Targeting Focal Adhesion Kinase Research Articles

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.

Targeting focal adhesion kinase (FAK) and spleen tyrosine kinase (SYK) in gastrointestinal neuroendocrine tumors: Unveiling mechanisms and anti-proliferative effects

Targeting focal adhesion kinase (FAK) and spleen tyrosine kinase (SYK) in gastrointestinal neuroendocrine tumors: Unveiling mechanisms and anti-proliferative effects

Abstract LB431: Direct comparison of focal adhesion kinase (FAK) therapeutic modalities in cancer: Kinase, FAT, and PROTAC

Abstract Introduction: Focal Adhesion Kinase (FAK) is a multifunctional non-receptor tyrosine kinase and scaffolding protein that has emerged as a potential therapeutic target for the treatment of cancer due to its upregulation and role in cancer cell survival. However, it remains unknown which cancer types are most susceptible to FAK modulation or the best pharmacological strategy to target FAK. FAK is endogenously regulated by an alternative transcript termed FAK-related non-kinase (FRNK), which inhibits FAK through the Focal Adhesion Targeting (FAT) domain. Current FAK-kinase inhibitors that block the catalytic site have shown limited efficacy in multiple Phase I/II clinical trials. FAK-degrading PROTACs have also shown inadequate induction of antiproliferative effects despite successful protein degradation. However, we have shown promising results with our novel FAK-FAT inhibitors, which target the scaffolding function of FAK to induce anoikis and inhibit proliferation in cancer cells. A direct comparison of these different FAK modulators in various cancer types has yet to be performed, creating a significant gap in understanding the best approach for targeted FAK inhibition for the treatment of cancer. Methods: The Broad Institute’s Cancer Dependency Map (DepMap) was utilized to select a cancer cell line cohort based on dependency on FAK for survival. Four cancer types, including breast, brain, lung, and skin cancers were selected based on the correlation of FAK expression for survival and previous literature evidence for dependency on FAK. Three FAK-dependent and one FAK-independent cell lines per cancer type were selected for a total of 16 cancer cell lines. Genetic adenoviral constructs that block the FAK-FAT domain (Ad-FRNK) and knockdown FAK levels (shRNA) were utilized to investigate the role of FAK in cancer cell survival. These constructs were used as they recapitulate the pharmacological strategy of our FAK-FAT inhibitor UA-2012 and FAK degrading PROTACs, respectively. Results: Cell viability was significantly decreased in 10/13 FAK-dependent cell lines after transduction with either adFRNK or FAK targeting shRNAs, six of which showed a statically significant decrease in cell viability upon transduction with adFRNK compared to shRNAs. No cell lines showed a greater response to FAK shRNAs compared to adFRNK. We then evaluated the ability of the FAK kinase inhibitor Defactinib, FAK degrading PROTAC BI-3663, and our novel FAK-FAT domain inhibitor UA-2012 to induce cancer cell death. Overall, FAK-FAT domain inhibition with UA-2012 resulted in the most potent reduction in viability of FAK-dependent cells, particularly in skin and breast cancer cell lines. Treatment with Defactinib resulted in low IC50 values regardless of FAK, and PROTAC BI-3663 displayed minimal effects with high IC50 values often above 200 µM despite successful protein degradation. Conclusion: This data suggests that the FAK-FAT domain may be the major modulator of FAK-mediated survival in breast and skin cancer cells. Citation Format: Lauren Reyes, Ruchi Khatiwala, Timothy Marlowe. Direct comparison of focal adhesion kinase (FAK) therapeutic modalities in cancer: Kinase, FAT, and PROTAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB431.

Abstract 4655: Targeting FAK improves the tumor penetration of antibody-drug conjugates to strengthen the anti-cancer responses

Abstract Antibody-drug conjugates (ADCs) such as HER2 ADC Enhertu and TROP2 ADC Trodelvy have been proved to be the promising therapeutic agents in portion of cancer diseases. However, there are still numerous patients refractory to the therapy, indicating that the efficacy of ADCs may need to be further optimized. Here, we report that the excessive cancer-associated fibroblasts (CAFs) can serve as a fibrotic barrier, to the detriment of anti-tumor effects from ADCs by alleviating their tissue penetration. Mechanistically, the cancer cells can transform the normal fibrotic cells into CAFs which are featured by the hyperactivated focal adhesion kinase (FAK) signaling. The fast-growing CAFs would decrease the tumor uptake of the big molecules, conferring drug resistance of ADCs to the cancer cells. Targeting FAK with a small molecule inhibitor IN10018 is capable to reduce CAFs associated tumor barrier, elevating the tissue penetration of different types of ADCs regardless their respective targets. The combination regimen comprising IN10018 and the ADCs targeting either HER2 or TROP2 outperformed each monotherapy with respect to antitumor outcomes in different animal experiments. The preclinical evidence for the dual regimen of IN10018 and ADCs warrants a further validation in clinical settings. Citation Format: Baoyuan Zhang, Zhixiang Zhang, Jiaming Gao, Shiqiang Lu, Ran Pang, Dongfang Li, Xun Huang, Natasha Qin, Leo Liu, Zaiqi Wang. Targeting FAK improves the tumor penetration of antibody-drug conjugates to strengthen the anti-cancer responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4655.

Abstract 1928: Targeting FAK to improve the therapy of KRAS G12C mutant cancers

Abstract Despite the pivotal role of KRAS G12C in tumor initiation and progression, currently available KRAS G12C inhibitors have relatively modest activity compared to other approved therapies targeting other classic oncogenic drivers. Fortunately, efforts to improve the anti-tumor response to KRAS G12C targeted therapy have benefited from the use of combination approaches. Here, we found that treatment with KRAS G12C inhibitors induces sustained activation of focal adhesion kinase (FAK) in KRAS G12C mutant cell lines from various cancer types, including non-small cell lung cancer, colorectal cancer, and pancreatic cancer. These results suggest a potential role for FAK in adaptive resistance to KRAS G12C inhibition and encourage us to explore the anticancer effects of combining the KRAS G12C inhibitor SY-5933 with a FAK inhibitor, CT-707. The cell viability and clonogenic assays using various KRAS G12C mutant cancer cell lines showed that the combination of SY-5933 and CT-707 synergistically inhibited cell growth. Mechanistically, this enhanced anti-proliferative effect is associated with a significant decrease in FAK activity and a more pronounced induction of cell cycle arrest and apoptosis. In vivo efficacy studies in multiple cell line derived xenograft (CDX) models, the combination of SY-5933 plus CT-707 consistently induced a more potent anti-tumor response than either monotherapy alone. In conclusion, our results demonstrate that CT-707 has the potential to enhance the efficacy of SY-5933 by suppressing adaptive FAK activation under KRAS G12C inhibition. This provides a rationale for an innovative combination therapy to improve outcomes in patients with KRAS G12C mutated cancers. Citation Format: Shuang Liu, Junfen Shi, Chang Lu, Miao Zhang, Ying Xin, Yan Zhu, Jijun Li, Hong Luo, Yinghui Sun. Targeting FAK to improve the therapy of KRAS G12C mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1928.

Targeting of focal adhesion kinase enhances the immunogenic cell death of PEGylated liposome doxorubicin to optimize therapeutic responses of immune checkpoint blockade

BackgroundsImmune checkpoint blockade (ICB) is widely considered to exert long-term treatment benefits by activating antitumor immunity. However, many cancer patients show poor clinical responses to ICB due in part to the lack of an immunogenic niche. Focal adhesion kinase (FAK) is frequently amplified and acts as an immune modulator across cancer types. However, evidence illustrates that targeting FAK is most effective in combination therapy rather than in monotherapy.MethodsHere, we used drug screening, in vitro and in vivo assays to filter out that doxorubicin and its liposomal form pegylated liposome doxorubicin (PLD) showed synergistic anti-tumor effects in combination with FAK inhibitor IN10018. We hypothesized that anti-tumor immunity and immunogenic cell death (ICD) may be involved in the treatment outcomes through the data analysis of our clinical trial testing the combination of IN10018 and PLD. We then performed cell-based assays and animal studies to detect whether FAK inhibition by IN10018 can boost the ICD of PLD/doxorubicin and further established syngeneic models to test the antitumor effect of triplet combination of PLD, IN10018, and ICB.ResultsWe demonstrated that the combination of FAK inhibitor IN10018, and PLD/doxorubicin exerted effective antitumor activity. Notably, the doublet combination regimen exhibited response latency and long-lasting treatment effects clinically, outcomes frequently observed in immunotherapy. Our preclinical study confirmed that the 2-drug combination can maximize the ICD of cancer cells. This approach primed the tumor microenvironment, supplementing it with sufficient tumor-infiltrating lymphocytes (TILs) to activate antitumor immunity. Finally, different animal studies confirmed that the antitumor effects of ICB can be significantly enhanced by this doublet regimen.ConclusionsWe confirmed that targeting FAK by IN10018 can enhance the ICD of PLD/doxorubicin, further benefiting the anti-tumor effect of ICB. The animal tests of the triplet regimen warrant further discovery in the real world.

Targeting focal adhesion kinase boosts immune response in KRAS/LKB1 co-mutated lung adenocarcinoma via remodeling the tumor microenvironment

BackgroundKRAS mutation is one of the most common oncogenic drivers in NSCLC, however, the response to immunotherapy is heterogeneous owing to the distinct co-occurring genomic alterations. KRAS/LKB1 co-mutated lung adenocarcinoma displays poor response to PD-1 blockade whereas the mechanism remains undetermined.MethodsWe explored the specific characteristics of tumor microenvironment (TME) in KL tumors using syngeneic KRASG12DLKB1−/− (KL) and KRASG12DTP53−/− (KP) lung cancer mouse models. The impact of focal adhesion kinase (FAK) inhibitor on KL lung tumors was investigated in vitro and in vivo through evaluation of both KL cell lines and KL lung cancer mouse models.ResultsWe identified KL tumors as “immune-cold” tumors with excessive extracellular matrix (ECM) collagen deposition that formed a physical barrier to block the infiltration of CD8+T cells. Mechanistically, abundant activated cancer-associated fibroblasts (CAFs) resulted from FAK activation contributed to the formation of the unique TME of KL tumors. FAK inhibition with a small molecular inhibitor could remodel the TME by inhibiting CAFs activation, decreasing collagen deposition and further facilitating the infiltration of anti-tumor immune cells, including CD8+ T cells, DC cells and M1-like macrophages into tumors, hence, converting “immune-cold” KL tumors into “immune-hot” tumors. The combined FAK inhibitor and PD-1 blockade therapy synergistically retarded primary and metastatic tumor growth of KL tumors.ConclusionsOur study identified FAK as a promising intervention target for KL tumors and provided basis for the combination of FAK inhibitor with PD-1 blockade in the management of KL lung cancers.

Targeting NANOG and FAK via Cx26-derived Cell-penetrating Peptides in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype with limited treatment options. We previously identified a protein complex unique to TNBC composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of the interaction region of Cx26 attenuated tumor growth in preclinical models. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Binding studies revealed that the Cx26 C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar-to-micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. Peptides with high affinity were engineered with a cell-penetrating antennapedia sequence and assessed in functional assays including cell proliferation, tumorsphere formation, and in vivo tumor growth, and downstream signaling changes were measured. The cell-penetrating Cx26 peptide (aCx26-pep) disrupted self-renewal while reducing nuclear FAK and NANOG and inhibiting NANOG target gene expression in TNBC cells but not luminal mammary epithelial cells. In vivo, aCx26-pep reduced tumor growth and proliferation and induced cell death. Here, we provide proof-of-concept that a Cx26 peptide-based strategy inhibits growth and alters NANOG activity specifically in TNBC, indicating the therapeutic potential of this targeting approach.

Development and Characterization of Selective FAK Inhibitors and PROTACs with In Vivo Activity.

Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.

Triple-negative mouse breast cancer initiating cells show high expression of beta1 integrin and increased malignant features.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that exhibits aggressive tumor phenotypes, including rapid metastasis and tumor recurrence. Integrins belong to the family of transmembrane glycoproteins involved in regulating cell adhesion, proliferation, and differentiation through cell-cell and cell-extracellular matrix interactions. Aberrant β1 integrin signaling has been implicated in cancer invasion and metastasis processes. The present work aimed to investigate the role of β1 integrin in TNBC cancer progression using a mouse 4T1 cell line as a model system. We have sorted a subset of tumor-initiating cells (TICs) from the 4T1 cell line based on CD133 positivity by flow cytometry. RT-PCR and protein analysis studies showed the transcriptional upregulation of β1 integrin and its downstream target focal adhesion kinase in 4T1-TICs compared to parental 4T1 cells. In addition, the expression of β1 receptors in TICs is significantly higher than in parental population cells. Furthermore, in vitro cellular assays revealed that CD133+ TICs have higher clonogenic ability, invasion, and sphere formation potential. These findings suggest that β1 integrin has a potential role in TNBC invasion and metastasis. Hence, β1 integrin could be a possible factor for future targeted cancer therapies.

Design, synthesis, and biological evaluation of diaminopyrimidine derivatives as novel focal adhesion kinase inhibitors.

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase that belongs to the family of focal adhesion complexes and is responsible for the development of various tumors. Herein, 24 diaminopyrimidine derivatives were designed and synthesized based on TAE-226. Several compounds with good activity were further evaluated regarding their antiproliferative activities against two cancer cells with high FAK expression. Compound A12 showed potent anticancer activity against A549 and MDA-MB-231 cell lines with IC50 values of 130 nM and 94 nM, respectively. In vitro metabolic stability and cytochrome P450 (CYP) inhibition assays showed that A12 exhibited favorable stability and weak inhibitory activity on CYP isoforms. Preliminary evaluation of kinase selectivity showed that A12 was a multi-kinase inhibitor. The acute toxicity in vivo indicated that A12 possessed acceptable safety. Compound A12 was also selected for molecular docking studies and the prediction of molecular properties and drug-like properties. These results indicated that compound A12 could be used as a potential lead compound targeting FAK for further development.

Abstract C065: Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer

Abstract Background: Unfortunately, the impact of immunotherapy in pancreatic cancer has, to date, been limited. Nonetheless, despite lack of success with checkpoint blockade alone, there is hope that combinations of agents may unlock the potential for immunotherapy success in pancreatic cancer. FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase that is elevated in human Pancreatic Ductal Adenocarcinoma (PDAC) tissue and correlates with high levels of fibrosis and poor CD8 cytotoxic T cell infiltration. We, and others, have already demonstrated a role for FAK in promoting tumor evasion by inducing an immunosuppressive microenvironment, specifically by regulation of cytokines such as CCL5. This has led to trials of the FAK inhibitor (defactinib) in conjunction with immunotherapy. Here we set out to improve our understanding of the role of FAK in regulating chemokine/cytokine mediated signaling in PDAC with a view to identify clinically targetable pathways with potential therapeutic benefit to pancreatic cancer patients. Methods: We combined CRISPR, NanoString, Forward Phase Protein Arrays (FPPA) and ELISA with in vivo experiments using a transplantable KrasG12Dp53R172H mouse model of Pancreatic Cancer and validated findings using human patient-derived PDAC cell lines. Results: We used CRISPR-Cas9 gene editing to deplete FAK gene expression in Panc47 cells, a syngeneic cell line isolated from PDAC arising in fully back-crossed C57BL/6 KPC mice, and reconstituted wild-type FAK (FAK-WT) expression into a clonal Panc47 FAK null (FAK-/-) cell line. Subsequent NanoString and FPPA analyses of secreted cytokines in Panc47 FAK-WT and Panc47 FAK-/- cell lines identified CXCL16 as one of the most significantly increased chemokines upon FAK depletion. Similar results were observed when CRISPR mediated FAK depletion was performed in patient-derived PDAC cell lines. Previous work in our lab has shown that depletion of Signal Transducer and Activator of Transcription-3 (STAT3), a key mediator of immune evasion, in combination with FAK depletion in orthotopically implanted PDAC tumors significantly increases CD8 T-cell infiltration into murine tumors when compared with FAK-/- tumors alone. Here we identify that dual FAK/STAT3 depletion further elevates CXCL16 expression and show that CXCR6, the receptor for CXCL16, is associated with both murine and human CD8 T-cells. Conclusions: These findings identify a FAK-dependent CXCL16:CXCR6 paracrine signaling axis associated with elevated recruitment of CD8 T-cells into PDAC tumors. This requires the dual targeting of FAK and STAT3, identifying a novel therapeutic strategy that warrants further investigation. Citation Format: Jane L. Parry, Marta Canel, Alan Serrels. Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C065.

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.

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.

Phase I dose escalation and expansion study of defactinib, pembrolizumab, and gemcitabine in patients with advanced treatment-refractory pancreatic cancer.

4146 Background: Targeting focal adhesion kinase (FAK) renders checkpoint immunotherapy effective in pancreatic ductal adenocarcinoma (PDAC) mouse models. Defactinib is a highly potent oral FAK inhibitor shown to have a tolerable safety profile. We evaluated the safety and recommended phase 2 dose (RP2D) of defactinib in combination with pembrolizumab and gemcitabine for PDAC patients. Methods: We conducted a multicenter, open-label, phase I study with dose escalation and expansion phases. In 3x3 dose escalation, patients with refractory solid tumors were treated at five escalating dose levels of defactinib and gemcitabine to identify a RP2D. In expansion phase, patients with metastatic PDAC who progressed on frontline treatment (refractory cohort) or had treatment response or stable disease (SD) on standard gemcitabine/nab-paclitaxel (maintenance cohort) were treated at RP2D. Pre- and post-treatment tumor biopsies were performed to evaluate changes in tumor immunity. Results: The triple drug combination was well-tolerated with no dose-limiting toxicities. Among 17 treated patients with refractory PDAC, the disease control rate (DCR) was 58.8% with one partial response (PR) and nine SDs and the median progression-free survival (PFS) and overall survival (OS) were 4.2 months and 9.1 months, respectively. Among the evaluable patients in the maintenance cohort, DCR was 63.6% with one PR and six SD. Three patients with SD came off study due to treatment- or disease-related complications. The median PFS and OS were 5.0 months and 8.3 months, respectively. Conclusions: The combination of defactinib, pembrolizumab, and gemcitabine was well-tolerated, had promising preliminary efficacy, and showed increased infiltrative T lymphocytes in post-treatment tumor biopsies. Incorporation of a more potent chemotherapy backbone should be considered to achieve better clinical response in future trial design. Clinical trial information: NCT02546531.

New Insights on the Nuclear Functions and Targeting of FAK in Cancer.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase over-expressed and activated in both adult and pediatric cancers, where it plays important roles in the regulation of pathogenesis and progression of the malignant phenotype. FAK exerts its functions in cancer by two different ways: a kinase activity in the cytoplasm, mainly dependent on the integrin signaling, and a scaffolding activity into the nucleus by networking with different gene expression regulators. For this reason, FAK has to be considered a target with high therapeutic values. Indeed, evidence suggests that FAK targeting could be effective, either alone or in combination, with other already available treatments. Here, we propose an overview of the novel insights about FAK’s structure and nuclear functions, with a special focus on the recent findings concerning the roles of this protein in cancer. Additionally, we provide a recent update on FAK inhibitors that are currently in clinical trials for patients with cancer, and discuss the challenge and future directions of drug-based anti-FAK targeted therapies.

FAK in Cancer: From Mechanisms to Therapeutic Strategies.

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is overexpressed and activated in many cancer types. FAK regulates diverse cellular processes, including growth factor signaling, cell cycle progression, cell survival, cell motility, angiogenesis, and the establishment of immunosuppressive tumor microenvironments through kinase-dependent and kinase-independent scaffolding functions in the cytoplasm and nucleus. Mounting evidence has indicated that targeting FAK, either alone or in combination with other agents, may represent a promising therapeutic strategy for various cancers. In this review, we summarize the mechanisms underlying FAK-mediated signaling networks during tumor development. We also summarize the recent progress of FAK-targeted small-molecule compounds for anticancer activity from preclinical and clinical evidence.

Protein tyrosine phosphatase 1B targets focal adhesion kinase and paxillin in cell-matrix adhesions.

Protein tyrosine phosphatase 1B (PTP1B, also known as PTPN1) is an established regulator of cell-matrix adhesion and motility. However, the nature of substrate targets at adhesion sites remains to be validated. Here, we used bimolecular fluorescence complementation assays, in combination with a substrate trapping mutant of PTP1B, to directly examine whether relevant phosphotyrosines on paxillin and focal adhesion kinase (FAK, also known as PTK2) are substrates of the phosphatase in the context of cell-matrix adhesion sites. We found that the formation of catalytic complexes at cell-matrix adhesions requires intact tyrosine residues Y31 and Y118 on paxillin, and the localization of FAK at adhesion sites. Additionally, we found that PTP1B specifically targets Y925 on the focal adhesion targeting (FAT) domain of FAK at adhesion sites. Electrostatic analysis indicated that dephosphorylation of this residue promotes the closed conformation of the FAT 4-helix bundle and its interaction with paxillin at adhesion sites.

Drug Discovery Targeting Focal Adhesion Kinase (FAK) as a Promising Cancer Therapy.

FAK is a nonreceptor intracellular tyrosine kinase which plays an important biological function. Many studies have found that FAK is overexpressed in many human cancer cell lines, which promotes tumor cell growth by controlling cell adhesion, migration, proliferation, and survival. Therefore, targeting FAK is considered to be a promising cancer therapy with small molecules. Many FAK inhibitors have been reported as anticancer agents with various mechanisms. Currently, six FAK inhibitors, including GSK-2256098 (Phase I), VS-6063 (Phase II), CEP-37440 (Phase I), VS-6062 (Phase I), VS-4718 (Phase I), and BI-853520 (Phase I) are undergoing clinical trials in different phases. Up to now, there have been many novel FAK inhibitors with anticancer activity reported by different research groups. In addition, FAK degraders have been successfully developed through “proteolysis targeting chimera” (PROTAC) technology, opening up a new way for FAK-targeted therapy. In this paper, the structure and biological function of FAK are reviewed, and we summarize the design, chemical types, and activity of FAK inhibitors according to the development of FAK drugs, which provided the reference for the discovery of new anticancer agents.

Focal adhesion kinase (FAK) promotes cholangiocarcinoma development and progression via YAP activation

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is upregulated in many tumor types and is a promising target for cancer therapy. Herein, we elucidated the functional role of FAK in intrahepatic cholangiocarcinoma (iCCA) development and progression. Expression levels and activation status of FAK were determined in human iCCA samples. The functional contribution of FAK to Akt/YAP murine iCCA initiation and progression was investigated using conditional Fak knockout mice and constitutive Cre or inducible Cre mice, respectively. The oncogenic potential of FAK was further examined via overexpression of FAK in mice. Invitro cell line studies and invivo drug treatment were applied to address the therapeutic potential of targeting FAK for iCCA treatment. FAK was ubiquitously upregulated and activated in iCCA lesions. Ablation of FAK strongly delayed Akt/YAP-driven mouse iCCA initiation. FAK overexpression synergized with activated AKT to promote iCCA development and accelerated Akt/Jag1-driven cholangiocarcinogenesis. Mechanistically, FAK was required for YAP(Y357) phosphorylation, supporting the role of FAK as a central YAP regulator in iCCA. Significantly, ablation of FAK after Akt/YAP-dependent iCCA formation strongly suppressed tumor progression in mice. Furthermore, a remarkable iCCA growth reduction was achieved when a FAK inhibitor and palbociclib, a CDK4/6 inhibitor, were administered simultaneously in human iCCA cell lines and Akt/YAP mice. FAK activation contributes to the initiation and progression of iCCA by inducing the YAP proto-oncogene. Targeting FAK, either alone or in combination with anti-CDK4/6 inhibitors, may be an effective strategy for iCCA treatment. We found that the protein FAK (focal adhesion kinase) is upregulated and activated in human and mouse intrahepatic cholangiocarcinoma samples. FAK promotes intrahepatic cholangiocarcinoma development, whereas deletion of FAK strongly suppresses its initiation and progression. Combined FAK and CDK4/6 inhibitor treatment had a strong anti-cancer effect in invitro and invivo models. This combination therapy might represent a valuable and novel treatment against human intrahepatic cholangiocarcinoma.