Focal Adhesion Kinase Inhibitor Research Articles

Schisandra chinensis Bee Pollen Extract Inhibits Proliferation and Migration of Hepatocellular Carcinoma HepG2 Cells via Ferroptosis-, Wnt-, and Focal Adhesion-Signaling Pathways.

Bee pollen possesses favorable anticancer activities. As a medicinal plant source, Schisandra chinensis bee pollen (SCBP) possesses potential pharmacological properties, such as reducing cisplatin-induced liver injury, but its anti-liver cancer effect is still rarely reported. This paper aims to investigate the effect and mechanism of SCBP extract (SCBPE) on hepatocellular carcinoma HepG2 cells. The effect of SCBPE on cell proliferation and migration of HepG2 cells was evaluated based on MTT assay, morphology observation, or scratching assay. Furthermore, tandem mass tag-based quantitative proteomics was used to study the effect mechanisms. The mRNA expression levels of identified proteins were verified by RT-qPCR. Tandem mass tag-based quantitative proteomics showed that 61 differentially expressed proteins were obtained in the SCBPE group compared with the negative-control group: 18 significantly downregulated and 43 significantly upregulated proteins. Bioinformatic analysis showed the significantly enriched KEGG pathways were predominantly ferroptosis-, Wnt-, and hepatocellular carcinoma-signaling ones. Protein-protein interaction network analysis and RT-qPCR validation revealed SCBPE also downregulated the focal adhesion-signaling pathway, which is abrogated by PF-562271, a well-known inhibitor of FAK. This study confirmed SCBPE suppressed the cell proliferation and migration of hepatocellular carcinoma HepG2 cells, mainly through modulation of ferroptosis-, Wnt-, hepatocellular carcinoma-, and focal adhesion-signaling pathways, providing scientific data supporting adjuvant treatment of hepatocellular carcinoma using SCBP.

SPP1 promotes tumor progression in esophageal carcinoma by activating focal adhesion pathway.

Recurrence and metastasis are the major obstacles affecting the therapeutic efficacy and clinical outcomes for patients with esophageal carcinoma (ESCA). Secreted phosphoprotein 1 (SPP1) is considered as a hub gene in ESCA and is negatively associated with disease-free survival (DFS) in ESCA. However, the exact roles and underlying mechanisms remain elusive. This study aims to examine the roles of SPP1 on ESCA, and elucidate the potential mechanisms. Bioinformatics were used to analyze the expression of SPP1 in ESCA tissues, and its relations with clinicopathological characteristics and clinical prognosis in patients with ESCA based on The Cancer Genome Atlas (TCGA) dataset. Loss-of-function was conducted to examine the roles of SPP1 on malignant behaviors of ESCA cells by cell counting kit-8 (CCK8), plate clone, wound healing, and transwell assays. Gene set enrichment analysis (GSEA) was conducted to screen the pathways associated with SPP1 in ESCA. Then, the enriched pathway and the underlying mechanism were elucidated by western blotting, cell adhesion, and cell spreading assays. Lastly, Y15 [a specific inhibitor of focal adhesion kinase (FAK)] was used to examine its potential to inhibit tumor growth in ESCA cells. SPP1 was upregulated in ESCA tissues compared to the adjacent nontumorous tissues, which was closely associated with clinical stage, lymph node metastasis, histological subtype, and p53 mutation. A high expression of SPP1 indicated a poor clinical prognosis in patients with ESCA. The knockdown of SPP1 inhibited cell proliferative, migratory, and invasive capacities in ESCA cells. GSEA indicated that the focal adhesion pathway was closely related with SPP1 in ESCA. Further studies confirmed that the knockdown of SPP1 suppressed cell adhesion ability and reduced the expression of p-FAK and p-Erk in ESCA cells. In addition, Y15 inhibited FAK autophosphorylation and dramatically inhibited cell proliferation, migration, and invasion in ESCA cells. SPP1 promotes tumor progression in ESCA by activating FAK/Erk pathway, and FAK is a potential therapeutic target to overcome tumor recurrence and metastasis of ESCA.

Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A.

Osteocytes are the primary mechano-sensitive cell type in bone. Mechanical loading is sensed across the dendritic projections of osteocytes leading to transient reductions in focal adhesion kinase (FAK) activity. Knowledge regarding the signaling pathways downstream of FAK in osteocytes is incomplete. We performed tyrosine-focused phospho-proteomic profiling in osteocyte-like Ocy454 cells to identify FAK substrates. Gsα, parathyroid hormone receptor (PTH1R), and phosphodiesterase 8A (PDE8A), all proteins associated with cAMP signaling, were found as potential FAK targets based on their reduced tyrosine phosphorylation in both FAK- deficient or FAK inhibitor treated cells. Real time monitoring of intracellular cAMP levels revealed that FAK pharmacologic inhibition or gene deletion increased basal and GPCR ligand-stimulated cAMP levels and downstream phosphorylation of protein kinase A substrates. Mutating FAK phospho-acceptor sites in Gsα and PTH1R had no effect on PTH- or FAK inhibitor-stimulated cAMP levels. Since FAK inhibitor treatment augmented cAMP levels even in the presence of forskolin, we focused on potential FAK substrates downstream of cAMP generation. Indeed, PDE8A inhibition mimicked FAK inhibition at the level of increased cAMP, PKA activity, and expression of cAMP-regulated target genes. In vitro kinase assay showed that PDE8A is directly phosphorylated by FAK while immunoprecipitation assays revealed intracellular association between FAK and PDE8A. Thus, FAK inhibition in osteocytes acts synergistically with signals that activate adenylate cyclase to increase intracellular cAMP. Mechanically-regulated FAK can modulate intracellular cAMP levels via effects on PDE8A. These data suggest a novel signal transduction mechanism that mediates crosstalk between mechanical and cAMP-linked hormonal signaling in osteocytes.

Adipocyte-specific FAK deletion promotes pancreatic β-cell apoptosis via adipose inflammatory response to exacerbate diabetes mellitus.

White adipose tissue (WAT) has a key role in maintaining energy balance throughout the body, and their dysfunction take part in the regulation of diabetes mellitus. However, the internal regulatory mechanisms underlying are still unknown. We generated adipocyte-specific FAK KO (FAK-AKO) mice and investigated their phenotype. The cascade of adipocyte, macrophage in adipocyte tissues, and pancreatic β-cells were proposed in FAK-AKO mice and validated by cell line studies using 3T3-L1, Raw264.7 and Min6. The FAK-AKO mice exhibited glucose intolerance, reduced adipose tissue mass and increased apoptosis, lipolysis and inflammatory response in adipose tissue. We further demonstrate that adipocyte FAK deletion increases β cell apoptosis and inflammatory infiltrates into islets, which is potentiated if mice were treated with STZ. In the STZ-induced diabetes model, FAK AKO mice exhibit less serum insulin content and pancreatic β cell area. Moreover, serum pro-inflammatory factors increased and insulin levels decreased after glucose stimulation in FAK AKO mice. In a parallel vitro experiment, knockdown or inhibition of FAK during differentiation also increased apoptosis, lipolysis and inflammatory in 3T3-L1 adipocytes, whereas the opposite was observed upon overexpression of FAK. Moreover, coculturing LPS-treated RAW264.7 macrophages with knockdown FAK of 3T3-L1 adipocytes increased macrophage pro-inflammatory response. Furthermore, conditioned medium from above stimulated Min6 cells apoptosis (with or without STZ), whereas the opposite was observed upon overexpression of FAK. Mechanistically, FAK protein interact with TRAF6 in adipocytes and knockdown or inhibition of FAK activated TRAF6/TAK1/NF-κB signaling, which exacerbates inflammation of adipocytes themselves. Adipocyte FAK deletion promotes both adipocyte apoptosis and adipose tissue inflammation. Pro-inflammatory factors released by the FAK-null adipose tissue further trigger apoptosis in pancreatic islets induced by the administration of STZ, thereby exacerbating the diabetes mellitus. This study reveals a link between FAK-mediated adipose inflammation and diabetes mellitus, a mechanism that has not been previously recognized.

Injectable hybrid hydrogels enable enhanced combination chemotherapy and roused anti-tumor immunity in the synergistic treatment of pancreatic ductal adenocarcinoma

Chemotherapy and immunotherapy have shown no significant outcome for unresectable pancreatic ductal adenocarcinoma (PDAC). Multi-drug combination therapy has become a consensus in clinical trials to explore how to arouse anti-tumor immunity and meanwhile overcome the poorly tumoricidal effect and the stroma barrier that greatly hinders drug penetration. To address this challenge, a comprehensive strategy is proposed to fully utilize both the ferroptotic vulnerability of PDAC to potently irritate anti-tumor immunity and the desmoplasia-associated focal adhesion kinase (FAK) to wholly improve the immunosuppressive microenvironment via sustained release of drugs in an injectable hydrogel for increasing drug penetration in tumor location and averting systematic toxicity. The injectable hydrogel ED-M@CS/MC is hybridized with micelles loaded with erastin that exclusively induces ferroptosis and a FAK inhibitor defactinib for inhibiting stroma formation, and achieves sustained release of the drugs for up to 12 days. With only a single intratumoral injection, the combination treatment with erastin and defactinib produces further anti-tumor performance both in xenograft and KrasG12D-engineered primary PDAC mice and synergistically promotes the infiltration of CD8+ cytotoxic T cells and the reduction of type II macrophages. The findings may provide a novel promising strategy for the clinical treatment of PDAC.

Plexin D1 negatively regulates macrophage-derived foam cell migration via the focal adhesion kinase/Paxillin pathway

BackgroundMacrophage-derived foam cell formation is a hallmark of atherosclerosis and is retained during plaque formation. Strategies to inhibit the accumulation of these cells hold promise as viable options for treating atherosclerosis. Plexin D1 (PLXND1), a member of the Plexin family, has elevated expression in atherosclerotic plaques and correlates with cell migration; however, its role in macrophages remains unclear. We hypothesize that the guidance receptor PLXND1 negatively regulating macrophage mobility to promote the progression of atherosclerosis. MethodsWe utilized a mouse model of atherosclerosis based on a high-fat diet and an ox-LDL- induced foam cell model to assess PLXND1 levels and their impact on cell migration. Through western blotting, Transwell assays, and immunofluorescence staining, we explored the potential mechanism by which PLXND1 mediates foam cell motility in atherosclerosis. ResultsOur study identifies a critical role for PLXND1 in atherosclerosis plaques and in a low-migration capacity foam cell model induced by ox-LDL. In the aortic sinus plaques of ApoE−/− mice, immunofluorescence staining revealed significant upregulation of PLXND1 and Sema3E, with colocalization in macrophages. In macrophages treated with ox-LDL, increased expression of PLXND1 led to reduced pseudopodia formation and decreased migratory capacity. PLXND1 is involved in regulating macrophage migration by modulating the phosphorylation levels of FAK/Paxillin and downstream CDC42/PAK. Additionally, FAK inhibitors counteract the ox-LDL-induced migration suppression by modulating the phosphorylation states of FAK, Paxillin and their downstream effectors CDC42 and PAK. ConclusionOur findings indicate that PLXND1 plays a role in regulating macrophage migration by modulating the phosphorylation levels of FAK/Paxillin and downstream CDC42/PAK to promoting atherosclerosis.

A reanalysis and integration of transcriptomics and proteomics datasets unveil novel drug targets for Mekong schistosomiasis

Schistosomiasis, caused by Schistosoma trematodes, is a significant global health concern, particularly affecting millions in Africa and Southeast Asia. Despite efforts to combat it, the rise of praziquantel (PZQ) resistance underscores the need for new treatment options. Protein kinases (PKs) are vital in cellular signaling and offer potential as drug targets. This study focused on focal adhesion kinase (FAK) as a candidate for anti-schistosomal therapy. Transcriptomic and proteomic analyses of adult S. mekongi worms identified FAK as a promising target due to its upregulation and essential role in cellular processes. Molecular docking simulations assessed the binding energy of FAK inhibitors to Schistosoma FAK versus human FAK. FAK inhibitor 14 and PF-03814735 exhibited strong binding to Schistosoma FAK with minimal binding for human FAK. In vitro assays confirmed significant anti-parasitic activity against S. mekongi, S. mansoni, and S. japonicum, comparable to PZQ, with low toxicity in human cells, indicating potential safety. These findings highlight FAK as a promising target for novel anti-schistosomal therapies. However, further research, including in vivo studies, is necessary to validate efficacy and safety before clinical use. This study offers a hopeful strategy to combat schistosomiasis and reduce its global impact.

Safety and efficacy of ifebemtinib (IN10018) combined with D-1553 in non-small-cell lung cancer (NSCLC) with KRAS G12C mutation: Results from a phase Ib/II study.

8605 Background: KRAS G12C mutation is present in about 12-14% of NSCLC population. Ifebemtinib (IN10018) is a highly potent and selective oral inhibitor of focal adhesion kinase (FAK). D-1553 (garsorasib) is a novel oral and potent KRAS G12C inhibitor. Preclinical data showed that ifebemtinib in combination with KRAS G12C inhibitors had synergistic anti-cancer effect in multiple KRAS G12C mutant cancer models. This study is to evaluate the safety and antitumor activity of ifebemtinib combined with D-1553 in solid tumors with KRAS G12C mutation. Here we report the preliminary data from phase II part of front-line NSCLCs with KRAS G12C mutation. Methods: Locally advanced or metastatic NSCLC subjects without prior systemic anti-cancer therapy were enrolled. Subjects were required to have KRAS G12C mutation in tumor tissues. All subjects were assigned to the recommended phase II dose (RP2D) of ifebemtinib (100mg qd) + D-1553 (600mg bid). Results: As of 31 January 2024, 33 front-line NSCLC subjects with KRAS G12C mutation were enrolled and received the combination therapy. Median age was 65yrs (range 58, 83), 93.9% were male, and 81.8% had stage IV metastatic disease. Median study follow-up (FU) was 2.2 months (range 1.1, 9.2). The safety profile of the combination therapy is comparable to each single agent without additive toxicities. No ifebemtinib- or D-1553-related death was reported. Four subjects (12.1%) had ifebemtinib-related SAEs (diarrhea, enteritis, oedema peripheral and proteinuria) as assessed by investigators, and all SAEs were also considered D-1553 related. Six subjects (18.1%) had ≥ Grade 3 ifebemtinib-related AEs as assessed by investigators, and all were also D-1553 related AEs. The majority of ifebemtinib-related AEs were CTCAE Grade 1 or 2. There was no event leading to study treatment discontinuation. We have observed continuing tumor shrinkage in almost all subjects who had at least 2 tumor assessments. Among 16 subjects with ≥ 3 months FU, preliminary efficacy found that 14 had best overall response of partial response (PR) including 12 confirmed PRs and 2 unconfirmed but still in treatment, 1 had stable disease (SD), and 1 had progressive disease (PD). The overall response rate (ORR) was 87.5% (95%CI: 56.6, 96.2), and disease control rate (DCR) was 93.8% (95%CI: 63.6, 98.5). The median duration of response (DOR), median progression-free survival (PFS) and overall survival (OS) have not been reached. Conclusions: The combination of ifebemtinib and D-1553, as a dual-oral regimen, showed promising antitumor activity and manageable safety profile in KRAS G12C mutant front-line NSCLC population. Long term follow-up is needed to assess the potential of such novel combo. Clinical trial information: NCT06166836 ; NCT05379946 .

Phase 1b/2a of narmafotinib (AMP945) in combination with gemcitabine and nab-paclitaxel in first-line patients with advanced pancreatic cancer (ACCENT trial): Interim analysis.

e16337 Background: Narmafotinib (AMP945) is a highly potent and selective, orally bioavailable inhibitor of Focal Adhesion Kinase (FAK). FAK is a non-receptor tyrosine kinase over-expressed in pancreatic cancer, where it contributes to cell survival, proliferation, migration and chemoresistance and thus plays a key role in tumor growth, FAK also contributes to multiple mechanisms underlying fibrosis and immunosuppression in the tumor microenvironment. Narmafotinib has successfully completed a Phase 1 study in healthy volunteers where the drug was safe and well tolerated with pharmacokinetics consistent with once-a-day dosing. A Phase 1b/2a trial of narmafotinib, in combination with gemcitabine and Abraxane, in advanced pancreatic cancer patients – the ACCENT trial – is currently underway in Australia and South Korea. Methods: The ACCENT trial (NCT05355298) is a Phase 1b/2a, open label study of the pharmacokinetics, safety and tolerability, and efficacy of narmafotinibin combination with gemcitabine and Abraxane (nab-paclitaxel) standard of care as first-line therapy in patients with advanced pancreatic cancer. The trial is a single-arm open label study conducted in two stages. In Part A, patients were enrolled in a 3+3 design to determine the narmafotinib recommended Phase 2 dose (RP2D), with narmafotinibdose-escalation (100, 200 and 400 mg). Safety and tolerability were assessed according to incidence and severity of adverse events. Narmafotinibplasma PK samples were collected on Days -8, -7, 1, 3, 8, and 10 of Run-In/Cycle 1. Imaging was conducted every 2 months. Part B of the trial is a Simon’s two stage design, with the primary objectives of assessing safety, tolerability, and efficacy of the combination using RECIST v1.1. Results: Preliminary analysis across all doses (14 patients) showed narmafotinib to be generally safe and well-tolerated and showed promising signs of efficacy. All patients who completed their first 28-day cycle of treatment elected to stay on narmafotinib and 9/14 patients have remained on trial for 5 months or more. There have been 6 confirmed PRs (4 in 400 mg cohort; 2 in 200 mg cohort) as best response. A single DLT was reported: uncontrolled grade 3 nausea (400 mg cohort). Fatigue, grade 3 or below related to narmafotinib occurred in more than one participant. The PK for narmafotinib is dose-proportional across the dose range tested, and the chemotherapy regimen used in this study did not impact narmafotinib PK. Safety, tolerability and efficacy data continues to be collected and will be presented. Conclusions: Based on this promising Phase 1b data, 400 mg of narmafotinib has been selected for the RP2D and Part B of the trial is now underway and recruiting patients in Australia and South Korea. The trial will enrol up to 50 patients as part of the Phase 2 expansion cohort. Clinical trial information: NCT05355298 .

Effect of targeting FAK by ifebemtinib and immunogenic cell death of Kras G12C/G12D inhibition on the efficacy of PD1 blockade.

e15102 Background: Focal adhesion kinase (FAK) is one major component of focal adhesion complex and is important in regulating cancer cell survival. Previously, we found that a clinical-stage FAK inhibitor, ifebemtinib (aka IN10018) can overcome drug resistance of Kras G12C inhibitors by down regulating the FAK-YAP signaling. Combination of Kras G12Ci and FAKi has demonstrated encouraging efficacy in NSCLC. Here, we reported that targeting Kras G12D by selective Kras G12Di can also be enhanced by ifebemtinib. Further, Kras G12C inhibition plus PD1 blockade has demonstrated clinical benefits in PD-L1 high expression patient population, but ≈30% of patients experienced early disease progression; this might be attributed to loss-of-function of LKB1 and others. Immunogenic cell death (ICD) of Kras inhibition is enhanced by ifebemtinib, ICD reportedly enhances anti-PD1 activity. Thus, Kras inhibition and PD1 blockade plus ifebemtinib may be an ideal combination for patients with Kras G12D. Methods: Cell killing and apoptosis assays were performed to test the combination of Kras G12D inhibition and ifebemtinib. FACs analysis evaluated exposure levels vs. ICD biomarkers. The mice inoculated with CRC GP2D and PDAC PC-07-0013 were used for evaluating the combinational effects of Kras G12Di and ifebemtinib. The tumor bearing mice with Kras G12D mutant/LKB1 loss, NSCLC KPL, and CRC CT26 with knock-in Kras G12C were used for testing the effects of Kras inhibition, ifebemtinib, and PD1 blockade. Results: Ifebemtinib enhances the drug effects of Kras G12D inhibition in vitro and in vivo. The dual regimen of ifebemtinib and Kras inhibition outperformed either monotherapy with significant ICD induction in vitro (calreticulin/GRP94 exposure). The triple regimen of Kras inhibition, FAK inhibition and PD1 blockade exhibited stronger anti-cancer effects compared to the other related therapies, especially in a Kras G12D mutant, LKB1 loss animal model which is intrinsically resistant to PD1 blockade/Kras G12D inhibition. Conclusions: We discovered that targeting FAK by ifebemtinib enhances the anti-cancer effects of Kras G12D inhibition. It promotes ICD by targeting Kras G12D or G12C, further primes the dual regimen of Kras inhibition and ifebemtinib towards a response to anti PD1 therapy. The preclinical results warrant a further evaluation in the clinic.

Avutometinib/defactinib and gemcitabine/nab-paclitaxel combination in first-line metastatic pancreatic ductal adenocarcinoma: Initial safety and efficacy of phase 1b/2 study (RAMP 205).

4140 Background: More than 90% of pancreatic ductal adenocarcinomas (PDAC) harbor an activating KRASmutation .Avutometinib (avuto) is an oral RAF/MEK clamp. Defactinib (defact) is an oral selective FAK inhibitor. Avuto/defact combined with gemcitabine/paclitaxel have shown synergistic antitumor activity in preclinical PDAC models. RAMP 205 (NCT05669482) is a Phase 1b/2 study assessing the safety and efficacy of avuto/defact + gemcitabine/nab-paclitaxel (GnP) in first-line metastatic PDAC. Methods: Eligible patients (pts) were enrolled and treated in 3+3 cohorts with escalating doses of avuto orally (PO) twice weekly (BIW) and defact PO twice daily (BID), both 3 out of 4 weeks, in combination with GnP dosed intravenously (IV) on a D1, D8, D15 or D1, D15 (modified) 4wk schedule (Table 1). Key eligibility criteria include histologically confirmed newly diagnosed metastatic PDAC with measurable disease, ECOG performance status (PS) ≤1, adequate organ function, and no prior treatment. Results: At the 24Jan2024 data cutoff, 18 pts were enrolled, 44% were male, 65 y median age, and 61% ECOG PS 1. Pts were enrolled to DL-1 (n=3), DL1 (n=6), DL1a (n=6), and DL2a (n=3). No dose-limiting toxicities (DLTs) were reported and the majority of treatment related adverse events (TRAEs) were mild to moderate. The most frequently reported TRAEs of any grade in the safety population (N=18) were nausea (50%), alopecia (44%), fatigue (39%), maculo-papular rash (33%), and peripheral edema (28%). The most common grade ≥3 TRAEs included alanine aminotransferase increase (17%), neutropenia (17%), peripheral edema (11%), anemia (11%), and sepsis (11%). Four patients experienced serious AEs (SAEs). Grade ≥3 treatment emergent SAEs included pulmonary embolism (n=2; unrelated), sepsis (n=2), febrile neutropenia (n=1; did not meet DLT criteria), and neutropenia (n=1). One patient discontinued treatment due to TRAE (febrile neutropenia and blood bilirubin increased). All efficacy evaluable pts (n=8) experienced a reduction in target lesions (100% disease control rate) and partial responses were observed in 6/8 (75%), 3 being confirmed at data cutoff. All efficacy evaluable pts with an elevated CA19-9 at baseline (n=7) had a ≥60% CA19-9 reduction from baseline. Conclusions: Avuto/defact and GnP are combinable and show notable preliminary efficacy in first-line metastatic PDAC based on RECIST v.1.1 criteria and CA19-9 levels. No DLTs were reported across the 4 dosing cohorts/schedules and safety signals were consistent with previous clinical data. Updated safety and efficacy will be reported. Clinical trial information: NCT05669482 . [Table: see text]

Updated study results of novel FAK/ALK/ROS1 inhibitor APG-2449 in patients (pts) with non-small-cell lung cancer (NSCLC) resistant to second-generation ALK inhibitors.

3124 Background: ALK inhibitors increase FAK pathway gene expression in ALK+ NSCLC cell lines, with the highest induced expression in drug-tolerant persister cells. This suggests that FAK pathway activation is involved in ALK inhibitor-induced early adaptive tolerance in ALK+ NSCLC. Investigational APG-2449 is a novel, orally active FAK inhibitor and a third-generation (3G) ALK/ROS1 tyrosine kinase inhibitor (TKI) with potent activity in preclinical models. APG-2449 is well tolerated at the recommended phase 2 dose (RP2D) and showed preliminary efficacy in pts resistant to 2G ALK TKIs. Here, we provide further safety and efficacy results and demonstrate associations between this therapy and FAK signaling pathways. Methods: After the RP2D was determined as 1,200 mg daily (QD), pts with NSCLC were enrolled into 2 dose expansion cohorts. Cohort 1 included pts who were resistant to 2G ALK TKIs. Cohort 2 included those who were ALK or ROS1 TKI naïve. Results: As of December 9, 2023, 144 pts (median [range] age, 53 [21-78] years; 53.5% female) with NSCLC, mesothelioma, or ovarian cancer were treated with APG-2449 at doses of 150 to 1,500 mg. A total of 129 (89.6%) pts had treatment-related adverse events (TRAEs), the most frequent of which were elevated serum creatinine (49.3%), ALT (42.4%), and AST (36.1%); nausea (28.5%); vomiting (23.6%); diarrhea (22.9%); and decreased leukocyte count (22.2%). In all, 20 (13.9%) TRAEs were grade ≥ 3. Of pts with TKI-naïve NSCLC (n = 36) treated at the RP2D, the overall response (ORR) and disease control rates (DCR = CR + PR + SD) were 68.2% (15/22) and 90.1% (20/22) in ROS1 TKI-naïve pts; and 78.6% (11/14) and 100% (14/14) in ALK TKI-naïve pts. Median progression-free survival (mPFS) in ALK TKI-naïve pts was not reached. Of 22 pts with NSCLC resistant to 2G ALK inhibitors and without targetable bypass gene mutations (e.g., KRAS G12C, BRAF V600E), 9 had PRs (9/22; 40.9%) and the mPFS during APG-2449 treatment at RP2D was 11.86 months. Nine of 12 pts in the RP2D group achieved intracranial PR, resulting in an intracranial ORR of 75.0%. Before APG-2449 administration, 17 tumor tissue samples were collected from pts with NSCLC resistant to 2G ALK inhibitors (in the RP2D group), and IHC was used to assess protein levels of phosphorylated FAK (pFAK). The PFS of pts treated with APG-2449 correlated with pFAK levels in baseline tumor tissue, and pts with higher pFAK levels were more likely to benefit from APG-2449 treatment. Conclusions: APG-2449 demonstrated preliminary efficacy in pts with NSCLC whose disease was TKI naïve and resistant to 2G ALK inhibitors, especially in brain metastases. High pFAK expression levels in baseline tumor tissue correlated with improved APG-2449 treatment responses in pts with NSCLC resistant to 2G ALK inhibitors. Internal study identifier: APG2449XC101. Clinical trial information: NCT03917043 .

Epithelial-mesenchymal transition status is a remarkable biomarker for the combination treatment with avutometinib and defactinib in KRAS-mutated non-small cell lung cancer.

Recent therapeutic strategies for KRAS-mutated cancers that inhibit the MAPK pathway have attracted considerable attention. The RAF/MEK clamp avutometinib (VS-6766/CH5126766/RO5126766/CKI27) is promising for patients with KRAS-mutated cancers. Although avutometinib monotherapy has shown clinical activity in patients with KRAS-mutated cancers, effective combination strategies will be important to develop. Using a phosphorylation kinase array kit, we explored the feedback mechanism of avutometinib in KRAS-mutated NSCLC cells, and investigated the efficacy of combining avutometinib with inhibitors of the feedback signal using in vitro and in vivo experiments. Moreover, we searched for a biomarker for the efficacy of combination therapy through an in vitro study and analysis using the The Cancer Genome Atlas Programme dataset. Focal adhesion kinase (FAK) phosphorylation/activation was increased after avutometinib treatment and synergy between avutometinib and FAK inhibitor, defactinib, was observed in KRAS-mutated NSCLC cells with an epithelial rather than mesenchymal phenotype. Combination therapy with avutometinib and defactinib induced apoptosis with upregulation of Bim in cancer cells with an epithelial phenotype in an in vitro and in vivo study. These results demonstrate that the epithelial-mesenchymal transition status may be a promising biomarker for the efficacy of combination therapy with avutometinib and defactinib in KRAS-mutated NSCLC.

CircMTO1/miR-30c-5p/SOCS3 axis alleviates oral submucous fibrosis through inhibiting fibroblast-myofibroblast transition.

circRNAs have been shown to participate in diverse diseases; however, their role in oral submucous fibrosis (OSF), a potentially malignant disorder, remains obscure. Our preliminary experiments detected the expression of circRNA mitochondrial translation optimization 1 homologue (circMTO1) in OSF tissues (n = 20) and normal mucosa tissues (n = 20) collected from Hunan Xiangya Stomatological Hospital, and a significant decrease of circMTO1 expression was showed in OSF tissues. Therefore, we further explored circMTO1 expression in OSF. Target molecule expression was detected using RT-qPCR and western blotting. The migration and invasion of buccal mucosal fibroblasts (BMFs) were assessed using wound healing and Transwell assays. The interaction between miR-30c-5p, circMTO1, and SOCS3 was evaluated using dual luciferase, RNA immunoprecipitation (RIP), and RNA pull-down assays. The colocalisation of circMTO1 and miR-30c-5p was observed using fluorescence in situ hybridisation (FISH). circMTO1 and SOCS3 expression decreased, whereas miR-30c-5p expression increased in patients with OSF and arecoline-stimulated BMFs. Overexpression of circMTO1 effectively restrained the fibroblast-myofibroblast transition (FMT), as evidenced by the increase in expression of Coll I, α-SMA, Vimentin, and the weakened migration and invasion functions in BMFs. Mechanistic studies have shown that circMTO1 suppresses FMT by enhancing SOCS3 expression by sponging miR-30c-5p and subsequently inactivating the FAK/PI3K/AKT pathway. FMT induced by SOCS3 silencing was reversed by the FAK inhibitor TAE226 or the PI3K inhibitor LY294002. circMTO1/miR-30c-5p/SOCS3 axis regulates FMT in arecoline-treated BMFs via the FAK/PI3K/AKT pathway. Expanding the sample size and in vivo validation could further elucidate their potential as therapeutic targets for OSF.

IGFBP2 induces podocyte apoptosis promoted by mitochondrial damage via integrin α5/FAK in diabetic kidney disease.

Podocyte apoptosis or loss is the pivotal pathological characteristic of diabetic kidney disease (DKD). Insulin-like growth factor-binding protein 2 (IGFBP2) have a proinflammatory and proapoptotic effect on diseases. Previous studies have shown that serum IGFBP2 level significantly increased in DKD patients, but the precise mechanisms remain unclear. Here, we found that IGFBP2 levels obviously increased under a diabetic state and high glucose stimuli. Deficiency of IGFBP2 attenuated the urine protein, renal pathological injury and glomeruli hypertrophy of DKD mice induced by STZ, and knockdown or deletion of IGFBP2 alleviated podocytes apoptosis induced by high concentration of glucose or in DKD mouse. Furthermore, IGFBP2 facilitated apoptosis, which was characterized by increase in inflammation and oxidative stress, by binding with integrin α5 (ITGA5) of podocytes, and then activating the phosphorylation of focal adhesion kinase (FAK)-mediated mitochondrial injury, including membrane potential decreasing, ROS production increasing. Moreover, ITGA5 knockdown or FAK inhibition attenuated the podocyte apoptosis caused by high glucose or IGFBP2 overexpression. Taken together, these findings unveiled the insight mechanism that IGFBP2 increased podocyte apoptosis by mitochondrial injury via ITGA5/FAK phosphorylation pathway in DKD progression, and provided the potential therapeutic strategies for diabetic kidney disease.

Platelet Spreading and Clot Retraction are Regulated by two Distinct αIIbβ3 Outside-in Signaling Pathways.

Bi-directional signaling through platelet integrin αIIbβ3 is essential in hemostasis and thrombosis. In quiescent platelets αIIbβ3 is in a low-affinity ligand binding state. However, upon platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the αIIbβ3 induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of PI3K, PLC, PKC, and FAK strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading. Whereas inhibition of Rac1, ROCK, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases (SFKs) are required for platelet spreading and FAK activation but suppresses clot retraction since their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through αIIbβ3 outside-in signaling and should not be used interchangeably as readout for αIIbβ3 outside-in signaling assessment. Significance Statement Current anti-platelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel anti-platelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of αIIbβ3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling is poorly understood. Our work included in this manuscript delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.

FBXO32 Stimulates Protein Synthesis to Drive Pancreatic Cancer Progression and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death worldwide, primarily due to its rapid progression. The current treatment options for PDAC are limited, and a better understanding of the underlying mechanisms responsible for PDAC progression is required to identify improved therapeutic strategies. In this study, we identified FBXO32 as an oncogenic driver in PDAC. FBXO32 was aberrantly upregulated in PDAC, and high FBXO32 expression was significantly associated with an unfavorable prognosis in patients with PDAC. FRG1 deficiency promoted FBXO32 upregulation in PDAC. FBXO32 promoted cell migration and invasion in vitro and tumor growth and metastasis in vivo. Mechanistically, FBXO32 directly interacted with eEF1A1 and promoted its polyubiquitination at the K273 site, leading to enhanced activity of eEF1A1 and increased protein synthesis in PDAC cells. Moreover, FBXO32-catalyzed eEF1A1 ubiquitination boosted the translation of ITGB5 mRNA and activated focal adhesion kinase (FAK) signaling, thereby facilitating focal adhesion assembly and driving PDAC progression. Importantly, interfering with the FBXO32-eEF1A1 axis or pharmaceutical inhibition of FAK by defactinib, an FDA-approved FAK inhibitor, substantially inhibited PDAC growth and metastasis driven by aberrantly activated FBXO32-eEF1A1 signaling. Overall, this study uncovers a mechanism by which PDAC cells rely on FBXO32-mediated eEF1A1 activation to drive progression and metastasis. FBXO32 may serve as a promising biomarker for selecting eligible patients with PDAC for treatment with defactinib. Significance: FBXO32 upregulation in pancreatic cancer induced by FRG1 deficiency increases eEF1A1 activity to promote ITGB5 translation and stimulate FAK signaling, driving cancer progression and sensitizing tumors to the FAK inhibitor defactinib.

Computational insights into allosteric inhibition of focal adhesion kinase: A combined pharmacophore modeling and molecular dynamics approach

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that modulates integrin and growth factor signaling pathways and is implicated in cancer cell migration, proliferation, and survival. Over the past decade various, FAK kinase, FERM, and FAT domain inhibitors have been reported and a few kinase domain inhibitors are under clinical consideration. However, few of them were identified as multikinase inhibitors. In kinase drug design selectivity is always a point of concern, to improve selectivity allosteric inhibitor development is the best choice. The current research utilized a pharmacophore modeling (PM) approach to identify novel allosteric inhibitors of FAK. The all-available allosteric inhibitor bound 3D structures with PDB ids 4EBV, 4EBW, and 4I4F were utilized for the pharmacophore modeling. The validated PM models were utilized to map a database of 770,550 compounds prepared from ZINC, EXIMED, SPECS, ASINEX, and InterBioScreen, aiming to identify potential allosteric inhibitors. The obtained compounds from screening step were forwarded to molecular docking (MD) for the prediction of binding orientation inside the allosteric site and the results were evaluated with the known FAK allosteric inhibitor (REF). Finally, 14 FAK-inhibitor complexes were selected from the docking study and were studied under molecular dynamics simulations (MDS) for 500 ns. The complexes were ranked according to binding free energy (BFE) and those demonstrated higher affinity for allosteric site of FAK than REF inhibitors were selected. The selected complexes were further analyzed for intermolecular interactions and finally, three potential allosteric inhibitor candidates for the inhibition of FAK protein were identified. We believe that identified scaffolds may help in drug development against FAK as an anticancer agent.

Preclinical in vitro and in vivo activity of the RAF/MEK clamp avutometinib in combination with FAK inhibition in uterine carcinosarcomas

ObjectivesUterine carcinosarcomas (UCS) are rare, biologically aggressive tumors. Since UCS may harbor mutations in RAS/MAPK pathway genes we evaluated the preclinical in vitro and in vivo efficacy of the RAF/MEK clamp avutometinib in combination with the focal adhesion kinase (FAK) inhibitors defactinib or VS-4718 against multiple primary UCS cell lines and xenografts. MethodsWhole-exome-sequencing (WES) was used to evaluate the genetic landscape of 5 primary UCS cell lines. The in vitro activity of avutometinib ± FAK inhibitor was evaluated using cell viability and cell cycle assays against primary UCS cell lines. Mechanistic studies were performed using western blot assays while in vivo experiments were completed in UCS tumor bearing mice treated with avutometinib ± FAK inhibitor by oral gavage. ResultsWES results demonstrated multiple UCS cell lines harbor genetic alterations including KRAS, PTK2, BRAF, MAP2K, and MAP2K1, potentially sensitizing to FAK and RAF/MEK inhibition. Four out of five of the UCS cell lines demonstrated in vitro sensitivity to FAK and/or RAF/MEK inhibition when used alone or in combination. By western blot assays, exposure of UCS cell lines to the combination of defactinib/avutometinib demonstrated decreased phosphorylated (p)-FAK as well as decreased p-ERK. In vivo, the combination of avutometinib/VS-4718 demonstrated superior tumor growth inhibition and longer survival compared to single agent treatment and controls starting at day 10 (p < 0.002) in UCS xenografts. ConclusionThe combination of avutometinib and defactinib demonstrates promising in vitro and in vivo anti-tumor activity against primary UCS cell lines and xenografts.

Acinar to β-like cell conversion through inhibition of focal adhesion kinase

Insufficient functional β-cell mass causes diabetes; however, an effective cell replacement therapy for curing diabetes is currently not available. Reprogramming of acinar cells toward functional insulin-producing cells would offer an abundant and autologous source of insulin-producing cells. Our lineage tracing studies along with transcriptomic characterization demonstrate that treatment of adult mice with a small molecule that specifically inhibits kinase activity of focal adhesion kinase results in trans-differentiation of a subset of peri-islet acinar cells into insulin producing β-like cells. The acinar-derived insulin-producing cells infiltrate the pre-existing endocrine islets, partially restore β-cell mass, and significantly improve glucose homeostasis in diabetic mice. These findings provide evidence that inhibition of the kinase activity of focal adhesion kinase can convert acinar cells into insulin-producing cells and could offer a promising strategy for treating diabetes.