibits MEK kinase activity and induces a dominant negative RAF-MEK complex preventing phosphorylation of MEK by ARAF, BRAF and CRAF. This unique mechanism allows VS-6766 to block MEK signaling without the compensatory re-activation of MEK that appears to limit the efficacy of MEKi. Material and Methods: In vitro assays were performed to evaluate the anti-proliferative activity of VS-6766 in human tumor cell lines with MAPK pathway alterations and in RAS-less mouse embryonic fibroblasts (MEFs) stably transfected with various KRAS variants. The anti-tumor activity of VS-6766 was assessed in genetically engineered mouse models (GEMM) of KRAS G12 V/Trp53 KO and KRAS G12C/Trp53 KO NSCLC. Results: VS-6766 showed strong anti-proliferative potency across tumor cell lines carrying KRAS, BRAF, CRAF, NRAS or NF1 alterations. Among KRAS mt cell lines, the anti-proliferative potency of VS-6766 differed by KRAS variant, with greatest potency observed with G12 V > G12C > G12D, which is consistent with G12 V signaling through CRAF/MEK/ERK and G12D signaling more through PI3 K/AKT/mTOR. Similarly, in MEFs stably transfected with different KRAS variants, VS-6766 showed best potency with KRAS G12 V and G12C, and least potency with G12D and KRAS wildtype. Accordingly, in the KRAS G12 V NSCLC GEMM model, previously shown to be CRAF-dependent, VS-6766 monotherapy induced stronger tumor regression than in a corresponding G12C NSCLC GEMM model. Strikingly, the combination of VS-6766 with FAK inhibition conferred tumor regressions in 87% (27/31) and 71% (36/51) of all tumors in the KRAS G12 V and KRAS G12C NSCLC models, respectively. Clinically, objective responses to VS-6766 monotherapy occurred mainly in patients with KRAS G12 V with 4 out of the 7 responders bearing KRAS G12 V (Guo, 2020). In KRAS mt NSCLC patients treated with VS-6766 in combination with the FAK inhibitor defactinib, a G12 V preference was also observed with confirmed responses in 2/2 patients with KRAS G12 V and tumor reduction in 4/6 patients with KRAS G12C (Krebs, 2021). Conclusions: These data support the ongoing registration-directed study evaluating VS-6766 ± defactinib for patients with KRAS G12 V NSCLC (NCT04620330), as well as the clinical combinations of VS-6766 with the KRAS G12C inhibitors sotorasib (NCT05074810) or adagrasib (NCT05375994) for KRAS G12C NSCLC. No conflict of interest.

Stromal Reprogramming by FAK Inhibition Overcomes Radiation Resistance to Allow for Immune Priming and Response to Checkpoint Blockade.

Checkpoint immunotherapeutics have not been effective in PDAC, even when combined with RT. One possible explanation is that RT fails to prime T-cell responses in PDAC. Here, we show that FAK inhibition allows RT to prime tumor immunity and unlock responsiveness to checkpoint immunotherapy. This article is highlighted in the In This Issue feature, p. 2711.

Combination Treatment of Retinoic Acid Plus Focal Adhesion Kinase Inhibitor Prevents Tumor Growth and Breast Cancer Cell Metastasis.

All-trans retinoic acid (RA), the primary metabolite of vitamin A, controls the development and homeostasis of organisms and tissues. RA and its natural and synthetic derivatives, both known as retinoids, are promising agents in treating and chemopreventing different neoplasias, including breast cancer (BC). Focal adhesion kinase (FAK) is a crucial regulator of cell migration, and its overexpression is associated with tumor metastatic behavior. Thus, pharmaceutical FAK inhibitors (FAKi) have been developed to counter its action. In this work, we hypothesize that the RA plus FAKi (RA + FAKi) approach could improve the inhibition of tumor progression. By in silico analysis and its subsequent validation by qPCR, we confirmed RARA, SRC, and PTK2 (encoding RARα, Src, and FAK, respectively) overexpression in all breast cells tested. We also showed a different pattern of genes up/down-regulated between RA-resistant and RA-sensitive BC cells. In addition, we demonstrated that both RA-resistant BC cells (MDA-MB-231 and MDA-MB-468) display the same behavior after RA treatment, modulating the expression of genes involved in Src-FAK signaling. Furthermore, we demonstrated that although RA and FAKi administered separately decrease viability, adhesion, and migration in mammary adenocarcinoma LM3 cells, their combination exerts a higher effect. Additionally, we show that both drugs individually, as well as in combination, induce the expression of apoptosis markers such as active-caspase-3 and cleaved-PARP1. We also provided evidence that RA effects are extrapolated to other cancer cells, including T-47D BC and the human cervical carcinoma HeLa cells. In an orthotopic assay of LM3 tumor growth, whereas RA and FAKi administered separately reduced tumor growth, the combined treatment induced a more potent inhibition increasing mice survival. Moreover, in an experimental metastatic assay, RA significantly reduced metastatic lung dissemination of LM3 cells. Overall, these results indicate that RA resistance could reflect deregulation of most RA-target genes, including genes encoding components of the Src-FAK pathway. Our study demonstrates that RA plays an essential role in disrupting BC tumor growth and metastatic dissemination in vitro and in vivo by controlling FAK expression and localization. RA plus FAKi exacerbate these effects, thus suggesting that the sensitivity to RA therapies could be increased with FAKi coadministration in BC tumors.

Activation of Autophagy Induces Monocrotaline-Induced Pulmonary Arterial Hypertension by FOXM1-Mediated FAK Phosphorylation.

It has been shown that activation of autophagy promotes the development of pulmonary arterial hypertension (PAH). Meanwhile, forkhead box M1 (FOXM1) has been found to induce autophagy in several types of cancer. However, it is still unclear whether FOXM1 mediates autophagy activation in PAH, and detailed mechanisms responsible for these processes are indefinite. PAH was induced by a single intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-smooth muscle actin (α-SMA) staining, and Ki67 staining were performed to evaluate the development of PAH. The protein levels of FOXM1, phospho-focal adhesion kinase (p-FAK), FAK, and LC3B were determined by immunoblotting or immunohistochemistry. FOXM1 protein level and FAK activity were significantly increased in MCT-induced PAH rats, this was accompanied with the activation of autophagy. Pharmacological inhibition of FOXM1 or FAK suppressed MCT-induced autophagy activation, decreased RVSP, RVHI and %MT in MCT-induced PAH rats, and inhibited the proliferation of pulmonary arterial smooth muscle cells and pulmonary vessel muscularization in MCT-induced PAH rats. FOXM1 promotes the development of PAH by inducing FAK phosphorylation and subsequent activation of autophagy in MCT-treated rats.

Topological Distribution of Wound Stiffness Modulates Wound-Induced Hair Follicle Neogenesis.

In the large full-thickness mouse skin regeneration model, wound-induced hair neogenesis (WIHN) occurs in the wound center. This implies a spatial regulation of hair regeneration. The role of mechanotransduction during tissue regeneration is poorly understood. Here, we created wounds with equal area but different shapes to understand if perturbing mechanical forces change the area and quantity of de novo hair regeneration. Atomic force microscopy of wound stiffness demonstrated a stiffness gradient across the wound with the wound center softer than the margin. Reducing mechanotransduction signals using FAK or myosin II inhibitors significantly increased WIHN and, conversely, enhancing these signals with an actin stabilizer reduced WIHN. Here, α-SMA was downregulated in FAK inhibitor-treated wounds and lowered wound stiffness. Wound center epithelial cells exhibited a spherical morphology relative to wound margin cells. Differential gene expression analysis of FAK inhibitor-treated wound RNAseq data showed that cytoskeleton-, integrin-, and matrix-associated genes were downregulated, while hair follicular neogenesis, cell proliferation, and cell signaling genes were upregulated. Immunohistochemistry staining showed that FAK inhibition increased pSTAT3 nuclear staining in the regenerative wound center, implying enhanced signaling for hair follicular neogenesis. These findings suggest that controlling wound stiffness modulates tissue regeneration encompassing epithelial competence, tissue patterning, and regeneration during wound healing.

Immunoglobulin superfamily 9 (IGSF9) is trans-activated by p53, inhibits breast cancer metastasis via FAK

Metastasis of breast cancer represents the major reason for its poor prognosis, leading to high mortality. In breast cancer, a tumor suppressor gene TP53 is commonly mutated. TP53 mutation leads to an altered expression of various genes, an event that is associated with aggressive tumor and is a strong independent marker for survival. In this study, we identified a novel p53 target gene, immunoglobulin superfamily 9 (IGSF9). IGSF9 is generally down-regulated in breast cancer tissues. Loss of IGSF9 is associated with frequent metastasis and poor prognosis of breast cancer patients. Wild-type p53, but not R175H mutant, trans-activates the transcription of IGSF9 via binding to its promoter (−137 to −131 bp), inhibits epithelial-mesenchymal transition (EMT), consequently the inhibition of breast cancer cells migration and invasion. IGSF9 interacts with focal adhesion kinase (FAK) and inhibits FAK/AKT signaling activity. PND1186, FAK inhibitor, inhibits breast cancer metastasis induced by IGSF9 knockdown in vitro and in vivo. Taken together, IGSF9 is trans-activated by p53 and inhibits breast cancer metastasis by modulating FAK/AKT signaling pathway. IGSF9 could serve as a prognostic marker and potential therapeutic target for breast cancer.

Connexins orchestrate progression of breast cancer metastasis to the brain by promoting FAK activation.

Brain metastasis is a complication of increasing incidence in patients with breast cancer at advanced disease stage. It is a severe condition characterized by a rapid decline in quality of life and poor prognosis. There is a critical clinical need to develop effective therapies to prevent and treat brain metastases. Here, we describe a unique and robust spontaneous preclinical model of breast cancer metastasis to the brain (4T1-BM2) in mice that has been instrumental in uncovering molecular mechanisms guiding metastatic dissemination and colonization of the brain. Key experimental findings were validated in the additional murine D2A1-BM2 model and in human MDA231-BrM2 model. Gene expression analyses and functional studies, coupled with clinical transcriptomic and histopathological investigations, identified connexins (Cxs) and focal adhesion kinase (FAK) as master molecules orchestrating breast cancer colonization of the brain. Cx31 promoted homotypic tumor cell adhesion, heterotypic tumor-astrocyte interaction, and FAK phosphorylation. FAK signaling prompted NF-κB activation inducing Lamc2 expression and laminin 332 (laminin 5) deposition, α6 integrin-mediated adhesion, and sustained survival and growth within brain parenchyma. In the MDA231-BrM2 model, the human homologous molecules CX43, LAMA4, and α3 integrin were involved. Systemic treatment with FAK inhibitors reduced brain metastasis progression. In conclusion, we report a spontaneous model of breast cancer metastasis to the brain and identified Cx-mediated FAK-NF-κB signaling as a mechanism promoting cell-autonomous and microenvironmentally controlled cell survival for brain colonization. Considering the limited therapeutic options for brain metastatic disease in cancer patients, we propose FAK as a therapeutic candidate to further pursue in the clinic.

Molecular Docking, Molecular Dynamics Simulations, and Free Energy Calculation Insights into the Binding Mechanism between VS-4718 and Focal Adhesion Kinase.

Focal adhesion kinase (FAK) is a 125 kDa nonreceptor tyrosine kinase that plays an important role in many carcinomas. Thus, the targeting of FAK by small molecules is considered to be promising for cancer therapy. Some FAK inhibitors have been reported as potential anticancer drugs and have entered into clinical development; for example, VS-4718 is currently undergoing clinical trials. However, the lack of crystal structural data for the binding of VS-4718 with FAK has hindered the optimization of this anticancer agent. In this work, the VS-4718/FAK interaction model was obtained by molecular docking and molecular dynamics simulations. The binding free energies of VS-4718/FAK were also calculated using the molecular mechanics generalized Born surface area method. It was found that the aminopyrimidine group formed hydrogen bonds with the C502 residue of the hinge loop, while the D564 residue of the T-loop interacted with the amide group. In addition, I428, A452, V484, M499, G505, and L553 residues formed hydrophobic interactions with VS-4718. The obtained results therefore provide an improved understanding of the interaction between human FAK and VS-4718. Based on the obtained binding mechanism, 47 novel compounds were designed to target the adenosine 5′-triphosphate-binding pocket of human FAK, and ensemble docking was performed to assess the effects of these modifications on the inhibitor binding affinity. This work is also expected to provide additional insights into potential future target design strategies based on VS-4718.

IGF-binding proteins secreted by cancer-associated fibroblasts induce context-dependent drug sensitization of lung cancer cells.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment are often linked to drug resistance. Here, we found that coculture with CAFs or culture in CAF-conditioned medium unexpectedly induced drug sensitivity in certain lung cancer cell lines. Gene expression and secretome analyses of CAFs and normal lung-associated fibroblasts (NAFs) revealed differential abundance of insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs), which promoted or inhibited, respectively, signaling by the receptor IGF1R and the kinase FAK. Similar drug sensitization was seen in gefitinib-resistant, EGFR-mutant PC9GR lung cancer cells treated with recombinant IGFBPs. Conversely, drug sensitivity was decreased by recombinant IGFs or conditioned medium from CAFs in which IGFBP5 or IGFBP6 was silenced. Phosphoproteomics and receptor tyrosine kinase (RTK) array analyses indicated that exposure of PC9GR cells to CAF-conditioned medium also inhibited compensatory IGF1R and FAK signaling induced by the EGFR inhibitor osimertinib. Combined small-molecule inhibition of IGF1R and FAK phenocopied the CAF-mediated effects in culture and increased the antitumor effect of osimertinib in mice. Cells that were osimertinib resistant and had MET amplification or showed epithelial-to-mesenchymal transition also displayed residual sensitivity to IGFBPs. Thus, CAFs promote or reduce drug resistance in a context-dependent manner, and deciphering the relationship between the differential content of CAF secretomes and the signaling dependencies of the tumor may reveal effective combination treatment strategies.

Netrin-1 induces the anti-apoptotic and pro-survival effects of B-ALL cells through the Unc5b-MAPK axis

BackgroundB-cell acute lymphoblastic leukemia (B-ALL) comprises over 85% of all acute lymphoblastic leukemia (ALL) cases and is the most common childhood malignancy. Although the 5 year overall survival of patients with B-ALL exceeds 90%, patients with relapsed or refractory B-ALL may suffer from poor prognosis and adverse events. The axon guidance factor netrin-1 has been reported to be involved in the tumorigenesis of many types of cancers. However, the impact of netrin-1 on B-ALL remains unknown.MethodsThe expression level of netrin-1 in peripheral blood samples of children with B-ALL and children without neoplasia was measured by enzyme-linked immunosorbent assay (ELISA) kits. Then, CCK-8 cell proliferation assays and flow cytometric analysis were performed to detect the viability and apoptosis of B-ALL cells (Reh and Sup B15) treated with exogenous recombinant netrin-1 at concentrations of 0, 25, 50, and 100 ng/ml. Furthermore, co-immunoprecipitation(co-IP) was performed to detect the receptor of netrin-1. UNC5B expression interference was induced in B-ALL cells with recombinant lentivirus, and then CCK-8 assays, flow cytometry assays and western blotting assays were performed to verify that netrin-1 might act on B-ALL cells via the receptor Unc5b. Finally, western blotting and kinase inhibitor treatment were applied to detect the downstream signaling pathway.ResultsNetrin-1 expression was increased in B-ALL, and netrin-1 expression was upregulated in patients with high- and intermediate-risk stratification group of patients. Then, we found that netrin-1 induced an anti-apoptotic effect in B-ALL cells, implying that netrin-1 plays an oncogenic role in B-ALL. co-IP results showed that netrin-1 interacted with the receptor Unc5b in B-ALL cells. Interference with UNC5B was performed in B-ALL cells and abolished the antiapoptotic effects of netrin-1. Further western blotting was applied to detect the phosphorylation levels of key molecules in common signaling transduction pathways in B-ALL cells treated with recombinant netrin-1, and the FAK-MAPK signaling pathway was found to be activated. The anti-apoptotic effect of netrin-1 and FAK-MAPK phosphorylation was abrogated by UNC5B interference. FAK inhibitor treatment and ERK inhibitor treatment were applied and verified that the FAK-MAPK pathway may be downstream of Unc5b.ConclusionTaken together, our findings suggested that netrin-1 induced the anti-apoptotic effect of B-ALL cells through activation of the FAK-MAPK signaling pathway by binding to the receptor Unc5b.-oqtjpF4fn996fXAD1ptLBVideo

Tumor-associated macrophage (TAM)-derived CCL22 induces FAK addiction in esophageal squamous cell carcinoma (ESCC)

Tumor cell dependence on activated oncogenes is considered a therapeutic target, but protumorigenic microenvironment-mediated cellular addiction to specific oncogenic signaling molecules remains to be further defined. Here, we showed that tumor-associated macrophages (TAMs) produced an abundance of C-C motif chemokine 22 (CCL22), whose expression in the tumor stroma was positively associated with the level of intratumoral phospho-focal adhesion kinase (pFAK Tyr397), tumor metastasis and reduced patient survival. Functionally, CCL22-stimulated hyperactivation of FAK was correlated with increased malignant progression of cancer cells. CCL22-induced addiction to FAK was demonstrated by the persistent suppression of tumor progression upon FAK-specific inhibition. Mechanistically, we identified that diacylglycerol kinase α (DGKα) acted as a signaling adaptor to link the CCL22 receptor C-C motif chemokine receptor 4 (CCR4) and FAK and promoted CCL22-induced activation of the FAK/AKT pathway. CCL22/CCR4 signaling activated the intracellular Ca2+/phospholipase C-γ1 (PLC-γ1) axis to stimulate the phosphorylation of DGKα at a tyrosine residue (Tyr335) and promoted the translocation of DGKα to the plasma membrane to assemble the DGKα/FAK signalosome, which critically contributed to regulating sensitivity to FAK inhibitors in cancer cells. The identification of TAM-driven intratumoral FAK addiction provides opportunities for utilizing the tumor-promoting microenvironment to achieve striking anticancer effects.

FAK Executes Anti-Senescence via Regulating EZH2 Signaling in Non-Small Cell Lung Cancer Cells.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase overexpressed in various cancer types that plays a critical role in tumor progression. Accumulating evidence suggests that targeting FAK, either alone or in combination with other agents, may serve as an effective therapeutic strategy for numerous cancers. In addition to retarding proliferation, metastasis, and angiogenesis, FAK inhibition triggers cellular senescence in lung cancer cells. However, the detailed mechanism remains enigmatic. In the present study, we found that FAK inhibition not only elicits DNA-damage signaling but also downregulates enhancer of zeste homolog 2 (EZH2) expression. The manipulation of FAK expression influences EZH2 expression and corresponding signaling in vitro. Immunohistochemistry shows that active FAK signaling corresponds with the activation of the EZH2-mediated signaling cascade in lung-cancer-cells-derived tumor tissues. We also found that ectopic EZH2 expression attenuates FAK-inhibition-induced cellular senescence in lung cancer cells. Our results identify EZH2 as a critical downstream effector of the FAK-mediated anti-senescence pathway. Targeting FAK-EZH2 axis-induced cellular senescence may represent a promising therapeutic strategy for restraining tumor growth.

Focal adhesion kinase priming in pancreatic cancer, altering biomechanics to improve chemotherapy.

The dense desmoplastic and fibrotic stroma is a characteristic feature of pancreatic ductal adenocarcinoma (PDAC), regulating disease progression, metastasis and response to treatment. Reciprocal interactions between the tumour and stroma are mediated by bidirectional integrin-mediated signalling, in particular by Focal Adhesion Kinase (FAK). FAK is often hyperactivated and overexpressed in aggressive cancers, promoting stromal remodelling and inducing tissue stiffness which can accelerate cancer cell proliferation, survival and chemoresistance. Therapeutic targeting of the PDAC stroma is an evolving area of interest for pre-clinical and clinical research, where a subtle reshaping of the stromal architecture prior to chemotherapy may prove promising in the clinical management of disease and overall patient survival. Here, we describe how transient stromal manipulation (or ‘priming’) via short-term FAK inhibition, rather than chronic treatment, can render PDAC cells exquisitely vulnerable to subsequent standard-of-care chemotherapy. We assess how our priming publication fits with the recent literature and describe in this perspective how this could impact future cancer treatment. This highlights the significance of treatment timing and warrants further consideration of anti-fibrotic therapies in the clinical management of PDAC and other fibrotic diseases.

Decreased FAK activity and focal adhesion dynamics impair proper neurite formation of medium spiny neurons in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin (HTT) [55]. While the final pathological consequence of HD is the neuronal cell death in the striatum region of the brain, it is still unclear how mutant HTT (mHTT) causes synaptic dysfunctions at the early stage and during the progression of HD. Here, we discovered that the basal activity of focal adhesion kinase (FAK) is severely reduced in a striatal HD cell line, a mouse model of HD, and the human post-mortem brains of HD patients. In addition, we observed with a FRET-based FAK biosensor [59] that neurotransmitter-induced FAK activation is decreased in HD striatal neurons. Total internal reflection fluorescence (TIRF) imaging revealed that the reduced FAK activity causes the impairment of focal adhesion (FA) dynamics, which further leads to the defect in filopodial dynamics causing the abnormally increased number of immature neurites in HD striatal neurons. Therefore, our results suggest that the decreased FAK and FA dynamics in HD impair the proper formation of neurites, which is crucial for normal synaptic functions [52]. We further investigated the molecular mechanism of FAK inhibition in HD and surprisingly discovered that mHTT strongly associates with phosphatidylinositol 4,5-biphosphate, altering its normal distribution at the plasma membrane, which is crucial for FAK activation [14, 60]. Therefore, our results provide a novel molecular mechanism of FAK inhibition in HD along with its pathological mechanism for synaptic dysfunctions during the progression of HD.

Design, synthesis and activity evaluation of isopropylsulfonyl-substituted 2,4- diarylaminopyrimidine derivatives as FAK inhibitors for the potential treatment of pancreatic cancer

A series of isopropylsulfonyl-substituted 2,4-diarylaminopyrimidine derivatives were designed and synthesized as FAK inhibitors to evaluate their biological activity against pancreatic cancer. One of the most promising compound, 9h, effectively interfered with FAK-mediated phosphorylation and suppressed the proliferation of human pancreatic cancer AsPC-1 cells with half maximal inhibitory concentration (IC50) values of 0.1165 nM and 0.1596 μM, respectively. In addition, 9h also exhibited relatively low toxicity against immortalized normal human liver L-02 cells, indicating its low hepatotoxicity at an equivalent dosage. Furthermore, the elucidation of the mechanism of action revealed that compound 9h effectively inhibited cell migration and inhibited the proliferation of AsPC-1 by blocking the cell cycle at the G2/M phase. Moreover, 9h also demonstrated efficacy in inhibiting tumor growth in a murine AsPC-1 cell xenograft model at the dosage of 10 mg/kg without losing noticeable body weight. All these findings provide important clues for the identification of potent FAK inhibitors.

Ras-Related Protein Rab-32 and Thrombospondin 1 Confer Resistance to the EGFR Tyrosine Kinase Inhibitor Osimertinib by Activating Focal Adhesion Kinase in Non-Small Cell Lung Cancer.

Simple SummaryOsimertinib is a third-generation EGFR tyrosine kinase inhibitor and the standard of care therapy for non-small cell lung cancer patients harboring EGFR-activating mutations. However, even for patients treated with osimertinib, resistance inevitably occurs leading to disease progression. Here, we utilized two osimertinib-resistant cell lines and investigated their RNA profiles. We found that Ras-related protein Rab-32 (RAB32) and thrombospondin 1 (THBS1) were upregulated and associated with resistance in osimertinib-resistant cells as well as in liquid biopsies from patients with disease progression following osimertinib treatment. Moreover, we found RAB32 and THBS1 to be mechanistically linked to activation of the focal adhesion pathway where combination of osimertinib with a FAK inhibitor resulted in a synergistic suppression of viability of osimertinib-resistant cells. Our findings propose a potential therapeutic strategy for overcoming acquired resistance to osimertinib in non-small cell lung cancer.Treatment with the tyrosine kinase inhibitor (TKI) osimertinib is the standard of care for non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR). Osimertinib is also used in T790M-positive NSCLC that may occur de novo or be acquired following first-line treatment with other EGFR TKIs (i.e., gefitinib, erlotinib, afatinib, or dacomitinib). However, patients treated with osimertinib have a high risk of developing resistance to the treatment. A substantial fraction of the mechanisms for resistance is unknown and may involve RNA and/or protein alterations. In this study, we investigated the full transcriptome of parental and osimertinib-resistant cell lines, revealing 131 differentially expressed genes. Knockdown screening of the genes upregulated in resistant cell lines uncovered eight genes to partly confer resistance to osimertinib. Among them, we detected the expression of Ras-related protein Rab-32 (RAB32) and thrombospondin 1 (THBS1) in plasmas sampled at baseline and at disease progression from EGFR-positive NSCLC patients treated with osimertinib. Both genes were upregulated in progression samples. Moreover, we found that knockdown of RAB32 and THBS1 reduced the expression of phosphorylated focal adhesion kinase (FAK). Combination of osimertinib with a FAK inhibitor resulted in synergistic toxicity in osimertinib-resistant cells, suggesting a potential therapeutic drug combination for overcoming resistance to osimertinib in NSCLC patients.