Identification of novel and potent PROTACs targeting FAK for non-small cell lung cancer: Design, synthesis, and biological study

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase. Consistent with its important roles in regulation of cell survival, migration and proliferation, FAK has been found to be up-regulated in a broad range of tumors. A large number of reports have shown that FAK is involved in cancer cell invasion, survival, metastasis, epithelial-to-mesenchymal transition (EMT), and maintenance of cancer stem cells. Additionally, FAK is important for vascular development and tumor angiogenesis. A recent study using an endothelial cell (EC)-specific tamoxifen-inducible FAK knockout mouse model has shown that endothelial FAK deletion in adult mice inhibits tumor growth and reduces tumor angiogenesis.1 Thus far, FAK has been considered an important drug target for cancer therapeutics. Several FAK inhibitors targeting FAK kinase activity have been developed, some of which have already been studied in clinical trials.Figure 1 Figure 1. The FAK scaffold inhibitor C4 inhibits melanoma growth by targeting both melanoma cells and endothelial cells. Recently, several studies have provided evidence that FAK can also regulate tumor growth in a kinase-independent manner through its scaffolding function. In one study, the disruption of FAK interaction with endophilin A2 through mutation of its Pro-878/881 motif suppressed mammary tumor growth and metastasis in a murine breast cancer model.2 In another example, Luo et al found that treatment with FAK inhibitor could only suppress the proliferation of luminal progenitor-like or basal subtype mammary tumor cells, but not MaSC-like or claudin-low subtype cells3. Moreover, expression of kinase-defective FAK was shown to rescue EC apoptosis resulting from FAK deletion in embryos, indicating that FAK scaffolding function is also important for EC survival.4 These data suggest that only targeting FAK kinase activity might not effectively block FAK involved pro-tumor signaling. Instead, targeting FAK scaffold function might be another promising approach. Several small molecules specifically targeting FAK scaffold function have been discovered by Cance's group, despite a big challenge in developing small molecules capable of disrupting protein-protein interactions.5,6 These inhibitors have shown significant anti-tumor activity in various human tumor cells, and poteintial for sensitizing tumors to chemotherapy. In a recent paper in Cell Cycle, Kurenova et al. investigated the activity of the FAK scaffold inhibitor C4, which targets the FAK/VEGFR3 complex, against melanoma.7 They found that C4 reduced phosphorylation of FAK and VEGFR3, decreasing complex formation. Importantly, C4 significantly reduced tumor growth in vivo and sensitized tumors to chemotherapy in both BRAF wild type and mutant melanomas. Interestingly, they showed that the anti-cancer activity of C4 was due to targeting of both melanoma cells and ECs. C4 inhibited FAK/VEGFR3 signaling in ECs and prevented vessel formation and sprouting. A significant reduction of tumor blood volume was seen following C4 treatment in a xenograft mouse model. This study by the Cance group further strengthens the evidence that blocking FAK-VEGFR3 scaffold signaling is highly effective in treating melanoma. More importantly, their data indicate that C4 may be a promising drug for treating patients harboring wild type BRAF, as prognoses are still very poor for these patients. This study also demonstrates the great therapeutical potential of targeting FAK scaffolding function. FAK interacts with different proteins, promising that future work in designing FAK scaffold inhibitors will contribute to the development of more effective cancer therapies.

Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96 h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology in vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.

Modeling of FAK-PROTAC candidates from GSK2256098 analogs for targeted protein degradation

Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that promotes cell migration, survival, and gene expression. Here we show that FAK signaling is important for tumor necrosis factor-alpha (TNFalpha)-induced interleukin 6 (IL-6) mRNA and protein expression in breast (4T1), lung (A549), prostate (PC-3), and neural (NB-8) tumor cells by FAK short hairpin RNA knockdown and by comparisons of FAK-null (FAK(-/-)) and FAK(+/+) mouse embryo fibroblasts. FAK promoted TNFalpha-stimulated MAPK activation needed for maximal IL-6 production. FAK was not required for TNFalpha-mediated nuclear factor-kappaB or c-Jun N-terminal kinase activation. TNFalpha-stimulated FAK catalytic activation and IL-6 production were inhibited by FAK N-terminal but not FAK C-terminal domain overexpression. Analysis of FAK(-/-) fibroblasts stably reconstituted with wild type or various FAK point mutants showed that FAK catalytic activity, Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNFalpha-stimulated MAPK activation and IL-6 production. Constitutively activated MAPK kinase-1 (MEK1) expression in FAK(-/-) and A549 FAK short hairpin RNA-expressing cells rescued TNFalpha-stimulated IL-6 production. Inhibition of Src protein-tyrosine kinase activity or mutation of Src phosphorylation sites on FAK (Tyr-861 or Tyr-925) did not affect TNFalpha-stimulated IL-6 expression. Moreover, analyses of Src(-/-), Yes(-/-), and Fyn(-/-) fibroblasts showed that Src expression was inhibitory to TNFalpha-stimulated IL-6 production. These studies provide evidence for a novel Src-independent FAK to MAPK signaling pathway regulating IL-6 expression with potential importance to inflammation and tumor progression.

Targeting focal adhesion kinase (FAK) for cancer therapy: FAK inhibitors, FAK-based dual-target inhibitors and PROTAC degraders

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr(407) was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr(407) increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr(397) phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr(397) and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr(407) phosphorylation negatively regulates the enzymatic and biological activities of FAK.

Focal adhesion kinase (FAK) is highly expressed in a variety of human cancers and is a target for cancer therapy. Since FAK kinase inhibitors only block the kinase activity of FAK, they are not highly effective in clinical trials. FAK also functions as a scaffold protein in a kinase-independent pathway. To effectively target FAK, it is required to block both FAK kinase-dependent and FAK-independent pathways. Thus, we tested a new generation drug FAK PROTAC for ovarian cancer therapy, which blocks both kinase and scaffold activity. We tested the efficacy of FAK PROTAC and its parent kinase inhibitor (VS-6063) in ovarian cancer cell lines in vitro by performing cell functional assays including cell proliferation, migration, invasion. We also tested in vivo activity in orthotopic ovarian cancer mouse models. In addition, we assessed whether FAK PROTAC disrupts kinase-dependent and kinase-independent pathways. We demonstrated that FAK PROTAC is highly effective as compared to its parent FAK kinase inhibitor VS-6063 in inhibiting cell proliferation, survival, migration, and invasion. FAK PROTAC not only inhibits the FAK kinase activity but also FAK scaffold function by disrupting the interaction between FAK and its interaction protein ASAP1. We further showed that FAK PROTAC effectively inhibits ovarian tumor growth and metastasis. Taken together, FAK PROTAC inhibits both FAK kinase activity and its scaffold protein activity by disrupting the interaction between FAK and ASAP1 and is highly effective in inhibiting ovarian tumor growth and metastasis.

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK*Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phosphorylated tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phosphorylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phosphorylation-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phosphorylation-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phosphorylation of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK*Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK*Grb7 complexes and Grb7 phosphorylation by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK*Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK*Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase, exerts kinase-dependent enzymatic functions and kinase-independent scaffolding functions, both of which are crucial in cancer development, early embryonic development, and reproduction. However, previous efforts for FAK blocking mainly focus on kinase inhibitors. Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that allow direct post-translational knockdown of proteins via ubiquitination of a target protein by E3 ubiquitin ligase and subsequent proteasomal degradation. Here, we designed and synthesized a FAK PROTAC library with FAK inhibitor (PF562271 or VS6063) and CRBN E3 ligand. A novel FAK-targeting PROTAC, FC-11, showed a rapid and reversible FAK degradation with a picomolar of DC50 in various cell lines in vitro, which imply that FAK-PROTACs could be useful as expand tools for studying functions of FAK in biological system and as potential therapeutic agents.

In our laboratory, we are interested in hyperosmolarity-induced apoptosis in neuronal cells. We have shown that high concentrations of glucose or mannitol induce apoptotic cell death in dorsal root ganglia in culture and in SH-SY5Y and SH-EP human neuroblastoma cells. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that has a critical role for transmitting integrin-mediated-signals. In this study, we report that hyperosmolar treatment mediates FAK dephosphorylation and cleavage, which is prevented by insulin-like growth factor I (IGF-I) treatment. Mannitol treatment of SH-EP cells transfected with vector (SH-EP/pSFFV) results in concentration- and time-dependent dephosphorylation and degradation of FAK. Dephosphorylation and degradation of FAK are tightly correlated with apoptotic morphological changes, including the disruption of actin stress fibers, the loss of focal adhesion sites, membrane blebbing, and cell detachment. Treatment of SH-EP/pSFFV cells with IGF-I or transfection of IGF-I receptor prevents these changes. Treatment of cells with pharmacologic inhibitors of the mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathways does not affect mannitol-induced FAK dephosphorylation and degradation. However, phosphatidylinositol 3-kinase is necessary for IGF-I-mediated protection against FAK alteration. Mannitol treatment also results in the degradation of Akt. Mannitol induces the activation of caspases-3 and -9 in a time course similar to the dephosphorylation and degradation of FAK. Treatment of the cells with ZVAD, a general caspase inhibitor, blocks the mannitol-induced FAK and Akt degradation as well as cell detachment and apoptosis. These results suggest that one of the pathways of mannitol-mediated apoptosis is through the degradation of FAK and Akt and that IGF-I protects the cells from apoptosis by blocking the activation of caspases, which may be responsible for the loss of FAK and Akt.

Background: Late stage thyroid cancers characterized by metastasis and extranodal invasion have a poor prognosis compared to those with localized disease. However, there are limited therapeutic options and few biomarkers to indicate which patients will develop aggressive disease. Our lab has identified Focal Adhesion Kinase (FAK) as a key regulator of thyroid cancer growth, invasion, and metastasis. FAK is a non-receptor tyrosine kinase that is auto-phosphorylated at tyrosine 397 (Y397) in response to integrin or growth factor receptor signaling resulting in the activation of downstream signaling pathways. While FAK is predominantly localized at the plasma membrane, FAK has also been shown to accumulate in the nucleus via a nuclear localization sequence (NLS) to promote cell survival. We have found that FAK localizes to the nucleus in a subset of thyroid cancer patient tumors and that phosphorylated Y397 FAK (pY397 FAK) specifically accumulates in the nucleolus. The nucleolus plays a key role in cancer progression through the synthesis of ribosomal RNA (rRNA) and subsequent increase in ribosome biogenesis, protein synthesis, and tumor growth. It is unclear how pY397 FAK localizes to the nucleolus and what the function of pY397 FAK is in the nucleolus. Hypothesis: Nuclear FAK drives thyroid cancer growth and survival through the phosphorylation of nucleolar proteins involved in rRNA transcription. Results: To address the functional role of FAK in the nucleus, we excluded FAK from the nucleus by mutating its NLS and found that it significantly decreased anchorage independent growth compared to wild type (WT) FAK in our thyroid cancer cells (BCPAP and 8505C). Importantly, non-phosphorylatable FAK mutant (Y397F) and kinase dead FAK mutant also decreased growth indicating that nuclear and pY397 FAK are required for anchorage independent growth. Furthermore, we found that forcing FAK into the nucleus with an SV40 NLS resulted in increased FAK nucleolar accumulation which was eliminated when FAK is forced into the nucleus with Y397F FAK. These data indicate that pY397 FAK is required for FAK nucleolar accumulation. To investigate the role of pY397 FAK in the nucleolus, we performed BioID to identify novel protein-protein interactions for WT FAK and Y397F FAK. We found that pY397 FAK interacts with a network of nucleolar proteins including NPM1, TOP1, and DDX46. Interestingly, the majority of these nucleolar proteins are involved in transcription of pre-rRNA which is essential for protein synthesis and growth. Of note, Nucleophosmin 1 (NPM1) is an endoribonuclease that regulates pre-rRNA synthesis through the cleavage of the 46S rRNA transcript. We confirmed that pY397 FAK specifically interacts with NPM1 by Proximity Ligation Assay. Conclusion: These data indicate that pY397 FAK interacts with a network of nucleolar proteins involved in rRNA transcription and that nuclear pY397 FAK drives growth and survival in thyroid cancer. Citation Format: Meghan Kellett, Vibha Sharma, Brittelle Kessler, Sharon Sams, Molishree Joshi, Rebecca E. Schweppe. Nuclear FAK drives thyroid cancer growth and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 669.

Focal adhesion kinase (FAK) has important functions in bone homeostasis but its role in early osteoprogenitor cells is unknown. We show herein that mice lacking FAK in Dermo1-expressing cells exhibited low bone mass and decreased osteoblast number. Mechanistically, FAK-deficient early osteoprogenitor cells had decreased proliferation and significantly reduced mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, a central regulator of cell growth and proliferation. Furthermore, our data showed that the pharmacological inhibition of FAK kinase-dependent function alone was sufficient to decrease the proliferation and compromise the mineralization of early osteoprogenitor cells. In contrast to the Fak deletion in early osteoprogenitor cells, FAK loss in Col3.6 Cre-targeted osteoblasts did not cause bone loss, and Fak deletion in osteoblasts did not affect proliferation, differentiation, and mTORC1 signaling but increased the level of active proline-rich tyrosine kinase 2 (PYK2), which belongs to the same non-receptor tyrosine kinase family as FAK. Importantly, mTORC1 signaling in bone marrow stromal cells (BMSCs) was reduced if FAK kinase was inhibited at the early osteogenic differentiation stage. In contrast, mTORC1 signaling in BMSCs was not affected if FAK kinase was inhibited at a later osteogenic differentiation stage, in which, however, the concomitant inhibition of both FAK kinase and PYK2 kinase reduced mTORC1 signaling. In summary, our data suggest that FAK promotes early osteoprogenitor cell proliferation by enhancing mTORC1 signaling via its kinase-dependent function and the loss of FAK in osteoblasts can be compensated by the upregulated active PYK2. © 2020 American Society for Bone and Mineral Research.

Focal Adhesion Kinase-Related Proline-Rich Tyrosine Kinase 2 and Focal Adhesion Kinase Are Co-Overexpressed in Early-Stage and Invasive ErbB-2-Positive Breast Cancer and Cooperate for Breast Cancer Cell Tumorigenesis and Invasiveness