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• W4376646473 abstract "Aberrant overexpression of nonreceptor tyrosine kinase FER (Fps/Fes Related) has been reported in various ovarian carcinoma–derived tumor cells and is a poor prognosis factor for patient survival. It plays an essential role in tumor cell migration and invasion, acting concurrently in both kinase-dependent and -independent manners, which is not easily suppressed by conventional enzymatic inhibitors. Nevertheless, the PROteolysis-TArgeting Chimera (PROTAC) technology offers superior efficacy over traditional activity–based inhibitors by simultaneously targeting enzymatic and scaffold functions. Hence in this study, we report the development of two PROTAC compounds that promote robust FER degradation in a cereblon-dependent manner. Both PROTAC degraders outperform a Food and Drug Administration–approved drug, brigatinib, in ovarian cancer cell motility suppression. Importantly, these PROTAC compounds also degrade multiple oncogenic FER fusion proteins identified in human tumor samples. These results lay an experimental foundation to apply the PROTAC strategy to antagonize cell motility and invasiveness in ovarian and other types of cancers with aberrant expression of FER kinase and highlight PROTACs as a superior strategy for targeting proteins with multiple tumor-promoting functions. Aberrant overexpression of nonreceptor tyrosine kinase FER (Fps/Fes Related) has been reported in various ovarian carcinoma–derived tumor cells and is a poor prognosis factor for patient survival. It plays an essential role in tumor cell migration and invasion, acting concurrently in both kinase-dependent and -independent manners, which is not easily suppressed by conventional enzymatic inhibitors. Nevertheless, the PROteolysis-TArgeting Chimera (PROTAC) technology offers superior efficacy over traditional activity–based inhibitors by simultaneously targeting enzymatic and scaffold functions. Hence in this study, we report the development of two PROTAC compounds that promote robust FER degradation in a cereblon-dependent manner. Both PROTAC degraders outperform a Food and Drug Administration–approved drug, brigatinib, in ovarian cancer cell motility suppression. Importantly, these PROTAC compounds also degrade multiple oncogenic FER fusion proteins identified in human tumor samples. These results lay an experimental foundation to apply the PROTAC strategy to antagonize cell motility and invasiveness in ovarian and other types of cancers with aberrant expression of FER kinase and highlight PROTACs as a superior strategy for targeting proteins with multiple tumor-promoting functions. Ovarian cancer is one of the most malignant gynecological cancers, with high morbidity and mortality among women (1Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2020.CA Cancer J. Clin. 2020; 70: 7-30Google Scholar). Despite substantial advances in surgery and chemical- and radiation therapy for ovarian cancer, medical challenges such as metastasis and resistance remain unresolved. An in-depth understanding of ovarian tumor progression and metastasis will be critical in identifying new therapeutic targets to intervene in this heterogeneous and lethal disease (2Binaschi M. Simonelli C. Goso C. Bigioni M. Maggi C.A. Maintenance therapy in ovarian cancer: molecular basis and therapeutic approach.Exp. Ther. Med. 2011; 2: 173-180Google Scholar). Tyrosine phosphorylation, precisely coordinated by tyrosine kinases and phosphatases, regulates multilayer signaling networks spatiotemporal dependently. Feline sarcoma (FES)–related kinase FER (Fps/Fes Related), along with FES, represents a distinct nonreceptor tyrosine kinase subfamily characterized by a functional N-terminal membrane-targeting F-BAR domain, a central SH2 domain, and a C-terminal kinase domain (3Greer P. Closing in on the biological functions of Fps/Fes and fer.Nat. Rev. Mol. Cell Biol. 2002; 3: 278-289Google Scholar) with essential roles in cell proliferation, motility, intercellular adhesion as well as the mediation of signal transmission from the cell surface to the cytoskeleton (3Greer P. Closing in on the biological functions of Fps/Fes and fer.Nat. Rev. Mol. Cell Biol. 2002; 3: 278-289Google Scholar, 4Craig A.W. FES/FER kinase signaling in hematopoietic cells and leukemias.Front. Biosci. (Landmark Ed.). 2012; 17: 861-875Google Scholar, 5Liu S. Xiong X. Zhao X. Yang X. Wang H. F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.J. Hematol. Oncol. 2015; 8: 47Google Scholar). Previous studies have strongly suggested that aberrantly high expression of FER as an independent prognostic indicator (6Ivanova I.A. Vermeulen J.F. Ercan C. Houthuijzen J.M. Saig F.A. Vlug E.J. et al.FER kinase promotes breast cancer metastasis by regulating alpha6- and beta1-integrin-dependent cell adhesion and anoikis resistance.Oncogene. 2013; 32: 5582-5592Google Scholar, 7Tavares S. Liv N. Pasolli M. Opdam M. Ratze M.A.K. Saornil M. et al.FER regulates endosomal recycling and is a predictor for adjuvant taxane benefit in breast cancer.Cell Rep. 2022; 39110584Google Scholar) is associated with tumor progression (8Zhang J. Wang Z. Zhang S. Chen Y. Xiong X. Li X. et al.Spatial regulation of signaling by the coordinated action of the protein tyrosine kinases MET and FER.Cell Signal. 2018; 50: 100-110Google Scholar, 9Allard P. Zoubeidi A. Nguyen L.T. Tessier S. Tanguay S. Chevrette M. et al.Links between Fer tyrosine kinase expression levels and prostate cell proliferation.Mol. Cell. Endocrinol. 2000; 159: 63-77Google Scholar, 10Lennartsson J. Ma H. Wardega P. Pelka K. Engstrom U. Hellberg C. et al.The Fer tyrosine kinase is important for platelet-derived growth factor-BB-induced signal transducer and activator of transcription 3 (STAT3) protein phosphorylation, colony formation in soft agar, and tumor growth in vivo.J. Biol. Chem. 2013; 288: 15736-15744Google Scholar, 11Oneyama C. Yoshikawa Y. Ninomiya Y. Iino T. Tsukita S. Okada M. Fer tyrosine kinase oligomer mediates and amplifies Src-induced tumor progression.Oncogene. 2016; 35: 501-512Google Scholar, 12Ivanova I.A. Arulanantham S. Barr K. Cepeda M. Parkins K.M. Hamilton A.M. et al.Targeting FER kinase inhibits melanoma growth and metastasis.Cancers (Basel). 2019; 11: 419Google Scholar, 13Zhang Y. Xiong X. Zhu Q. Zhang J. Chen S. Wang Y. et al.FER-mediated phosphorylation and PIK3R2 recruitment on IRS4 promotes AKT activation and tumorigenesis in ovarian cancer cells.Elife. 2022; 11e76183Google Scholar, 14Li H. Ren Z. Kang X. Zhang L. Li X. Wang Y. et al.Identification of tyrosine-phosphorylated proteins associated with metastasis and functional analysis of FER in human hepatocellular carcinoma cells.BMC Cancer. 2009; 9: 366Google Scholar, 15Zirngibl R. Schulze D. Mirski S.E. Cole S.P. Greer P.A. Subcellular localization analysis of the closely related Fps/Fes and Fer protein-tyrosine kinases suggests a distinct role for Fps/Fes in vesicular trafficking.Exp. Cell Res. 2001; 266: 87-94Google Scholar) and metastasis (6Ivanova I.A. Vermeulen J.F. Ercan C. Houthuijzen J.M. Saig F.A. Vlug E.J. et al.FER kinase promotes breast cancer metastasis by regulating alpha6- and beta1-integrin-dependent cell adhesion and anoikis resistance.Oncogene. 2013; 32: 5582-5592Google Scholar, 16Ahn J. Truesdell P. Meens J. Kadish C. Yang X. Boag A.H. et al.Fer protein-tyrosine kinase promotes lung adenocarcinoma cell invasion and tumor metastasis.Mol. Cancer Res. 2013; 11: 952-963Google Scholar, 17Arregui C. Pathre P. Lilien J. Balsamo J. The nonreceptor tyrosine kinase fer mediates cross-talk between N-cadherin and beta1-integrins.J. Cell Biol. 2000; 149: 1263-1274Google Scholar, 18Sangrar W. Gao Y. Scott M. Truesdell P. Greer P.A. Fer-mediated cortactin phosphorylation is associated with efficient fibroblast migration and is dependent on reactive oxygen species generation during integrin-mediated cell adhesion.Mol. Cell. Biol. 2007; 27: 6140-6152Google Scholar) in several cancer types. In particular, FER is significantly upregulated in ovarian carcinoma samples and carcinoma-derived cell lines. Downregulation of FER substantially inhibits tumor cell migration, invasion, and metastasis (19Zoubeidi A. Rocha J. Zouanat F.Z. Hamel L. Scarlata E. Aprikian A.G. et al.The Fer tyrosine kinase cooperates with interleukin-6 to activate signal transducer and activator of transcription 3 and promote human prostate cancer cell growth.Mol. Cancer Res. 2009; 7: 142-155Google Scholar, 20Fan G. Zhang S. Gao Y. Greer P.A. Tonks N.K. HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer.Genes Dev. 2016; 30: 1542-1557Google Scholar), indicating the urgent need and market potential for developing antagonists against FER kinase to benefit ovarian cancer patients. So far, only one small-molecule inhibitor targeting the kinase domain of FER has been reported (21Elkis Y. Cohen M. Yaffe E. Satmary-Tusk S. Feldman T. Hikri E. et al.A novel Fer/FerT targeting compound selectively evokes metabolic stress and necrotic death in malignant cells.Nat. Commun. 2017; 8: 940Google Scholar). Of note, several studies also revealed the kinase-independent function of FER in regulating cell motility (20Fan G. Zhang S. Gao Y. Greer P.A. Tonks N.K. HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer.Genes Dev. 2016; 30: 1542-1557Google Scholar, 21Elkis Y. Cohen M. Yaffe E. Satmary-Tusk S. Feldman T. Hikri E. et al.A novel Fer/FerT targeting compound selectively evokes metabolic stress and necrotic death in malignant cells.Nat. Commun. 2017; 8: 940Google Scholar, 22Pasder O. Shpungin S. Salem Y. Makovsky A. Vilchick S. Michaeli S. et al.Downregulation of Fer induces PP1 activation and cell-cycle arrest in malignant cells.Oncogene. 2006; 25: 4194-4206Google Scholar, 23Salem Y. Shpungin S. Pasder O. Pomp O. Taler M. Malovani H. et al.Fer kinase sustains the activation level of ERK1/2 and increases the production of VEGF in hypoxic cells.Cell Signal. 2005; 17: 341-353Google Scholar). Therefore, targeting kinase activity alone with the conventional inhibitor seems to need improvement to block the whole spectrum of the enzyme’s function. Recently, we have witnessed revolutionary paradigm shifts in the landscape of drug design from traditional enzymatic inhibition strategy to more challenging small-molecule–induced protein degradation technology. PROTAC (PROteolysis-TArgeting Chimera), as one of the cores of this technology, is a bifunctional small-molecule compound with one ligand that binds to the target protein and another ligand that binds to E3 ubiquitin ligase, with a linker in between, which allows target protein to irreversibly enter the ubiquitin–proteasome pathway for degradation to affect all functions of the protein (24Benowitz A.B. Jones K.L. Harling J.D. The therapeutic potential of PROTACs.Expert Opin. Ther. Pat. 2021; 31: 1-24Google Scholar, 25Burslem G.M. Crews C.M. Proteolysis-targeting chimeras as therapeutics and tools for biological discovery.Cell. 2020; 181: 102-114Google Scholar, 26Qi S.M. Dong J. Xu Z.Y. Cheng X.D. Zhang W.D. Qin J.J. Protac: an effective targeted protein degradation strategy for cancer therapy.Front. Pharmacol. 2021; 12692574Google Scholar, 27Wang P. Zhou J. Proteolysis targeting chimera (PROTAC): a paradigm-shifting approach in small molecule drug discovery.Curr. Top. Med. Chem. 2018; 18: 1354-1356Google Scholar, 28Chamberlain P.P. Hamann L.G. Development of targeted protein degradation therapeutics.Nat. Chem. Biol. 2019; 15: 937-944Google Scholar). Compared with small-molecule inhibitors and macromolecular antibodies, PROTACs have many distinct advantages, including low dosage, low toxicity, and high selectivity (26Qi S.M. Dong J. Xu Z.Y. Cheng X.D. Zhang W.D. Qin J.J. Protac: an effective targeted protein degradation strategy for cancer therapy.Front. Pharmacol. 2021; 12692574Google Scholar). Of most importance, PROTAC technology can expand its client reservoir to traditionally undruggable targets and overcome drug resistance caused by mutation or overexpression (20Fan G. Zhang S. Gao Y. Greer P.A. Tonks N.K. HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer.Genes Dev. 2016; 30: 1542-1557Google Scholar, 27Wang P. Zhou J. Proteolysis targeting chimera (PROTAC): a paradigm-shifting approach in small molecule drug discovery.Curr. Top. Med. Chem. 2018; 18: 1354-1356Google Scholar). Furthermore, this protein degradation–oriented strategy could simultaneously eliminate the target's enzymatic-dependent and -independent functions (29Cromm P.M. Samarasinghe K.T.G. Hines J. Crews C.M. Addressing kinase-independent functions of Fak via PROTAC-mediated degradation.J. Am. Chem. Soc. 2018; 140: 17019-17026Google Scholar), resulting in complete inhibition and a lower chance for acquired resistance. Given the critical role of FER in ovarian cancer and both kinase-dependent and -independent functions (20Fan G. Zhang S. Gao Y. Greer P.A. Tonks N.K. HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer.Genes Dev. 2016; 30: 1542-1557Google Scholar), we aimed to employ protein degradation technology to design a PROTAC degrader of FER and conduct an in-depth activity evaluation and mechanism study of this compound. This will lay a solid experimental foundation for the ultimate development of the FER-targeting PROTAC drug. Meanwhile, it will also provide substantial evidence for supporting FER as an essential target for ovarian cancer and the scientific significance of degrading FER protein for treating ovarian cancer patients. TAE684 is a small-molecule compound screened as the first-generation inhibitor of anaplastic lymphoma kinase (ALK) (Fig. 1A) (30Galkin A.V. Melnick J.S. Kim S. Hood T.L. Li N. Li L. et al.Identification of NVP-TAE684, a potent, selective, and efficacious inhibitor of NPM-ALK.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 270-275Google Scholar). Interestingly, it also exhibits inhibitory activity against FES kinase, another member of the FER kinase family (31Hellwig S. Miduturu C.V. Kanda S. Zhang J. Filippakopoulos P. Salah E. et al.Small-molecule inhibitors of the c-Fes protein-tyrosine kinase.Chem. Biol. 2012; 19: 529-540Google Scholar). However, TAE684 fails to enter clinical research because of its potential oxidative and metabolic toxicity (30Galkin A.V. Melnick J.S. Kim S. Hood T.L. Li N. Li L. et al.Identification of NVP-TAE684, a potent, selective, and efficacious inhibitor of NPM-ALK.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 270-275Google Scholar, 32Karachaliou N. Santarpia M. Gonzalez Cao M. Teixido C. Sosa A.E. Berenguer J. et al.Anaplastic lymphoma kinase inhibitors in phase I and phase II clinical trials for non-small cell lung cancer.Expert Opin. Investig. Drugs. 2017; 26: 713-722Google Scholar). The emergence of drug resistance and increased demand for better medicines have led to the development of second- and third-generation ALK inhibitors (33Kong X. Pan P. Sun H. Xia H. Wang X. Li Y. et al.Drug discovery targeting anaplastic lymphoma kinase (ALK).J. Med. Chem. 2019; 62: 10927-10954Google Scholar). Brigatinib (Fig. 1A) has been approved for treating ALK-positive metastatic non–small cell lung cancer that has deteriorated after crizotinib treatment or is intolerant to crizotinib in 2017 as for the first-line treatment of ALK-positive metastatic non–small cell lung cancer in 2020. Meanwhile, the KINOSMEScan profiling showed that brigatinib has a strong binding affinity to FER, second only to ALK (34Zhang S. Anjum R. Squillace R. Nadworny S. Zhou T. Keats J. et al.The potent ALK inhibitor Brigatinib (AP26113) overcomes mechanisms of resistance to first- and second-generation ALK inhibitors in preclinical models.Clin. Cancer Res. 2016; 22: 5527-5538Google Scholar). As a starting point of the project, we assessed the inhibitory effect of TAE684 and brigatinib on FER kinase activity by monitoring the phosphorylation of Tyr402, one of its autophosphorylation sites, upon activation. Indeed, both TAE684 and brigatinib effectively inhibited the phosphorylation of FER at Tyr402, with IC50 reaching 0.4106 and 0.1375 μM, respectively (Fig. 1, B and C). According to the value of IC50, both brigatinib and TAE684 exhibited a superior inhibitory effect on the kinase activity of FER than E260 (21Elkis Y. Cohen M. Yaffe E. Satmary-Tusk S. Feldman T. Hikri E. et al.A novel Fer/FerT targeting compound selectively evokes metabolic stress and necrotic death in malignant cells.Nat. Commun. 2017; 8: 940Google Scholar), the only reported FER kinase inhibitor with IC50 around 2 μM. In pursuing PROTAC compounds that degrade ALK, with brigatinib as the warhead, we consistently noticed that one of the lead compounds, SIAIS164018 (Fig. 1A), could degrade ALK and FER simultaneously (35Ren C. Sun N. Liu H. Kong Y. Sun R. Qiu X. et al.Discovery of a brigatinib degrader SIAIS164018 with destroying metastasis-related oncoproteins and a reshuffling kinome profile.J. Med. Chem. 2021; 64: 9152-9165Google Scholar). SIAIS164018 consisted of demethylated brigatinib as ALK or FER binder, pomalidomide as cereblon (CRBN) E3 ligase ligand, and a short acetyl linker. Interestingly, the KINOMEScan profiling revealed that SIAIS164018 inhibited FER kinase more preferentially than ALK (35Ren C. Sun N. Liu H. Kong Y. Sun R. Qiu X. et al.Discovery of a brigatinib degrader SIAIS164018 with destroying metastasis-related oncoproteins and a reshuffling kinome profile.J. Med. Chem. 2021; 64: 9152-9165Google Scholar). Nevertheless, the degradation capability of SIAIS164018 on FER still needs to be improved (35Ren C. Sun N. Liu H. Kong Y. Sun R. Qiu X. et al.Discovery of a brigatinib degrader SIAIS164018 with destroying metastasis-related oncoproteins and a reshuffling kinome profile.J. Med. Chem. 2021; 64: 9152-9165Google Scholar). After multiple rounds of a structure optimization, including using lenalidomide to replace pomalidomide and screening the length and type of linkers, we finally obtained SIAIS352008 and SIAIS262039 (hereinafter referred to as 008 and 039, Fig. 1A) with better degradation efficacy. We evaluated their biochemical and biological properties in the context of ovarian cancer. Previous studies have demonstrated the upregulation of FER proteins in multiple ovarian carcinoma–derived cell lines (20Fan G. Zhang S. Gao Y. Greer P.A. Tonks N.K. HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer.Genes Dev. 2016; 30: 1542-1557Google Scholar). We evaluated the protein degradation efficacy of PROTAC compounds 008 and 039 first on OVCAR-5 and CAOV4 ovarian cancer cell lines, which have the highest FER protein expression (20Fan G. Zhang S. Gao Y. Greer P.A. Tonks N.K. HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer.Genes Dev. 2016; 30: 1542-1557Google Scholar). Compared with the dimethyl sulfoxide (DMSO) control, 008 or 039 treatment robustly degraded the kinase, and the effective concentration was as low as 1 nM (Fig. 2, A and B). In addition, TAE684 and brigatinib demonstrated no degradation capability of FER kinase even at a high concentration of 1000 nM (Fig. 2, A and B). Next, we assessed whether the 008- and 039-mediated dynamic FER protein turnover could be recapitulated in the other seven ovarian cancer cell lines with high FER expression. Indeed, both PROTAC compounds universally disrupt the endogenous FER proteins in these cell lines (Fig. 2, C and D). Third, we evaluated the capability of these two compounds to degrade the ectopically overexpressed FER proteins in human embryonic kidney 293FT (HEK293FT) cells. As shown in Figure 2, E and F, both 008 and 039 could also degrade the exogenous FER protein in a dose-dependent manner. In summary, these two PROTAC compounds effectively disrupted the expression of FER proteins in cells. To accurately quantify the efficacy of both PROTAC compounds on FER protein degradation, we measured their DC50 values in OVCAR-5 and CAOV4 cell lines. The results indicated that 008 and 039 degraded endogenous FER protein in a concentration-dependent manner without any sign of hook effect (Fig. 3, A and C). The DC50 values of 008 and 039 in the OVCAR-5 cell line were 0.2883 nM and 0.4113 nM, respectively (Fig. 3B), and in the CAOV4 cell line, were 0.7839 nM and 0.4356 nM, respectively (Fig. 3D). To explore the time trajectory of endogenous FER degradation by PROTAC compounds, we treated OVCAR-5 or CAOV4 cells with 10 nM 008, respectively, and collected samples at indicated time points for immunoblotting analysis. The PROTAC 008 started to degrade FER proteins in OVCAR-5 cells 20 min after treatment, and the degradation reached the maximum level in 3 h. The FER protein in CAOV4 cells began to be degraded 30 min after the treatment and reached the maximum degradation in 6 h, respectively (Fig. 4, A and B). Meanwhile, we applied the cycloheximide (CHX) treatment to estimate the half-life of the FER proteins in OVCAR-5 cells. The kinase was relatively stable in the DMSO group, with no significant degradation occurring in 24 h (Fig. 4C). However, the expression level of FER proteins was significantly reduced upon 100 nM 008 treatment, with a half-life of less than 1 h (Fig. 4C). These results indicated that the PROTAC compounds' addition largely destroyed the FER protein's stability. To evaluate the degradation selectivity of our PROTAC compound, we performed a mass spectrometry–based quantitative proteomics analysis in the OVCAR-5 cell line in the absence and presence of the 008 compounds. A total of 4458 proteins were identified by mass spectrometry analysis, among which eight proteins could be degraded by 008 in three independent repeated experiments (AAK1 [AP2-associated kinase 1], BTF3 [basic transcription factor 3], PARS2 (prolyl-TRNA synthetase 2), GAK (cyclin G-associated kinase), FER, YEATS2 (YEATS domain containing 2), DRAM2 (DNA damage–regulated autophagy modulator 2), and PPIL2 [peptidylprolyl isomerase like 2]), as illustrated by the volcano plot (Fig. 4D). It is worth noting that AAK1, GAK, and FER are kinases. Compared with DMSO-treated control samples, 008-treated cells showed a 99.56% (p = 1.776 × 10−8) reduction in FER protein level (Fig. 4D). Subsequently, we verified the top candidate kinases shown in the high-confidence intervals of the volcano plot. The immunoblotting analysis confirmed that AAK1, GAK, and FER protein levels in OVCAR-5 and CAOV4 cells were significantly downregulated upon 008 or 039 treatment for 16 h (Fig. 4, E and F). IRAK1 and CHK2, two negative control kinases we included, showed no protein level change in the same treatment condition (Fig. 4, E and F). These data indicated that the downregulation of AAK1, GAK, and FER induced by 008 and 039 is selective. Two major systems within cells are responsible for protein quality control: the ubiquitin–proteasome system (UPS) and the lysosome-mediated protein degradation system (36Wang Y. Le W.D. Autophagy and ubiquitin-proteasome system.Adv. Exp. Med. Biol. 2019; 1206: 527-550Google Scholar). Usually, the PROTAC technology harnesses the UPS system for target protein clearance (25Burslem G.M. Crews C.M. Proteolysis-targeting chimeras as therapeutics and tools for biological discovery.Cell. 2020; 181: 102-114Google Scholar). Hence, we attempted to determine whether 008- or 039-induced FER degradation depends on the UPS route. As shown in Figure 4G, 008 triggered robust degradation of FER as well as AAK1 and GAK1 in the liver carcinoma cell line Bel7404; however, CRISPR–Cas9-meditated E3 ligase CRBN knockout significantly blocked turnover of these kinase proteins, indicating that the E3 ligase CRBN is required for PROTAC compound–mediated kinase degradation. Adding the proteasome inhibitor MG132, but not lysosome inhibitor chloroquine, effectively prevented the degradation of FER protein initiated by these two compounds (Fig. 4, H–K), confirming the necessity of proteasome machinery in FER clearance. Given the superior degradation effect on FER kinase, we conducted a series of cell-based assays to assess their anticancer properties. CTG-based cell proliferation assay showed no significant growth delay upon treating PROTACs in four ovarian cancer cell lines, including CAOV4, OVK18, PA-1, and COLO316 (Fig. S1, A–D). Consistently, no significant difference was observed neither in cell cycle distribution (Fig. S1, E–F) nor in cell apoptosis (Fig. S1, G–H) between DMSO or PROTAC-treated CAOV4 cells, indicating these compounds have minimal capability in inhibiting ovarian cancer cell proliferation and survival. Cancer metastasis often leads to treatment failure. The FER kinase has been reported to regulate cell migration and invasion in many types of cancers (6Ivanova I.A. Vermeulen J.F. Ercan C. Houthuijzen J.M. Saig F.A. Vlug E.J. et al.FER kinase promotes breast cancer metastasis by regulating alpha6- and beta1-integrin-dependent cell adhesion and anoikis resistance.Oncogene. 2013; 32: 5582-5592Google Scholar, 16Ahn J. Truesdell P. Meens J. Kadish C. Yang X. Boag A.H. et al.Fer protein-tyrosine kinase promotes lung adenocarcinoma cell invasion and tumor metastasis.Mol. Cancer Res. 2013; 11: 952-963Google Scholar, 17Arregui C. Pathre P. Lilien J. Balsamo J. The nonreceptor tyrosine kinase fer mediates cross-talk between N-cadherin and beta1-integrins.J. Cell Biol. 2000; 149: 1263-1274Google Scholar, 18Sangrar W. Gao Y. Scott M. Truesdell P. Greer P.A. Fer-mediated cortactin phosphorylation is associated with efficient fibroblast migration and is dependent on reactive oxygen species generation during integrin-mediated cell adhesion.Mol. Cell. Biol. 2007; 27: 6140-6152Google Scholar). To evaluate the efficacy of the FER-targeting PROTAC compounds on ovarian cancer cell motility suppression, we performed the wound healing assay in CAOV4 cells, using brigatinib as a control. As shown in Figure 5, A and B, whereas brigatinib exhibited mild suppression at a high concentration (500 nM), both 008 and 039 inhibited cell wound healing to a greater extent at a relatively low concentration (50 and 100 nM). Indeed, this inhibitory effect on cell motility was ready to be observed at 10 nM (Fig. S2, A and B), the concentration of which caused effective FER protein degradation. Consistently, we observed similar results in the transwell assay. Compared with brigatinib, which exhibited slight inhibition on cell migration, 008 and 039 significantly inhibited cell migration through the Boyden chamber cassette at both low (100 nM) and high (500 nM) concentrations (Fig. 5, C and D). Actually, we started to observe a significant delay in cell migration from 10 nM compound concentration (Fig. S2, C and D). Therefore, both PROTAC compounds demonstrated superior function than brigatinib on ovarian cancer cell motility suppression. To verify that the PROTAC-induced cell motility decrease is due to the degradation of FER protein, we established a FER-knockdown CAOV4 cell line using shRNA technology (Fig. 6A), followed by the wound healing assay. Indeed, the wound-healing ability of CAOV4shFER cells was lower than that of CAOV4shCon cells (Fig. 6, B and C). We also observed that 008 significantly dampened the wound-healing capacity of CAOV4 cells (Fig. 6, B and C). Interestingly, there was little difference in the migration ability of 008-treated CAOV4shCon and CAOV4shFER cells up to 12 h of treatment, indicating the suppressive effect of PROTAC compounds was due to the on-target degradation of FER protein, at least under the current experimental conditions (Fig. 6, B and C). To further evaluate the efficacy of these two PROTAC compounds in vivo, we intraperitoneally injected CAOV4shCon or CAOV4shFER cells in NSG mice, followed by either vehicle or 008 treatment and counted the number of tumor nodules of metastasis on the peritoneal wall. Compared with the mice injected with CAOV4shCon cells, the pharmacological degradation of FER kinase or the genetical knockdown of FER expression showed an equivalent decrease in tumor nodules on the peritoneal wall (Fig. 6D). Interestingly, FER knockdown synergized with the administration of the PROTAC degrader 008 to show a more reduced number of tumor nodules on the peritoneal wall, implying either incomplete suppression of FER expression by shRNA or the potential degradation of other targets may also contribute to the reduced metastasis phenotype (Fig. 6D). Nevertheless, the PROTAC compound 008 significantly suppressed ovarian cancer cell motility and invasiveness in vivo. Alike known oncogenic fusion ITK (interleukin-2-induced T-cell kinase)-spleen tyrosine kinase in peripheral T-cell lymphomas, ITK–FER has also been identified as another potential oncogene in peripheral T-cell lymphomas by paired DNA and RNA next-generation sequencing analysis (Fig. 7A, ITK–FER fusion 1) (37Boddicker R.L. Razidlo G.L. Dasari S. Zeng Y. Hu G. Knudson R.A. et al.Integrated mate-pair and RNA sequencing identifies novel, targetable gene fusions in peripheral T-cell lymphoma.Blood. 2016; 128: 1234-1245Google Scholar). The ITK–FER protein retains the ITK N-terminal pleckstrin homology and FER C-terminal kinase domains (37Boddicker R.L. Razidlo G.L. Dasari S. Zeng Y. Hu G. Knudson R.A. et al.Integrated mate-pair and RNA sequencing identifies novel, targetable gene fusions in peripheral T-cell lymphoma.Blood. 2016; 128: 1234-1245Google Scholar). A very similar ITK–FER fusion mutation (Fig. 7A, ITK–FER fusion 2), caused by t (1, 5) (p34; q21.3) chromosome rearrangement, was also reported in a patient suffering from the follicular T-cell lymphoma (38Debackere K. van der Krogt J.A. Tous" @default.
• W4376646473 created "2023-05-17" @default.
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• W4376646473 title "Development of the nonreceptor tyrosine kinase FER-targeting PROTACs as a potential strategy for antagonizing ovarian cancer cell motility and invasiveness" @default.
• W4376646473 cites W1978154545 @default.
• W4376646473 cites W1985601287 @default.
• W4376646473 cites W1990598699 @default.
• W4376646473 cites W1994170326 @default.
• W4376646473 cites W1997632499 @default.
• W4376646473 cites W1998661425 @default.
• W4376646473 cites W2007058107 @default.
• W4376646473 cites W2014639631 @default.
• W4376646473 cites W2017430826 @default.
• W4376646473 cites W2049896044 @default.
• W4376646473 cites W2052140448 @default.
• W4376646473 cites W2080905329 @default.
• W4376646473 cites W2084054884 @default.
• W4376646473 cites W2090271522 @default.
• W4376646473 cites W2097132638 @default.
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• W4376646473 cites W2164999320 @default.
• W4376646473 cites W2404359304 @default.
• W4376646473 cites W2442366440 @default.
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• W4376646473 cites W2533300620 @default.
• W4376646473 cites W2590932802 @default.
• W4376646473 cites W2594488303 @default.
• W4376646473 cites W2611654128 @default.
• W4376646473 cites W2763290628 @default.
• W4376646473 cites W2803393870 @default.
• W4376646473 cites W2808971736 @default.
• W4376646473 cites W2895912954 @default.
• W4376646473 cites W2901669535 @default.
• W4376646473 cites W2913179893 @default.
• W4376646473 cites W2922827447 @default.
• W4376646473 cites W2967295339 @default.
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• W4376646473 cites W2990651874 @default.
• W4376646473 cites W2998130894 @default.
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