Effective degradation of EGFRL858R+T790M mutant proteins by CRBN- based PROTACs through both proteosome and autophagy/lysosome degradation systems
Xiaojuan Qu a, b, f, 1, Haixia Liu a, c, f, 1, Xiaoling Song a, *, 1, Ning Sun a, 2, Hui Zhong a, d, 2, Xing Qiu a, e, Xiaobao Yang a, ***, Biao Jiang a, e, **
A B S T R A C T
Targeted therapy of treating patients with specific tyrosine kinase inhibitors (TKIs) is currently the standard care for epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer. However, the inevitably developed drug resistance in patients to EGFR TKIs is the biggest obstacle for cancer targeted therapy. About 60% of drug resistance to the 1st generation of EGFR TKIs was resulted from an acquired T790M mutation in the kinase domain of EGFR protein. Proteolysis targeting chimera (PROTAC) is a lately-developed technology to target point of interest proteins for degradation. Because EGFR- mutant lung cancers are highly dependent on EGFR proteins, designing specific PROTAC molecules to degrade EGFR proteins from cancer cells provides a very promising strategy to treat such patients and eradicate drug resistance. Currently, there is no cereblon (CRBN)-based PROTAC reported able to degrade T790M-containing EGFR resistant proteins. In this study, we synthesized two novel CRBN-based EGFR PROTACs, SIAIS125 and SIAIS126, based on EGFR inhibitor canertinib and cereblon ligand pomalidomide. These two degraders displayed potent and selective antitumor activities in EGFR TKI resistant lung cancer cells. Firstly, they could selectively degrade EGFRL858R+T790M resistant proteins in H1975 cells at the concentration of 30e50 nM, and EGFREx19del proteins in PC9 cells. But they did not degrade EGFREx19del+T790M mutant proteins in PC9Brca1 cells or wild type EGFR in A549 lung cancer cells. They could also selectively inhibit the growth of EGFR mutant lung cancer cells but not that of normal cells or A549 cells. Secondly, the degradation of EGFRL858R+T790M proteins was long lasting up to 72 h. Thirdly, these degraders displayed better inhibition of EGFR phosphorylation in H1975 cells and PC9Brca1 cells comparing to canertinib. Finally, these degraders could also induce significant apoptosis and cell cycles arrest in H1975 cells. Pre-incubation with canertinib, pomalidomide or ubiquitination inhibitor MLN4924 totally blocked EGFR degradation by PROTACs. Mechanistic studies showed that PROTAC could induce autophagy in lung cancer cells. PROTAC-induced EGFR degradation acted through both ubiquitin/pro- teosome system and ubiquitin/autophagy/lysosome system. Elevating autophagy activities enhanced EGFR degradation and cell apoptosis induced by PROTACs. Our research not only offered a novel PROTAC tool to target EGFR TKI drug resistance in lung cancer, but also firstly demonstrated that the involvement of autophagy/lysosome system in PROTAC- mediated target protein degradation.
Keywords:
EGFR PROTAC
Resistance
Non-small cell lung cancer T790M
Autophagy/ lysosome Degradation
1. Introduction
Lung cancer is the leading cause of cancer-related death in the worldwide [1], of which about 90% is non-small cell lung cancer (NSCLC) [2,3]. About 15%e45% of NSCLC patients carrying EGFR activating mutations, and currently, targeted therapy is the stan- dard care for such patients [4e7]. Treating patients with EGFR specific tyrosine kinase inhibitors greatly improved the life quali- ties of NSCLC patients comparing to traditional chemotherapy [8e11]. However, drug resistance to these kinase inhibitors even- tually occurs a period after initial treatment even when the latest FDA approved EGFR TKI drug osimertinib is used [12e14]. The drug resistanceproblem is currently a big obstacle for targeted therapy in clinic. Effective therapeutic strategies that can overcome drug resistance are in great need to treat such patients. Because a big proportion of drug resistance is caused by alterations in EGFR proteins [15], eradicating EGFR proteins from cancer cells provides a promising way to treat such patients and overcome drug resistance.
Proteolysis targeting chimera (PROTAC) is a brand new technology that can degrade oncogenic proteins and has great potential to overcome drug resistance. Each product of PROTAC is a bi- functional molecule that can bind both the protein of interest (POI) and an E3 ubiquitin ligase simultaneously [16]. After entering cells, it can recruit the E3 ubiquitin ligase to the proximity of the target protein, leading to the ubiquitination of its target proteins followed by protein degradation by proteasome [17e19]. Because many onco-proteins act as the critical drivers to promote cancer formation, eliminating these onco-proteins by PROTAC molecules will lead to the death of these cancer cells and theoretically provide an ideal way to treat such diseases. Many PROTACs have been developed to target onco-proteins now, and the most successful PROTAC compound is ARV110. Latest data from ASCO2020 had shown that ARV110 was very effective to treat cancer patients in clinic and it could induce tumor regression in drug resistant cancer patients [20].
EGFR mutant lung cancer is found in about 15% of Caucasion NSCLC patients and about 40e50% of Asian NSCLC patients [5e7]. This type of cancer is driven by EGFR onco-genic activating muta- tions, and the majority of these mutations are either EGFR exon 21 point mutations (L858R) or exon 19 deletion mutations (Exdel19). The majority of drug resistance to 1st generation EGFR TKIs is caused by an acquired T790M mutation in the EGFR kinase domain (about 60%) [15]. Effectively targeting EGFR proteins with acquired T790M mutations is critical to evade such drug resistance. Several EGFR-targeted PROTAC molecules have been developed to degrade EGFR oncogenic proteins in lung cancer in the past couple of years [17,21e24]. In these studies, some EGFR PROTACs could degrade
EGFRL858R+T790M mutant proteins. These PROTACs utilized either VHL ligands or MDM2 ligands to recruit E3 ubiquitin ligase. So far, there is no CRBN-based EGFR PROTAC reported able to degrade EGFR proteins with acquired T790M mutations. Although three different groups had tried to generate EGFR degraders using CRBN ligands, all these CRBN-based PROTACs fail to degrade EGFRL858R+T790M mutant proteins [21e23]. Herein, we successfully synthesized two novel CRBN-recruiting EGFR PROTACs that could effectively degrade EGFRL858R+T790M mutant proteins at the con- centration of 30e50 nM. These degraders could also effectively degrade EGFREx19del mutants in PC9 cells, but not EGFR wild type protein in A549 lung cancer cells. The degradation mechanisms of these PROTACs were also investigated in this study. Our study provided the first evidence that both the ubiquitin/proteosome system and ubiquitin/autophagy/lysosome system were involved in the degradation of target proteins by PROTACs.
2. Results
2.1. Synthesis of CRBN-based EGFR-targeting PROTACs
To generate EGFR PROTACs, pomalidomide was used in our study as the ligand of E3 ubiquitin ligase. It is the second generation of immuno-modulatory drug (IMiDs) and was approved by FDA to treat multiple myeloma in 2013. It is orallyavailable and can induce significant degradation of essential ikaros transcription factors through interacting with cereblon E3 ubiquitin ligase [25]. Comparing to two other cereblon ligands, thalidomide and its de- rivative lenalidomide, pomalidomide is the most potent immuno- modulatory drug [26]. Previously, it has been successfully used to design PROTACs (such as BRD4 targeting PROTAC ARV-825), leading to fast and potent degradation of target proteins [27]. In our pre- screening, CRBN-based PROTACs generated from canertinib could effectively inhibit the growth of cancer cells and induce the degradation of EGFRL858R+T790M mutant proteins. Canertinib (Fig. 1A) is an effective and irreversible EGFR inhibitor that was previously developed for cancer treatment by University of Auck- land and Pfizer [28]. It is water soluble, orally-available, and highly potent in repressing the growth of tumor in xenograft mouse models [28]. Carbon linker is a very commonly used linker for PROTAC technology. So it was used in our PROTAC design to generate a series of canertinib-CRBN based PROTACs with different lengths of linkers (Scheme 1).
The scheme of synthetic routes for generating these EGFR de- graders was displayed in Scheme 1 and Experimental section 4.1. All the starting materials were commercially available. Firstly, tert- butyl piperazine-1-carboxylate and 3-bromopropan-1-ol form compound 3 by nucleophilic substitution. Then, compound 3 was reacted with N-(3-chloro-4-fluorophenyl)-7-fluoro-6- nitro- quinazolin-4-amine which was purchased from the company to get the key intermediate 5. The compound 5 was following hydroge- nation and condensation with Acryloyl chloride and removed Boc protection under TFA condition to afford Canertinib derivative 7 (SIAIS092). Meanwhile, 2-(2,6-dioxopiperidin-3-yl)- 4- fluo- roisoindoline-1,3-dione was reacted with various amines(9a~9f) by nucleophilic substitution following hydrolyzation and removed tert-butyl protection to form intermediate 10a~10f. Finally, the compounds 10a~10fcondensed with compound 7 (SIAIS092) to afford the desired compounds SIAIS121~SIAIS126.
The degradation effects of our synthesized PROTACs on EGFRL858R+T790M proteins were examined in H1975 cells (Fig. 1F), and the effects on cell proliferation were evaluated in a bunch of EGFR mutant or wild type cell lines (Table 1). Two PROTACs SIAIS125 and SIAIS126 were identified to have the best degradation capabilities for EGFRL858R+T790M mutants (Fig. 1F) and best anti- proliferation of EGFR mutant cancer cells (Table 1) in our pre- screening. Further increasing the length of protac linker did not achieve better effects than SIAIS126. So, these two PROTACs were mainly investigated in the following studies. The structures of canertinib, pomalidomide and PROTACs, SIAIS125 and SIAIS126 were displayed in Fig. 1.
2.2. SIAIS125 & SIAIS126 induced effective degradation of mutant EGFR proteins in lung cancer cells
The degradation capabilities of SIAIS125 and SIAIS126 were evaluated in a bunch of EGFR mutant or wild type lung cancer cell lines. Results showed that turning canertinib into PROTAC de- graders endowed it with great capabilities to degrade mutant EGFR protein (Fig. 2). Firstly, SIAIS125 and SIAIS126 could degrade oncogenic EGFR proteins. In PC9 cells, SIAIS125 and SIAIS126 induced significant degradation of EGFREx19del proteins after treating cells for 48 h, and the degradation occurred at various concentrations above 10 nM (Fig. 2A).
Secondly, these two degraders could very effectively degrade EGFRL858R+T790M mutant proteins in H1975 lung cancer cells. After treating H1975 cells for 16 h, SIAIS125 and SIAIS126 degraded EGFRL858R+T790M mutant proteins at the minimum concentration of about 50 nM and 30 nM respectively (Fig. 2B). The degradation capability of SIAIS126 was stronger than that of SIAIS125. Treating cells with EGFR warhead inhibitor canertinib did not reduce EGFR protein level, but instead, it slightly increased EGFR total protein by about 15e31% (Fig. 2B). The maximum extents of degradation for these two degraders reachedabout 95e100%. Thirdly, the degra- dation of EGFR proteins occurred in a dose- and time-dependent manner (Fig. 2AeD). With the increase in PROTAC concentrations, EGFR degradation effects were elevated. After treating H1975 cells with PROTACs for 48 h, almost all EGFR proteins were degraded at the concentrations above 30 nM (Fig. 2D). At the same time, the degradation degrees were enhanced with the increase of incuba- tion time, and the maximum degradation reached about 98%e100% after 48 h degraders’ treatment (Fig. 2C).
Finally, the degradation effects on EGFR proteins were mutant- specific. SIAIS125 and SIAIS126 did not degrade EGFREx19del+T790M proteins after 16 h treatment in PC9Brca1 cells (Fig. 2E). Even after treating cells with SIAIS125 or SIAIS126 for 48 h, EGFREx19del+T790M mutant proteins were barely degraded (Fig. 2F). Additionally, SIAIS125 and SIAIS126 barely affected the total level of wild type EGFR protein in A549 human cancer cells (Suppl. Figure 1A). In contrast, in Ba/F3 cells that were stably expressed human EGFRL858R+T790M mutant proteins, the levels of EGFR protein were also significantly reduced by SIAIS125 and SIAIS126 at 100 nM concentration (Supplemental Figure 1B). A slightly weaker degra- dation of EGFR protein in Ba/F3 cells than H1975 cells might be caused by species difference when a mouse cell line instead of a human cell line was used.
In summary, SIAIS125 and SIAIS126 could selectively induce the degradation of EGFREx19del activating mutants and EGFRL858R+T790M resistant proteins. These data also suggested that turning inhibitor canertinib into a PROTAC could improve its target specificity on EGFR mutant proteins and potentially reduce its toxic side effects.
2.3. Evaluation of the anti-proliferative activity in vitro
The effects of PROTACs on the proliferation of EGFR-mutant lung cancer cells were also examined (Table 1). Results showed that SIAIS125 and SIAIS126 not only inhibited the proliferation of non- small cell lung cancer cells expressing EGFREx19del mutant proteins such as PC9 and HCC827, but also inhibited the growth of H1975 and PC9Brca1 cells that express EGFRL858R+T790M or EGFRex19del+T790M mutants respectively (Table 1). SIAIS125 and SIAIS126 displayed a slightly weaker inhibition of the proliferation comparing to parent inhibitor canertinib, but were more potent than canertinib derivative SIAIS092. For example, in H1975 cells, the IC50s for SIAIS125 and SIAIS126 were 19.41 and 30.76 nM respectively, which were close to that of canertinib (11.34 nM)but better than that of canertinib derivative SIAIS092 (162.1 nM). SIAIS125 and SIAIS126 also inhibited the proliferation of EGFRL858R+T790M expressing Ba/F3 cells. However, they did not affect the proliferation of A549 cells which express wild type EGFR. We also examined the effects of SIAIS125 and SIAIS126 on the proliferation of two normal cell lines: human embryo kidney cell line 293T cells and human normal foreskin fibroblast BJ cells (Table 1). Both degraders did not inhibit the proliferation of these two cell lines. These data indicated that these two degraders had potent and selective anti-tumor activities in EGFR mutant lung cancer cells with very low side effects on EGFR wild type cells.
2.4. Better inhibition of EGFR activation by degraders in EGFR mutant lung cancer cells
EGFR promotes cell proliferation through phosphorylation itself followed by the activation of downstream signaling pathways. Our data showed that both canertinib and EGFR degraders could inhibit the phosphorylation of EGFR in H1975 cells (Fig. 3A). Interestingly, SIAIS125 and SIAIS126 exhibited stronger inhibition of EGFR phosphorylation than canertinib did (Fig. 3A). Similar effects were observed in PC9Brca1 cells that express EGFREx19del+T790M mutants (Fig. 3B). Thus, turning an inhibitor canertinib into a PROTAC degrader endowed the inhibitor with stronger inhibition capabilities.
2.5. The degradation effects on EGFR proteins were long lasting
To study how long the effects on EGFR protein degradation could last, a drug wash-out experiment was performed according to the experimental scheme shown in Fig. 4A. H1975 cells were firstly treated with 300 nM either SIAIS125 or SIAIS126 for 24 h. Then, the condition medium was discarded and cells were washed with PBS for 3 times. Fresh complete 1640 culture medium with 10% fetal calf serum was added to cells for maintenance. Finally, cells were harvested at indicated time points and EGFR total protein levels were evaluated by Western blot (Fig. 4A). Results showed that EGFR proteins were degraded completely by 300 nM of SIAIS125 or SIAIS126 after 24-h treatment (Fig. 4B). The degrada- tion effects of SIAIS125 and SIAIS126 on EGFR proteins were long lasting. After changing with fresh complete culture medium, EGFR protein levels did not recover even at the time of 72 h after drug removal (Fig. 4B).
2.6. degraders induced cell cycle arrest in cancer cells
The effects of SIAIS125 and SIAIS126 on cell doubling were tested in H1975 cells by labeling cells with a fluorescent dye CFSE (Fig. 5A). After cell duplication, the dye will split into two daughter cells and the intensities of fluorescent dye will reduce by half. Re- sults showed that in H1975 cells that were treated with 100 nM or 300 nM EGFR degraders, the intensities of fluorescence were about similar to that in the DMSO treatment on day 1; but, on day 4 the fluorescence levels were significantly higher than that of DMSO control. This indicated that after PROTAC treatment, cell doubling time was greatly increased comparing to DMSO control, and consequently the intensities of cell fluorescence decreased comparing to those treated with DMSO (Fig. 5A). SIAIS126 exhibi- ted stronger repression effects on cell doubling than SIAIS125 did. The proliferation of cells was slower in SIAIS126 treatment than that in SIAIS125 treatment when both were used to treat cells at 300 nM.
In the meanwhile, PROTAC induced cell cycle arrest in H1975 cells (Fig. 5B). Results of flow cytometry experiment showed that PROTAC treatment increased the proportion of cells in G0/G1 phase, and dramatically reduced the proportion of cells in S phase (Fig. 5B). In summary, degrader treatment induced cell cycle arrest and inhibited cell proliferation.
2.7. degraders promoted cell apoptosis in cancer cells
To see whether SIAIS125 and SIAIS126 induced cell apoptosis, H1975 cells were firstly incubated with PROTACs for 72 h, and then were collected and analyzed by flow cytometry with Annexin V- FITC apoptosis detection kit. Results showed that treating cells with SIAIS125 or SIAIS126 induced the apoptosis of H1975 cells (Fig. 5C). With the increase in the degrader concentrations, less live cells were presented and more apoptosis events were observed. Stron- ger apoptosis effects were observed in cells treated with higher concentration of SIAIS125 or SIAIS126 comparing to those treated with canertinib (Fig. 5C).
2.8. degraders affected cell migration
The effects of SIAIS125 and SIAIS126 on cell migration were evaluated via wound healing assay (Fig. 5D). Briefly, H1975 cells were seeded in a 24-well-plate. Cells were scratched at confluence with the tip of a 0.2 mL pipette 24 h later, and then were washed for 3 times with PBS. Followed by incubating cells with PROTACs at the concentration of 300 nM or 500 nM, six photos were taken from each well at the indicated time points as shown in supplementary figure 2. ImageJ software was used to quantify the areas that were not covered by cells. Results revealed that SIAIS126 affected cell migration at the concentration of both 300 nM and 500 nM, while SIAIS125 could only affect cell migration at 500 nM (Fig. 5D).
2.9. Degrader SIAIS126 blocked cancer cell invasion
The effects of degraders on cell invasion were evaluated via transwell assay (Fig. 5E). H1975 cells that stably expressed GFP proteins were seeded inside of the matrigel-coated transwell chamber, and EGFR degraders were then added to cells at indicated concentrations. Five photos were randomly taken from the lower side of the chamber at 18-h and 42-h time points after initial drug treatment. Three biological replicates were set up for each treat- ment. Cell numbers were counted and were used to compare the difference among different treatments (Fig. 5E). Results showed that 18-h after drug treatment, both canertinib and SIAIS125 did not show much effects on cell invasion comparing to DMSO control. SIAIS126 significantly reduced the number of invaded cells at the concentration of 500 nM (P < 0.05). There was about 38% of reduction in the number of invaded cells after 18 h treatment, and 40% after 42-h drug treatment in SIAIS126 treatment (Fig. 5E). Because both SIAIS125 and SIAIS126 could inhibit cell proliferation and induced cell apoptosis at about similar extents, but only SIAIS126 at high concentration affected cell migration and invasion, we speculated that SIAIS126 might have the ability to block cell invasion. 2.10. PROTACs degrade EGFR proteins through both ubiquitin/ proteosome system and autophagy/lysosome system The mechanisms of EGFR degradation by PROTACs were studied in H1975 cells. Results revealed that the degradation acted through not only the ubiquitin/proteosome system, but also the ubiquitin/ autophagy/lysome degradation system. Firstly, the degradation of EGFR proteins relied on the recruit- ment of E3 ubiquitin ligase to EGFR protein. In this experiment, two components of EGFR PROTAC molecules, canertinib (1 mM) and CRBN ligand pomalidomide (10 mM), were added to cells before treatment to compete with PROTACs (0.3 mM) to bind to EGFR and E3 ubiquitin ligase respectively (Fig. 6A and B). The aim was to validate that the requirement of both EGFR ligand binding and E3 ubiquitin ligase binding for EGFR degradation. Results showed that both canertinib and pomalidomide were able to out-compete EGFR PROTAC molecules to bind to either EGFR protein or E3 ubiquitin ligase (Fig. 6B). In the presence of these two ligands, the degrada- tion effects on EGFR protein by degraders were eliminated. This suggested that both EGFR ligand and E3 ligase ligand were required for EGFR degradation by degraders SIAIS125 and SIAIS126. Secondly, PROTAC-induced EGFR degradation relied on the ubiquitination of EGFR protein (Fig. 6C). An inhibitor pevonedistat (MLN4924) was added to cells to block the process of ubiquitina- tion prior PROTAC treatment, and then the effects on EGFR degra- dation were evaluated by Western blot (Fig. 6C). Results showed that treating cells with degrader SIAIS125 or SIAIS126 reduced EGFR protein levels. However, the effects were totally blocked by the inhibitor MLN4924 (1 mM), which in turn led to a recovery at EGFR total protein levels. This indicated that the process of ubiq- uitination was an essential procedure for EGFR degradation by PROTACs. Thirdly, ubiquitin/proteosome system (UPS) was partially responsible for EGFR degradation by PROTACs. Previous studies showed that ubiquitin/proteosome/system was the major mecha- nism responsible for PROTAC-induced EGFR degradation. So, we used a reversible proteosome inhibitor MG132 to stop the proteosome-mediated protein degradation, and then evaluated the impacts on EGFR degradation (Fig. 6A and B). Pretreating cells with 100 nM MG132 only led to a partial recovery of EGFR protein level in degrader treatments, which indicated that there might be other mechanisms involved in PROTAC-induced EGFR degradation (Fig. 6B). To further test this hypothesis, a second-generation irre- versible proteosome inhibitor, carfilzomib, was used to examine the impacts on EGFR degradation (Fig. 6C). Treating cells with 5 nM of carfilzomib alone increased endogenous EGFR protein levels, which indicated that proteosome-mediated pathway was involved in endogenous EGFR protein degradation. When treated together with EGFR PROTACs, carfilzomib also could not totally block the degradation of EGFR by SIAIS125 or SIAIS126 (Fig. 6C). In summary, above data indicated that other protein degradation pathways might be also present and were equally important for EGFR degradation by PROTACs. Fourthly, an autophagy/lysosome-mediated protein degradation system was required for EGFR protein degradation by PROTACs. It has been demonstrated that inhibiting proteosome function could induce a switch in the balance of target protein degradation from an ubiquitination/proteosome -dependent pathway to an ubiquitination/lysosomal-dependent protein degradation pathway [29]. To examine whether lysosome pathway was involved in the ubiquitin/proteosome independent degradation of EGFR, H1975 cells were incubated with different concentrations of bafi- lomycin A1, a lysosome inhibitor, for 16 h before treatment with PROTACs (Fig. 7A). Western blot analysis showed that endogenous EGFR protein levels were increased. Dramatically after bafilomycin A1 treatment (Fig. 7B). This result was consistent with literature that lysosome-mediated degradation played an important role in endogenous EGFR protein degradation [30,31]. Interestingly, when cells were pretreated with bafilomycin A1 for 2 h and then treated with 0.3 mM SIAIS125 or SIAIS126 for 16 h, the degradation effects on EGFR protein were partially reversed with increase in the concentration of lysosome inhibitor (Fig. 7B). These data indicated that lysosome-mediated pathway also took part in the PROTAC-induced EGFR degradation. Previous studies by Zhao et al. showed that autophagy might be involved in the degradation of EGFR by PROTAC molecules P3 [24]. So, we examined the activation of autophagy by EGFR degraders (Fig. 7C). Both serum free treatment and rapamycin were used as a positive control for the activation of autophagy, and the relative protein levels of autophagy markers LC3B II to LC3B I were used to evaluate the occurrence of autophagy. Results showed that both serum starvation and rapamycin treatment greatly increased the ratio of LC3B II to LC3B I protein level, which indicated that auto- phagy occurred in these cases (Fig. 7C). Interestingly, treating cells with 30, 100 or 300 nM of EGFR degrader SIAIS126 also increased the ratio of LC3B II/LC3B I (Fig. 7C). These results indicated that EGFR degraders induced autophagy in lung cancer cells. Combi- nation treatment of cells with rapamycin and degrader SIAIS126 further increased the degradation of EGFR protein comparing to SIAIS126 alone (Fig. 7C). Additionally, under serum starvation condition that promoted autophagy, the number of live cells was dramatically reduced when treated with SIAIS125 or SIAIS126 for either 12 h or 24 h (Fig. 7D&E). While PROTACs barely induced apoptosis after 24-h drug treatment in the complete medium with 10% FBS, under the condition of serum starvation, SIAIS125 and SIAIS126 induced significant apoptosis in H1975 lung cancer cells (Fig. 7F&G). These data suggested that autophagy could enhance the apoptosis of lung cancer cells induced by PROTACs. Because Bafilomycin A1 inhibited the function of both autophagy and lysosome by preventing their fusion, it was possible that EGFR degradation by PROTACs might act through the autophagy/ lysosome pathway. To examine whether both proteosome system and autophagy/lysosome pathway were both required in EGFR degradation by PROTACs, we inhibited the function of both together to see the effects on PROTAC-mediated EGFR degradation (Fig. 7H). We found out that the inhibition of both pathways led to a signif- icant recovery of EGFR protein amount (Fig. 7H). To study the relationships among EGFR ubiquitination, the proteosome system and the autophagy/lysosome system, the in- hibitor of ubiquitin ligase MLN4924 was treated together with the inhibitors of proteosome or autophagy/lysosome, and then to evaluate the effects on EGFR degradation by PROTACs (Fig. 8A). Results showed that treating H1975 cells with SIAIS126 induced EGFR degradation, treating cells with MLN4924 alone slightly increased EGFR protein level (Fig. 8B). Inhibiting the function of ubiquitin ligase alone or in combination with lysosome inhibition totally blocked EGFR degradation by PROTACs (Fig. 8B). Because Bafilomycin only slightly reversed the degradation effect of SIAIS126 as shown in Fig. 7B - D, it suggested that ubiquitination was an upstream event for lysosome degradation of EGFR. Similarly, ubiquitination was also an upstream event for proteosome- mediated degradation of EGFR protein by PROTAC (Fig. 8B). Taken together, our data suggested that PROTAC-mediated EGFR degradation acted through both ubiquitin/proteosome system and ubiquitin/autophagy lysosome degradation system. 3. Conclusions and discussion Drug resistance is a big clinical problem in EGFR-targeted therapy, and acquired T790M secondary mutation in EGFR is responsible for about 60% of drug-resistance to the first generation of EGFR TKIs [15]. EGFR T790M-targeting PROTACs provide poten- tial tools to overcome the drug resistance to EGFR tyrosine kinase inhibitors. Right now, EGFRL858R+T790M mutant protein was the only form of resistant proteins that were able to be degraded by PROTACs. EGFREx19del+T790M mutants are as important as EGFRL858R+T790M mutants in drug resistance. In addition, there was no PROTAC developed against EGFR with acquired C797S mutation yet. In the long run, developing PROTACs that are able to degrade EGFR with these different resistant mutations is still in great need to overcome drug resistance. Both cereblon ligands and VHL ligands are E3 ubiquitin ligases that respectively mediate the degradation of IKZF proteins and HIF1a proteins in vivo [32,33]. They had also been successfully used in PROTAC design to induce the degradation of many proteins. Previously developed T790M-targeted EGFR PROTACs used either VHL ligands or MDM2 ligands to recruit E3 ubiquitin ligase [17,22,24]. None of reported CRBN-based EGFR PROTAC was able to degrade EGFR with T790M mutation [22e24]. In these reports, most of CRBN-based PROTACs used lenalidomide as the ligand to recruit E3 ubiquitin ligase. In our experiments, pomalidomide was used to develop EGFR PROTACs. However, because different war- heads were used in these EGFR PROTACs, we cannot conclude that pomalidomide is better than the other ligands in EGFR PROTAC design. For example, Jin’s group found that a thalidomide-based compound MS154 exhibited better EGFR degradation activities comparing to pomalidomide-based compound 9 when the same warhead was used [21]. Rational design of PROTACs with different warheads and different ligands of E3 ubiquitin ligase is crucial for fast developing a successful PROTAC compound. Computer-aided PROTAC design might be helpful to speed up the process of designing of a good PROTAC. Our data suggested that turning inhibitor canertinib into a PROTAC degrader not only could endow it with extra degradation capability, but also could improve its target specificity. For example, SIAIS126 could inhibit the phoshorylation of both EGFRL858R+T790M and EGFREx19del+T790M mutant proteins, however, it only could degrade EGFR L858R+T790M proteins very well. The underline reason is not clear yet. Knowing the mechanisms may help later to design a better EGFR PROTAC to target both T790M containing resistant proteins and the C797S-containing triple mutant proteins. Most targets of developed PROTACs are intracellular proteins such as BCR-ABL [34], EML4-ALK [19], and bromodomain and extraterminal (BET) proteins [35]. The degradation mechanisms of such PROTACs acted mainly through the proteosome system. Some technologies such as LYTAC (lysosome-targeting chimeras) were developed to specifically target membrane proteins for degrada- tion, and reported successful targets included EGFR and PDL1 [36]. Although EGFR is mainly expressed on cell membrane, previous studies showed it could also be effectively degraded by PROTACs through the ubiquitin/proteosome system. In this study, we found that EGFR PROTAC activated autophagy in cancer cells. We firstly demonstrated that PROTACs could induce EGFR degradation through the ubiquitin/autophagy/lysosome degradation system in addition to the well known ubiquitin/proteosome system. Our study will be very helpful for later PROTAC design and future PROTAC applications. 4. Experimental section 4.1. Chemistry general procedure All chemicals were obtained from commercial suppliers (Ada- mas and Alfa), and used without further purification, unless otherwise indicated. N,N-diisopropylethylamine (DIPEA),dichloro- methane (DCM), Tetrahydrofuran (THF) and N,N- dimethylformamide (DMF) were dried by a 4 Å MS. Flash chro- matography was carried out on silica gel (200e300 mesh). Analytical TLC was performed on Haiyang ready-to-use plates with silica gel 60 (F254). All new compounds were characterized by 1H NMR, HRMS. 1H NMR spectra were recorded on Bruker AVANCE III 500 MHZ (operating at 500 MHz for 1H NMR), chemical shifts were reported in ppm relative to the residual DMSO‑d6 (2.50 ppm 1H), and coupling constants (J) are given in Hz. Multiplicities of signals are described as follows: s — singlet, br. s — broad singlet, d — doublet, t — triplet, m — multiple. High Resolution Mass spectra were recorded on AB Triple 4600 spectrometer with acetonitrile and water as solvent. 4.2. Biology 4.2.1. Cell lines and agents Non-small cell lung cancer cell line HCC827(EGFR exon 19 deletion), H1975 (EGFR L858R + T790M) and A549 (EGFR WT) were purchased from ATCC. EGFR deletion mutant lung cancer cell line PC9 (EGFR exon 19 deletion), PC9Brc1 (EGFR exon 19 deletion + T790M) were received as previously [37]. All lung cancer cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. Cells were cultured in a humidified atmosphere containing 5% CO2 at 37 ◦C. All cells were tested asmycoplasma negative regularly with correct cell identification (STR analysis). The antibodies against EGFR (#4267S, 1:1000), P-EGFR (#3777S, 1:1000) alpha-tubulin (#9099S, 1:1000), beta-actin(#1260S, 1:1000) and GAPDH (#2118S, 1:1000) were purchased from Cell Signaling Technology. LC3 antibodies were purchased from Sigma Aldrich (#L7543, 1:1000). MLN4924 (#S7109) was purchased from Selleck. MG132 (#M832899-5 mg), Cell Counting Kit-8 (CCK-8)(#C0005) and Car- filzomib (#T1795, 5 mg) were purchased from Targetmol. Bafilo- mycin A1(#88899-55-2, 1 mg) was obtained from Bide Pharmatech Ltd. CellTrace™ CFSE Cell Proliferation Kit for flow cytometry was purchased from ThermoFisher Scientific (#C34554). 4.2.2. Generation of stable cell line Lentiviral vector containing human EGFR cDNA was mutated at the sites of L858 and T790 to generate EGFR L858R/T790M double mutant plasmid with KOD plus mutagenesis kit (Toyobo). EGFRL858R/T790M plasmid was firstly packed in 293T cells to generate lentivirus, and then transduced Ba/F3 cells to establish stable Ba/F3 LT (EGFRL858R/T790M) cell line. Ba/F3 LT cells were cultured in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS), and 1% penicillin/streptomycin. A GFP containing lenti-vector was firstly packed in 293T cells to make lentivirus. Then, the lentiviruses were added to culture me- dium with polybrene (8 mg/mL) to infect H1975 cells. Cells that stably expresedGFP proteinswere sorted by flow cytometry. 4.2.3. Cell proliferation inhibition assay The anti-proliferative activities of compounds in different cell lines were determined by CCK-8 reagent. Different cells were firstly seeded in 96-well plates at the density of 3000 cells/well supple- mented with 100 mL culture medium per well. One day later, cells were treated with different concentrations of compounds and incubated for further 72 h. Then, cells were incubated with 10 mL of CCK-8 solution for 1e4 h, and the optical density of each well was determined by EnVision multi-mode plate reader at 450 nm wavelength. 4.2.4. Western blot Cells (1e3X105/well) were seeded in 12-well plates. One day later, different concentrations of compounds were added to cells for indicated time, and then cells lysates were collected with RIPA lysis buffer containing protease inhibitors. Equal amount of protein from each treatment (15 mg) was separated by 8% SDS-PAGE gel and then transferred to nitrocellulose membrane (AmershamTM ProtranTM 0.4 mm NC, A21886265). Then, membranes were sequentially incubated with primary and secondary antibodies (Cell Signaling). Chemistar ECL western blotting substrates were used to visualize protein levels with an Amersham Imager 600 imaging system. 4.2.5. CFSE staining 3X 10^6 H1975 cells were re-suspended in 1 mL pre- warmed(37 ◦C) phosphate-buffered saline (PBS), then mixed with 1 mL CFSE(10 mM/mL: 2 mL 5 mM CFSE stock solution diluted with 1 mL PBS) and incubated for 10 minutesat room temperature (protected from light). Added 8 mL completemedium to the cells and incubated for 5 minuteson ice. Pelleted the cells by centrifu- gation and re-suspended them in fresh, ice-cold complete medium, repeated the procedure for 3 times. Planted the cells in a 12-well- plate with 1X10^5 cells/well. 24 h after cell seeding, treated above cells with degraders at indicated concentrations and detected the CFSE Fluorescence with the CytoFLEX at indicated time (H1975 cells without CFSE staining were used as the negative control). Data were analyzed with FlowJo software (BD). 4.2.6. Cell apoptosis assay H1975 cells (8 × 104/well) were seeded in 12-well plates, and one day later different compounds were added at indicated concentrations. After 72 h-incubation, cells were collected and evalu- ated with an Annexin V-FITC apoptosis detection kit (Vazyme) according to the instructions. The extent of apoptosis was deter- mined by Flow Cytometry. 4.2.7. Cell cycle assay H1975 cells (1 × 105/well) were seeded into 6-well plates. One day later, different compounds were added to cells at indicated concentrations. 48 h later, cells were collected and washed twice with pre-cooled PBS. Cells were then re-suspended in 250 mL pre- cooled PBS with dropwise addition of 50 mL cold ethanol. Cells were then fixed at —20 ◦C for over 24 h. Samples were then pelleted at 5000 rpm, and the supernatant was discarded completely. After treated with 400 mL PI/RNase (4A Bioteck Co., Ltd, FXP0211-200), cells were kept in dark place and incubated at 37 ◦C for 30 min. The proportion of cells at different phase were analyzed on a flow cytometer (CytoFLEX, Beckman Coulter). 4.2.8. Cell migration assay H1975 cells (3 × 105/well) were firstly seeded into 24-well plates, and then treated with different compounds at indicated concentrations. After 12 h and 24 h compound-administration, photos were randomly taken from different areas for evaluation and three biological repeats were set for each treatment. The scratch areas in each photo were analyzed with Image J software. 4.2.9. Transwell assay Matrigel (Biocoat 354234) was firstly diluted 10 times with serum free culture medium to make coating buffer. 100 mL coating buffer was then added into transwell insert (Corning FluoroBlok™ 24 well plate permeable Support with 8.0 mm Colored PET mem- brane, #351152) and incubated at 37 ◦C for 1 h. H1975 cells were digested with tripsin and washed with PBS. After re-suspended in 150 mL serum-free medium with different compounds, cells were then seeded in Transwell inserts. 200 mL RPMI1640 full growth medium were added into the lower chambers. The 24-well-plates were incubated in a humidified atmosphere containing 5% CO2 at 37 ◦C. InvasedH1975-GFP cells were visualized with Fluorescence inverted microscope. Five areas were randomly chosen to take photos from each Transwell. Image J software was used to quantify the number of cells for each treatment. 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