A subset of flavaglines inhibits KRAS nanoclustering and activation

RAS proteins are small GTPases that function as molecular switches regulating the transmission of extracellular signals from the outside of the cell to the nucleus by various effector proteins (Hobbs et al., 2016; Ostrem and Shokat, 2016; Simanshu et al., 2017). Their activation cycle is regulated by the binding of GDP or GTP, which in turn is controlled by GTPase-activating proteins (GAPs) or guanine nucleotide exchange factors (GEFs). In their GTP-bound form, RAS proteins bind to several effectors and trigger multiple signalling pathways that control various fundamental cellular processes. The most common oncogenic RAS mutations occur in codons 12, 13 and 61 and prevent GAP-mediated GTP hydrolysis of RAS thus keeping it constitutively in the GTP-bound, active state (Prior et al., 2012). Through alternative splicing, two KRAS isoforms are generated (KRAS4A and KRAS4B), with KRAS4B being predominantly expressed in most cancers (Tsai et al., 2015). Most of the mutations in RAS isoforms are confined to three hotspot residues (G12, G13 and Q61) whose mutations lead to distinct biological consequences (COSMIC; https://cancer.sanger.ac.uk/cosmic; Ihle et al., 2012).

Recently drugs directly targeting specifically the KRAS G12C mutant have entered clinical trials (Khan et al., 2020; Ostrem and Shokat, 2016). The three RAS isoforms exhibit 82–90% sequence identity and most of the differences are confined to the C-terminal HVR region. While KRAS mutations are frequently identified in pancreatic, lung and colon carcinomas, NRAS mutations predominate in melanomas, and HRAS mutations in head and neck cancers. The molecular consequences caused by the isoform-specific RAS mutations and their tissue-specific roles are currently unclear. There are also significant differences between the RAS isoforms with respect to their posttranslational modifications and their intracellular localization. KRAS4A and KRAS4B have polybasic stretches that are responsible for their affinity to phospholipids in lipid nanoclusters of the plasma membrane (Zhou and Hancock, 2015). KRAS4B can also be phosphorylated at S181, which might dictate its localization to endomembranes. While HRAS is palmitoylated at two residues, KRAS4A and NRAS are palmitoylated at a single residue in the HVR region. These modifications determine their distribution within the plasma membrane microdomains, which in turn dictates the downstream signalling (Hancock and Parton, 2005).

Here, we show that targeting plasma membrane-associated prohibitins with a subset of flavaglines inhibits the GTP loading of RAS, thereby leading to inactivation of this GTPase. Flavaglines are natural anti-tumour drugs isolated from plants of the genus Aglaia that are characterized by a cyclopenta[b]benzofuran ring that directly targets prohibitins 1 and 2 (PHB1, also known as PHB, and PHB2, respectively) and eukaryotic initiation factor-4A (eIF4A) (Chu et al., 2020; Ebada et al., 2011; Ribeiro et al., 2012). PHB1 and PHB2 are evolutionarily conserved proteins predominantly known for their roles in the regulation mitochondrial function and cristae morphogenesis (Merkwirth and Langer, 2009; Mishra et al., 2010). Prohibitins are members of the SPFH family of membrane proteins with PHB domains (Browman et al., 2007). They are often detected in distinct subdomains of the plasma membrane, and they form functional oligomers, which dictate the formation of a signalling and functional unit at the plasma membrane (Kim et al., 2013; Yurugi et al., 2012). Previous studies have shown that PHB1 directly binds to kinase CRAF (also known as RAF1) and is required for its activation (Rajalingam et al., 2005). Several biological and chemical ligands of prohibitins that target the prohibitin complex (PHB1 and PHB2) at the plasma membrane have been identified (Thuaud et al., 2013). Two PHB ligands, fluorizoline and rocaglamide, inhibit the interaction between PHB1 and CRAF and inhibit KRAS-mediated tumorigenesis (Yurugi et al., 2017). However, activated BRAF [BRAF(V600E)], which can directly phosphorylate MEK1 and MEK2 (MEK1/2, also known as MAP2K1 and MAP2K2) and activate the MAPK pathway, overcomes the anti-tumour effects of fluorizoline and rocaglamide (Yurugi et al., 2017). Here, we investigated the underlying mechanisms of flavagline action and show that only a subset of flavaglines with defined side chains inhibit RAS activation in cells. We identify that rocaglamide prevented the interaction between prohibitins, KRAS, and specific phospholipids in the plasma membrane. Furthermore, rocaglamide inhibits the growth of KRAS-driven tumours in autochthonous mouse models.

We have previously shown that treatment with rocaglamide inhibits RAS-GTP loading in cells upon EGF stimulation (Yurugi et al., 2017). In those experiments, we primarily employed the CRAF-RBD domain for precipitating active GTP-bound RAS. To rule out the possibility that rocaglamide primarily inhibits the interaction between KRAS and the RAS-binding domain (RBD) of CRAF, we detected GTP-loaded RAS upon pulldown with CRAF-RBD and the Ral-GDS-RA domains. Activation of RAS in response to EGF was inhibited upon pre-treatment of HeLa cells with rocaglamide at 200 nM concentration (Fig. S1A). In the early time points post treatment (up to 1 h), we could not detect any significant changes in the levels of cyclin D1 a known substrate of eIF4F complex (Fig. S1A). Furthermore, these results also suggest that the inhibition is not dependent on the RBD employed in the assay. Despite the extremely high affinity of abundant GTP to RAS in cells, rocaglamide treatment inhibited this interaction in cells.

Next, we established a NanoBiT assay (live cell Nano-luciferase complementation assay) (Oh-Hashi et al., 2016) with KRAS and the CRAF-RBD to quantify activation of KRAS (Fig. 1A). We validated the sensitivity of this NanoBiT assay with wild-type, active and inactive mutants of KRAS that confirms the GTP-driven binding between KRAS and CRAF-RBD (Fig. S1B). We then tested the effect of rocaglamide on gain-of-function KRAS mutants. Rocaglamide strongly reduced the interaction between each of the KRAS mutants and CRAF-RBD (Fig. 1B; Fig. S1C,D). These data and those from the RBD pulldown experiment (Fig. S1A) suggested that rocaglamide inhibits KRAS activation and, thus, the effector binding in cells.

Compared to the RBD pulldown assay, the NanoBit assay is quantitative and can be used to calculate affinity and kinetic parameters. We determined with the NanoBit assay that rocaglamide inhibits KRAS activation with an IC₅₀ of 24.8 nM (Fig. 1C), and that 100 nM of rocaglamide produced 50% inhibition by 50 min (Fig. 1D; Fig. S1E). The results also show that rocaglamide inhibits KRAS4A activated either by EGF stimulation or through gain-of-function mutations (Fig. S1F,G). Recent studies have led to the successful development of a KRAS G12C inhibitor, which has entered clinical trial (https://clinicaltrials.gov/ct2/show/NCT03600883). For further validation of our NanoBiT assay, we included the KRAS G12C inhibitor (ARS-1620) in our experiments (Janes et al., 2018). As shown in Fig. 1E, ARS-1620 strongly inhibited KRAS G12C mutants while the G12V mutant was only modestly inhibited by the treatment. In contrast, treatment with rocaglamide inhibited both KRAS mutants to the same extent (Fig. 1F). This data confirms that rocaglamide is not biased towards specific KRAS mutants, which is valuable as recent studies have called for combined inhibition of both wild-type and mutant KRAS due to feedback reactivation of wild-type RAS through activation of receptor tyrosine kinases in a panel of KRAS^G12C cell lines treated with ARS-1620 and AMG 510 (Ryan et al., 2019). We then tested whether rocaglamide treatment can inhibit other RAS isoforms by NanoBit assays and found that rocaglamide, but not the closely related flavagline FL1 inhibited all the three RAS isoforms (Fig. 1G).

To further evaluate the inhibition of KRAS with other flavaglines, we selectively employed 17 different flavaglines, which differ in their side chains (Fig. S2). Using NanoBiT assays, we detected that, apart from rocaglamide, only a subset of flavaglines was able to inhibit KRAS (FL3, FL10, FL13, FL15, FL19, FL23, FL32, FL37, FL40 and FL42). While the degree of inhibition slightly varied among the different flavaglines, some members (like FL1, FL6, FL26 and FL30) failed completely to inhibit KRAS activation, indicating that the substituents on the cyclopenta[b]benzofuran skeleton are critical in the ability to inhibit KRAS (Fig. 2A,B). As we expected, the pattern of KRAS inhibition aligned with the inhibition of the downstream MEK1/2 kinases (Fig. 2C,D). More precisely, the suppression of the methoxy in position 8 was highly detrimental, while the hydroxyl in position 1 could be replaced by a formamide or dimethylurea with the opposite configuration. The introduction of a dimethylcarboxamide, methylcarboxamide or methyl ester promoted the anti-KRAS activity. The introduction of a fluorine in position 3′ enhanced or lowered the anti-KRAS activity depending upon the other substituents in position 1′ and 4′. The replacement of the methoxy in position 4′ by a bromine or a chlorine promoted the anti-KRAS activity (Fig. 2E). We performed validation experiments with specific flavaglines for the inhibition of different KRAS mutations and RAS isoforms. As expected, apart from rocaglamide, FL3, FL10, FL23 and FL42, but not FL1 and FL6, inhibited activation of KRAS and other RAS isoforms (Fig. 2F–H).

Because rocaglamide inhibits the interaction between CRAF and PHB1 (Polier et al., 2012; Yurugi et al., 2017), we tested whether the inhibition of RAS activation was dependent on CRAF. As expected, depletion of CRAF failed to prevent EGF-mediated RAS activation, suggesting that inhibition of CRAF is not influencing RAS activation under these settings (Fig. S3A). Furthermore, rocaglamide treatment failed to prevent the activating phosphorylation of EGFR in response to EGF stimulation, suggesting that the effects observed are downstream of EGFR activation (Fig. S3B). We then tested whether rocaglamide treatment induces structural changes to RAS in cells. Indeed, addition of GTPγS to RAS precipitated from rocaglamide-treated cells rescued GTP loading (Fig. S3C,D), indicating that rocaglamide treatment does not cause irreversible structural and/or functional modifications to RAS.

We then tested whether treatment with selected flavaglines (rocaglamide and FL42) inhibits the growth of cancer cells in vitro by employing soft agar colony formation assays. Apart from rocaglamide, we selected FL42 for subsequent experiments as FL42 has been previously shown to have no effect on eIF4A function, the other proposed target of rocaglamide (Boussemart et al., 2014). In MTT assays, both flavaglines were very effective in blocking the growth of HCT-116, ASPC-1 and Calu-1 cells, which carry KRAS mutations, with an IC₅₀ in the low nanomolar range (6–20 nM, Fig. 3A). The treatment also inhibited growth of HCT-116 and ASPC-1 cells in soft agar (Fig. 3B,C). We then expanded the panel of flavaglines in soft agar colony forming assays, including ones that failed to inhibit KRAS activation. These results were consistent with the results obtained with RAS inhibition (Figs 2F–H, 3D). Taken together, these data suggest that a subset of flavaglines can inhibit KRAS activation and prevent the oncogenic growth of tumour cells in vitro, although the latter effect could also be attributed to the additive inhibition of eIF4F complex.

To test the effects of rocaglamide in vivo, we employed an autochthonous KRAS G12D-driven NSCLC mouse model as detailed in the Materials and Methods section. The expression of KRAS G12D was induced with doxycycline treatment for 2 months followed by treatment of the mice with rocaglamide at a concentration of 2.5 mg/kg body weight intraperitoneally (i.p.) three times a week for 6 weeks. Rocaglamide treatment reduced the number of lung nodules suggesting a successful inhibition of the growth and maintenance of KRAS G12D-driven NSCLC model (Fig. 3D; Fig. S4). To address potential toxic effects of long-term rocaglamide treatment, we also tested liver and kidney toxicity in two different mouse strains. We did not detect any toxic effects as measured by liver enzyme activities and the blood creatine levels in two different mouse strains at the concentrations employed (Fig. S5). As rocaglamide also inhibits eIF4A, we tested the effect of rocaglamide in influencing the growth of BRAF-mutated cell lines, where the MAPK pathway is constitutively activated. As expected, the growth of BRAF mutated cells was also inhibited by rocaglamide treatment, although with varying efficiency (Fig. S6). These data suggest that the long-term phenotypic effects on cell growth by rocaglamide can possibly be attributed to the inhibition of both RAS and the eIF4F complex, although further experiments are clearly warranted with other flavaglines that do not inhibit eIF4A, like FL42.

We then explored the molecular mechanisms behind rocaglamide-mediated inhibition of KRAS activation in cells. Nanoclustering of KRAS is required for the activation of downstream effectors like the RAF kinases (Abankwa et al., 2010; Zhou and Hancock, 2015). By performing FLIM-FRET imaging of RAS isoform FRET pairs (Šolman et al., 2015), we detected that treatment with nanomolar concentrations of rocaglamide prevents KRAS nanoclustering-associated FRET but not FRET indicative of HRAS or NRAS nanoclusters (Fig. 4A–C). Consistent with these studies, immunogold labelling coupled to electron microscopic analysis of RAS proteins revealed that rocaglamide inhibited nanoclustering of KRAS but not NRAS or HRAS (Fig. 4D–G). These results are intriguing because we detected rocaglamide-mediated inhibition of HRAS and NRAS in the NanoBiT assay, which primarily measures GTP-driven binding to its effector.

To study the dynamics of KRAS activation, we cultured cells onto nanobar substrates to generate patterns on plasma membrane and to measure curvature response. Consistent with a recent study (Liang et al., 2019), activated KRAS formed clusters preferentially at the end of the nanobars, unlike the wild-type KRAS (Fig. 5A–C). Treatment with rocaglamide reversed the bar end preference (marked by bar end to centre ratio) of activated KRAS on these nanobars, phenocopying the KRAS wild-type distribution pattern (Fig. 5D–G). Interestingly, PHB1 was also enriched at the end of the nanobars like KRAS (Fig. S7). Together with the FLIM-FRET and electron microscopic analyses, these data indicate that rocaglamide treatment inhibits the formation of KRAS clusters at the plasma membrane leading to KRAS inactivation.

Because PHB1 and KRAS showed a similar distribution pattern on nanobars, we tested the interaction between activated KRAS G12D and PHB1 by performing biochemical assays in cells with crosslinking agents. As expected, PHB1 coimmunoprecipitated with KRAS G12D protein and the interaction was increased after crosslinking (Fig. 6A). Thus, we performed a bimolecular fluorescence complementation assay (BiFC) (Fig. 6B) which confirmed the interaction between PHB1 and KRAS in living cells (Fig. 6C).

Finally, we performed a cellular thermal shift assay (CETSA) to test whether PHBs serve as the direct targets of rocaglamide and active flavaglines, like FL42. These experiments revealed that the PHB1–RAS complex is stabilized even at 50°C upon treatment with rocaglamide and FL42, but not with FL1, thus confirming the specificity of the observed effects (Fig. 6D). Under these settings, we could not detect any significant changes to the levels of eIF4A (Fig. 6D). To further corroborate these observations we employed PHB1 siRNAs. As expected transient depletion of PHB1 in HeLa cells prevented RAS activation upon EGF stimulation as shown by western blotting (Fig. 6E,F) and in a NanoBiT assay (Fig. 6G). These data suggest that PHB1 is required for interaction of active RAS with the kinase CRAF.

KRAS, but not HRAS and NRAS, nanoclustering is driven by phospholipids like phosphatidic acid (PA) and phosphatidylserine (PS) (Ryan et al., 2019; Zhou et al., 2017). Previous studies have shown that prohibitins can function as scaffolds for phospholipids, such as cardiolipin, in the mitochondoria (Osman et al., 2009). We confirmed that PHB1 precipitated from cells specifically binds to PS and PA, and that this can be reversed by rocaglamide treatment (Fig. 7A). Using FLIM-FRET analysis employing PS/PA biosensors (LactadherinC2 domain, Lact-C2, for PS, and PASS, phosphatidic acid biosensor with superior sensitivity, for PA), we revealed that treatment with rocaglamide in fact inhibited clustering of KRAS with both PS and PA (Fig. 7B). These results indicate that rocaglamide specifically inhibits KRAS nanoclustering and effector protein activation, possibly by influencing the prohibitin-dependent segregation of lipids within the plasma membrane.

Here, we found that treatment with a subset of flavaglines at low nanomolar concentrations (6–20 nM), directly inhibited KRAS GTP loading when cells were stimulated with EGF or when cells harboured gain-of-function KRAS mutants thus functioning as potent KRAS inhibitor. Our results confirm that flavaglines inhibit KRAS but not HRAS and NRAS nanoclustering, suggesting that PHB1-mediated segregration of phospholipids (PS and PA in specific) is probably required for maintaining the active KRAS conformation in cells. Although the presence of membrane or lipids are not required for the GTP loading to KRAS, which is indeed a high-affinity interaction in the picomolar range, flavagline treatment prevented KRAS GTP loading, which was reversed by exogenous addition of GTPγS in solution. These effects are reproduced by the depletion of PHB1, which suggests a direct role for PHB1 in the regulation of KRAS activation.

Treatment with a subset of flavaglines also inhibited NRAS and HRAS activation, as measured by the Nanobit assay, although rocaglamide treatment failed to inhibit the nanoclustering of these two RAS isoforms. Previous studies have shown that NRAS and HRAS bind to different phospholipids than does KRAS during their nanoclustering (Ryan et al., 2019; Zhou et al., 2017). Further studies are clearly warranted to clarify this phenotype and the underlying mechanisms. KRAS is one of the most frequently mutated oncogenes and several efforts are being made to target this oncogene in tumours. Our observations suggest treatment with rocaglamide disrupts the PHB–phospholipid–KRAS complex, thus preventing effector protein activation (Fig. 8). Our study revealed a natural anti-tumour drug that potently inhibits KRAS at nanomolar concentrations irrespective of the mutations both in in vitro cell culture models and in autochthonous mouse models. These observations suggest that flavaglines should be further pursued for clinical development.

Calu-1 cells were obtained from Sigma-Aldrich and cultured in McCoy's 5A medium with 10% heat-inactivated fetal bovine serum (FBS). HeLa S3 (DSMZ), HEK-293T and HCT-116 cells (a gift from Ulf Rapp, University of Wurzburg, Germany) were authenticated by Eurofin genomics, and these cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated FBS. ASPC-1 cells and NCI-H2122 cells were purchased from DSMZ and ATCC, respectively. These cells were cultured in RPMI-1640 (10% heat-inactivated FBS). HEK 293 EBNA (ATCC) and U-2OS cells (ATCC) were cultured in DMEM (10% FBS). BHK cells were purchased from ATCC and maintained in DMEM supplemented with 10% bovine calf serum (BCS). HeLa cells were starved in serum-free medium with rocaglamide or other flavaglines for various time points and stimulated with EGF (100 ng/ml) for 30 min. KRAS mutation-carrying cells were treated with rocaglamide or FL1 in complete growth medium for 24 h.

Rocaglamide was a gift from Marcus Dobler (Syngenta, Basel, Switzerland). Large-scale synthesis was performed by Activ Biochem. The synthetic flavaglines were prepared as previously described (Ribeiro et al., 2012; Thuaud et al., 2009, 2011).

After stimulation or treatment, active RAS pulldown buffer (25 mM Tris-HCl pH 7.2, 150 mM NaCl, 5 mM MgCl₂, 1% NP-40, 5% glycerol with protease inhibitor cocktail) was added to each well, and plates were incubated on ice for 30 min. Cells were sonicated for 3 s and the lysate was centrifuged for 15 min at 4°C, 18,000 g. Protein concentration was measured by 660 nm protein assay reagent (Thermo Scientific), and adjusted so that each sample had an equal concentration. Before the affinity pulldown-based RAS activation assay, 20% of the lysates was taken for the total cell lysate. CRAF-RBD-immobilised agarose beads (20 µl; GE Healthcare) were added to the rest of the lysate and rotated at 4°C for 60 min. After incubation, the beads were washed with binding buffer twice, and 50 µl of SDS-PAGE sample buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 10% glycerol and Bromophenol Blue) were added.

Samples were subjected to SDS-PAGE (14% gels) followed by western blotting. After transfer, the membrane was blocked with 3% BSA in TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h at room temperature. The membrane was incubated with primary antibody diluted in 1% BSA in TBST and incubated overnight at 4°C. After the overnight incubation, the membrane was washed with TBST (five times for 5 min each time) and incubated with HRP-conjugated secondary antibody in TBST for 1 h at room temperature. After the secondary antibody treatment, the membrane was washed and the signal was visualized by chemiluminescence substrate (Millipore) and Chemi Doc touch (Bio-Rad). A list of antibodies is provided in Table S1.

The N-terminal LgBit and C-terminal smBit construct was purchased from Promega and KRAS (full length) was cloned with Xho I and Bgl II to LgBit and the CRAF RBD (amino acids 1–149) was cloned with EcoRI and BglII to SmBit. The construct was transfected into HeLa cells by employing transfection reagent (Polyethilenimine, PEI 25000, Sigma-Aldrich). For transfections, 1 µg or 2 µg of plasmids (12-well or 6-well) were transfected into cells with 0.5 mM of PEI reagent in 100 µl or 200 µl PBS. At 1 day after transfection, cells were harvested and seeded into 96-well white plates (Greiner). After an additional day, the medium was changed to serum-free DMEM for 0 to 4 h, containing DMSO, rocaglamide (Active biochem), Flavaglines or ARS-1620 (Selleckchem, S8707). After pre-treatment, wild-type KRAS-transfected cells were stimulated with EGF for 30 min. The NanoGlo assay was then performed for the EGF-stimulated wild-type KRAS-transfected cells or mutated KRAS-transfected cells according to the manufacturer's instructions. The luminescence was measured using a Tecan infinite reader (Tecan). For co-transfection of plasmids and siRNAs, Lipofectamine 2000 reagent (Invitrogen) was employed. 1 µl of siRNA (100 µM), 0.25 µg of LgBit and smBit plasmid was mixed in 125 µl of opti-MEM and the mixture was added to 3 µl Lipofectamine 2000 containing Opti-MEM (125 µl). After incubation, the solution was used for transfection. siRNAs used for PHB1 were: 5′-CCCAGAAAUCACUGUGAAA-3′ and 5′-UUUCACAGUGAUUUCUGGG-3′.

HCT-116, Calu-1 and ASPC-1 cells were seeded in 96-well plates at a concentration of 5×10⁴ cells/ml. A 50 µl cell suspension was added into 96-well cell culture plates and cultured for 1 day. Then, 50 µl of compound containing growth medium was added to the well and the cells were cultured for an additional 48 h, before 10 µl of MTT solution was added to the wells followed by incubation for 2 to 3 h. After incubation with MTT, solubilisation buffer was added and followed by incubation overnight. MTT level was measured at an absorbance of 570 nm.

A 1.5% agarose solution was mixed with 2× growth medium (20% FBS, with or without 100 nM rocaglamide) and placed with 1.5 ml of 0.75% agarose/1× growth medium in 6-well plates. To solidify agarose, plates were incubated at room temperature for at least 10 min. HCT-116 and ASPC-1 cells were diluted in 2× growth medium (20% FBS, with or without 100 nM rocaglamide) and mixed with 0.9% agarose solution. 1.5 ml of cell suspension in 0.45% agarose in 1× growth medium was added to the bottom agarose layer. The cells seeded in soft agar were cultured for 2 to 4 weeks followed by Crystal Violet staining. The images were taken using a ChemiDoc Touch (Bio-Rad), and the number of colonies was counted with Image J software. Soft agar colony formation assays were also performed in 96-well plates to simplify the quantification of colonies. In short, 50 µl of 0.75% agarose/1× growth medium was added to the 96-well plate. A cell suspension was prepared in 1× growth medium and mixed with 0.75% agarose/1× growth medium (1:2 ratio, 75 µl). A total of 2500–5000 cells were seeded in each well, and 100 µl of growth medium was added to the solidified layer with compounds. After 1 week, 20 µl of MTT solution was added to the well and incubated for 4 h. After incubation, the medium was removed and 175 µl of solubilisation buffer was added and the plate was heated at 70°C followed by absorbance measurement.

SP-C/rtTA (SP-C) mice (Tichelaar et al., 2000) were crossed to TetO-KRAS4bG12D (KRAS G12D) mice (Fisher et al., 2001). For transgene expression, mice were fed a doxycycline (DOX) diet for a total of 3.5 months. After 2 months, treatment with rocaglamide (2.5 mg/kg body weight, three times/week) was started and continued for 6 weeks. The DOX diet was purchased from ssniff Spezialdiäten GmbH. C57BL/6J and B6129SF1/J mice (The Jackson Laboratory) were treated with rocaglamide (2.5 mg/kg body weight, three times/week) for 2 or 4 weeks respectively to address potential cytotoxicity on liver and kidney. For toxicity testing, blood from mice treated with rocaglamide for 2–4 weeks was subjected to analysis of liver transaminases and kidney parameters. All animal experiments were approved by local authorities (National Investigation Office Rheinland-Pfalz, Approval ID: G15-1-064) and conducted according to the German Animal Protection Law.

HEK293 EBNA cells were seeded in 6-well or 12-well plates onto 16 mm sterile coverslips. The next day, cells were transfected by FuGENE HD transfection reagent (E2311, Promega) or jetPRIME (114-15, Polyplus) using a total of 2 μg plasmids for 6-well or 0.8 μg of plasmids for 12-well plates. For donor fluorophore lifetime samples, cells were transfected with only mGFP-tagged RAS G12V plasmid. In FRET pairs, cells were transfected with mGFP-tagged and mCherry-tagged RAS G12V plasmids at a ratio of 1:3. To monitor FRET between KRAS and PS, cells were co-transfected with pmGFP-Lact C2 and pmcherry-KRAS G12V at a ratio of 1:3. To monitor FRET between KRAS and PA, cells were co-transfected with pmGFP-KRAS G12V and pmRFP-Pass at a ratio of 1:3. At 24 h after transfection, cells were treated with either 0.1% DMSO control or 25 or 50 nM rocaglamide for 24 h and fixed in 4% PFA for 12 min before mounting with Mowiol 4-88 (Sigma-Aldrich, Cat. No. 81381). The donor fluorophore lifetime was measured using a Lambert-Fluorescence Lifetime Imaging instrument (Groningen, The Netherlands) attached to a fluorescence microscope (Zeiss AXIO Observer D1) as previously described (Guzmán et al., 2014). The percentage of the apparent FRET efficiency (E_app), was measured using the lifetimes of donor–acceptor pairs (τDA) of samples and the average donor lifetime (τD), based on the equation: E_app=(1−τDA/τD)×100%.

The nanobar arrays with 250 nm width, 2 μm length, 300 nm height and 5 μm pitch were fabricated on a square quartz wafer by using electron-beam lithography (FEI Helios NanoLab) as previously reported (Zhao et al., 2017). For cell culture and live-cell imaging, the nanobar chips were immersed in Chromium Etchant (Sigma-Aldrich) overnight to remove the Cr mask on the nanobars, then attached to hole-punched 40×11 mm tissue culture dishes (TPP). Prior to cell culture, the nanobar-chip bottom dish was treated with air plasma for 5 min and coated with 2 mg/ml gelatine (Sigma-Aldrich) for 30 min at room temperature. 5×10⁴ U-2OS cells were seeded in the chip bottom dish and maintained in complete DMEM supplemented with GlutaMAX™ until the 70–90% confluency for DNA transfection, drug treatment, live cell imaging and immunostaining was reached. Imaging of KRAS-transfected cells without rocaglamide treatment on nanobar arrays was performed with a laser scanning confocal microscopy (Zeiss LSM 800 with Airyscan) at 100×/1.4 oil objective. Each image had a resolution of 512×512 pixels, with a pixel size of 124 nm and a bit depth of 16. To quantify the curved nanobar-end preferred distribution of each protein with or without drug treatment, the background intensity of each image was subtracted with a rolling ball algorithm at a 3.5-pixel radius in Fiji NIH (Schindelin et al., 2012) and the intensity ratio of nanobar-end to nanobar-cencer was measured and calculated using a custom-written MATLAB code derived from previously reported work (Zhao et al., 2017).

HeLa cells were seeded at the density of 5×10⁴ cells/ml in a 6-well plate (2 ml). After transfection of the FLAG-KRAS, NRAS and HRAS G12D plasmids, cells were cultured for 2 days followed by DSP crosslink. RAS-overexpressing HeLa cells were washed with PBS twice and 750 µl of PBS was added to the 6 well plate with DSP (1 mM, 4% DMSO) at room temperature for 30 min. After incubation, the reaction was stopped with TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) and washed with TBS. The cell lysate was prepared in active RAS pull-down buffer and the lysate was used for FLAG immunoprecipitation assay. 10 µl of anti-FLAG M2 Affinity Gel (Sigma-Aldrich, A2220-5ML) was added to the lysate and rotated at 4°C for 1 h. After washing with binding buffer, SDS-PAGE and western blotting was performed as described above.

Full-length PHB1 and KRAS G12D were cloned into the BiFC plasmid pair (Addgene #73636 and #73637) deposited by Darren Saunders (Croucher et al., 2016). The plasmid was transfected into HeLa cells as described for the NanoBit assay and the cells were harvested with 0.05% trypsin/0.02% EDTA in PBS solution. The cells were used for FACS analysis.

HEK-293T cells were harvested in 10% glycerol/PBS with protease inhibitor (Roche, EDTA-free). The protein concentration was adjusted to 0.5 µg protein/ml and aliquoted to a 1.5 ml tube at 30 µl/tube with 200 nM of rocaglamide, FL42 or FL1, followed by an incubation on ice for 30 min. After incubation, the tubes were heated on a heat block for 6 min and then transferred to an icebox. The lysate was centrifuged (18,000 g, 4°C, 15 min) and the supernatant was employed for SDS-PAGE and western blotting.

For siRNA transfection, 2 µl of siRNA (100 µM) was mixed with 10 µl of SAINT-sRNA (Synvolux) in 200 µl of PBS and incubated for 10 min at room temperature. HeLa cells were harvested with 0.05% trypsin/0.02% EDTA in PBS and seeded in 6- or 12-well cell culture plates at a concentration of 5×10⁴ cells/ml in complete DMEM (2 ml for 6-well plate and 1 ml for 12-well plate). After 1 day, transfection reagent was added to the well, and the cells were maintained in the incubator for 1 to 2 days. The medium was changed to serum-free DMEM and incubated at 37°C for 4 h with compound (100 nM). After starvation of the cells, they were stimulated with EGF (100 ng/ml) for 30 min. The cells were washed with PBS and used for the active RAS pulldown assay. siRNA for PHB1 was 5′-CCCAGAAAUCACUGUGAAA-3′ and 5′-UUUCACAGUGAUUUCUGGG-3′; siRNA for CRAF was 5′-GGAUGUUGAUGGUAGUACATT-3′ and 5′-UGUACUACCAUCAACAUCCAC-3′.

3xFLAG-CMV-14 (Sigma-Aldrich) with a full-length PHB1 insert was transfected into HEK-293T for 2 days in 15 cm diameter cell culture dishes. After overexpression, the cells were lysed in active RAS pulldown assay buffer and 300 µl of anti-FLAG-M2 antibody immobilized agarose (Sigma-Aldrich) was added to the lysate. After 4 h of incubation at 4°C, the beads were washed with TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) followed by elution with 100 µg/ml of 3× FLAG peptide in TBS. The elution fraction was used for further experiments. The lipid strip (Echelon, P-6002) was blocked with 3% BSA/TBST overnight at 4°C. The membrane was incubated in the same buffer with either 1:500 diluted FLAG-tagged PHB1 elution fraction with 10 µM of rocaglamide or with the same amount of DMSO followed by overnight incubation at 4°C. Finally, the membrane was washed and the FLAG–PHB1 was detected by HRP–conjugated anti-FLAG M2 antibody. The signal intensity was quantified with ImageJ software.

K.R. would like to thank the excellent technical support of Stefanie Wenzel. We thank Prof. Frank McCormick for critical advice, Ulf Rapp for providing cell lines and Dr Schonfeld for critical reading of the manuscript. Technical support for the nanobar assay was provided by the Centre for Disruptive Photonic Technologies (CDPT) and and Nanyang NanoFabrication Centre (N2FC) of Nanyang Technological University.

The peer review history is available online at https://jcs.biologists.org/lookup/doi/10.1242/jcs.244111.reviewer-comments.pdf