Dual mechanisms of action of the 5-benzylidene-hydantoin UPR1024 on lung cancer cell lines

Lung cancer is the leading cause of death from malignant disease in the developed world, but only a minority of patients is eligible for radical treatment with curative intent. The availability of new cytotoxic drugs has led to incremental improvements, but a paradigm shift is required to make a significant effect on the poor prognosis for most patients. Due to major advances in cancer biology, many potential therapeutic targets have been identified. Most of these targets are components of signaling pathways or metabolic processes contributing to one of the hallmarks of the cancer phenotype (1).

One of these targets is the epidermal growth factor (EGF) receptor (EGFR). EGFR (ErbB1, HER1) is the prototypic member of the ErbB family of receptor tyrosine kinase, which also includes ErbB2-4 (HER2-4). Upon specific binding of EGF-like ligands to the extracellular domain, ErbB receptors dimerize either as homodimers or as heterodimers. After this dimerization, the receptor undergoes autophosphorylation at specific tyrosine residues within the intracellular domain. These phosphorylated tyrosines serve as docking sites for adapter molecules, such as growth factor receptor binding protein 2 and the p85 subunit of phosphoinositide 3-kinase, which activate a complex network of downstream signaling pathways. These pathways, including the Ras/mitogen-activated protein kinase and Akt/mTOR kinase cascades, in turn regulate transcription factors and other proteins involved in cell proliferation, survival, motility, and differentiation (2).

The overexpression of EGFR has been well documented in a variety of human tumors, including non–small cell lung cancer (NSCLC; ref. 3), and has been associated with poor prognosis and worse clinical outcome (4).

The 4-aniloquinazolines gefitinib (Iressa) and erlotinib (Tarceva) are the two most important anticancer drugs developed to target the EGFR tyrosine kinase activity and are used in the treatment of NSCLC (5).

However, tumor cells often acquire resistance to EGFR inhibitors through several different mechanisms. Certain mutations in the EGFR gene (T790M mutation; refs. 6, 7), other mechanisms, such as drug efflux or protein binding, or additional oncogenic changes downstream of the receptor have been associated with resistance to EGFR-targeting drugs (8, 9).

New EGFR tyrosine kinase inhibitors, structurally different from 4-anilinoquinazolines having additional mechanisms of cytotoxicity, need to be designed to circumvent acquired resistance to gefitinib and erlotinib therapy.

On the other hand, several studies have shown that in combination with radiation (10) or classic cytotoxic drugs (11, 12), gefitinib provided synergistic, antiproliferative, and proapoptotic actions.

We have recently shown that hydantoin derivatives designed to interact at the ATP site of EGFR inhibited the EGFR kinase activity and exerted an antiproliferative effect on the A431 cell line overexpressing EGFR (13). Among a panel of these compounds, the one originally termed 2e and now called UPR1024 showed the highest potency. UPR1024 belongs to a new class of EGFR inhibitors characterized by a hydantoin nucleus and a 5-benzylidene substituent having a double bond conjugated with a carbonyl group on the hydantoin ring.

In this study, we further investigated the effects of UPR1024 on a panel of NSCLC cell lines showing a wide range of sensitivity to gefitinib. The compound induced a significant inhibition of cell proliferation with a preferential accumulation of the cells in the S phase of the cell cycle. Apoptosis was also detectable in cells exposed to the drug as indicated by an increase in the sub-G₁ cell population, release of cytochrome c from mitochondria and activation of caspases 3, 8, and 9. Moreover, we found that UPR1024, a drug designed to act as tyrosine kinase inhibitor, had a genotoxic effect suggesting an additive mechanism of action. The presence of wild-type p53 improved drug efficacy, although the effect was also detectable in p53 null cells.

Overall our results suggest that UPR1024, a 1,5-disubstituted hydantoin, possesses chemical properties that confer multiple mechanisms of action and can be considered a novel combi-molecule capable of both blocking EGFR tyrosine kinase activity and inducing genomic DNA damage.

The human NSCLC cell lines A549, H460, and H1299 were cultured in RPMI 1640; Calu-3 and Lx-1 were cultured in DMEM with 1 mmol/L sodium pyruvate and 0.1 mmol/L nonessential amino acids; SKLU-1 was cultured in MEM supplemented with 1 mmol/L sodium pyruvate and 0.1 mmol/L nonessential amino acids. All media were supplemented with 2 mmol/L glutamine, 10% fetal bovine serum. Cell lines were from the American Type Culture Collection and maintained under standard cell culture conditions at 37°C in a water-saturated atmosphere of 5% CO₂ in air.

Inducible p53 transfectans of cell line Calu-1 were generated from the NSCLC Calu-1 cell line as previously described (14). These stable clones, conditionally expressing the p53 gene from the Ponasterone A–inducible promoter, were maintained in RPMI 1640 in the presence of 50 μg/mL zeocin and 400 μg/mL G418.

UPR1024 was synthesized in the Pharmaceutical Department of Parma University as described elsewhere (13). In all assays, the drug was dissolved in DMSO immediately before the addition to cell cultures. The concentration of DMSO never exceeded 0.1% (v/v) and equal amounts of the solvent were added to control cells. Gefitinib was synthesized as previously reported (13).

Media were from Euroclone; fetal bovine serum were purchased from Life Technologies-Bethesda Research Laboratories. Monoclonal anti–cytochrome c (7H8), monoclonal anti-p53 (DO-1), monoclonal anti-p21^WAF1 antibodies were obtained from Santa Cruz Biotechnology. Monoclonal anti–caspase-8 (1C12), polyclonal anti–caspase-9, polyclonal anti–caspase-3, monoclonal anti-EGFR, polyclonal anti–phospho-EGFR (Tyr¹⁰⁶⁸) antibodies were from Cell Signaling Technology. Monoclonal anti-actin (AC-40) antibody was from Sigma Aldrich. Horseradish peroxidase–conjugated secondary antibodies and the enhanced chemiluminescence system were from Millipore. Reagents for electrophoresis and blotting analysis were obtained from BIO-RAD Laboratories. Caspase-8 inhibitor z-IETD-FMK was purchased from Enzyme Systems Products.

Procedures for protein extraction, solubilization, and protein analysis by one-dimensional PAGE are described elsewhere (15). Briefly, cells were lysed, and protein concentration was determined by the dye-binding method (Bio-Rad) with bovine serum albumin as standard (16). Proteins (50–100 μg) from lysates were resolved by 5% to 15% SDS-PAGE and transferred to nitrocellulose membranes. Cytosolic and mitochondrial fractions were generated using a digitonin-based subcellular fractionation technique (17) as previously described (18). Equal volumes of cytosolic and mitochondrial fractions were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then incubated with primary antibody, washed, and then incubated with horseradish peroxidase anti-mouse or horseradish peroxidase anti-rabbit antibodies. Immunoreactive bands were visualized using an enhanced chemiluminescence system.

Inhibition of EGFR autophosphorylation in A549 cells was determined using a specific anti-phosphotyrosine antibody by Western blot analysis. Briefly, serum-starved cells were preincubated for 1 h with the indicated concentrations of UPR1024 before stimulation with 0.1 μg/mL EGF for 5 min. Equal amounts of cell lysates were analyzed by Western blotting using anti–phosphorylated EGFR (Tyr¹⁰⁶⁸) antibody. Membranes were stripped and reprobed with anti-EGFR antibody.

Cell growth and survival were evaluated by cell counting, clonogenic assay, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Briefly, cells were detached from the plates by trypsinization and counted in a Burker hemocytometer by trypan blue exclusion.

Colony formation by viable cells was determined by seeding them at a density of 400 cells per dish. After 10 days of incubation, colonies were fixed with 95% ethanol, stained with 0.1% crystal violet and counted. Colonies containing at least 50 cells were scored, and all data on viability are given as percentage versus untreated control.

Cell viability was also assessed by tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma] assay as previously described (15).

Distribution of the cells in the cell cycle was determined by propidium iodide staining and flow cytometry analysis (EPICS XL-MCL cytometer; Beckman Coulter). Cells (5 × 10⁵) were harvested, washed in PBS, fixed in cold 70% ethanol, and then stained with 40 μg/mL propidium iodide while treating with RNase. Analysis was done with a Beckman Coulter EPICS XL-MCL cytometer (Beckman Coulter). Cell cycle distributions were analyzed by MultiCycle DNA Content and Cell Cycle Analysis Software (Phoenix Flow Systems, Inc.) as previously described (15, 18).

Apoptosis was assessed by (a) morphology on stained (Hoechst 33342, propidium iodide) or unstained cells using light, phase contrast, and fluorescence microscopy; (b) analysis of phosphatidylserine exposure on the cell surface by the Annexin V–FITC assay [Bender MedSystems; briefly, 2.5 × 10⁵ cells were harvested, washed with PBS, incubated with Annexin V–FITC and propidium iodide according to the supplier's protocol, and analyzed by flow cytometry, and data were collected using logarithmic amplification of both the FL1 (FITC) and FL3 (propidium iodide) channels and quantitated by quadrant analysis of coordinate dot plots]; (c) quantitative analysis of hypodiploid sub-G₁ cells by flow cytometry done after the procedure described for cell cycle analysis; (d) activity of caspase-3 by a colorimetric assay (Caspase Colorimetric Assay kit, MBL International Corp.) and inhibition by specific tetrapeptides (z-IETD-FMK for caspase-8); (e) activation of caspase-3, caspase-8, and caspase-9 (detection of cleavage products) and release of cytochrome c by Western Blotting procedure as previously described (18); and (f) dissipation of mitochondrial membrane potential by the MitoCapture Assay kit (MBL) according to published procedures (19).

The fast halo assay was carried out as previously described (20). Briefly, after drug treatment, cells were resuspended at 4 × 10⁴/100 μL in ice-cold PBS containing 5 mmol/L EDTA: this cell suspension was diluted with an equal volume of 2% low-melting agarose in PBS and immediately sandwiched between an agarose-coated slide and a coverslip. After complete gelling on ice, the coverslips were removed and the slides were immersed in 0.3 mol/L NaOH for 15 min at room temperature. Ethidium bromide (10 μg/mL) was directly added during the last 5 min of incubation. The slides were then washed and destained for 5 min in distilled water.

The ethidium bromide–labeled DNA images were acquired using a Digital Net Camera (DN100, Nikon Corporation) and processed with image analysis software (Scion Image). The slides were numerically coded before reading to minimize operator bias. The extent of strand scission was quantified by calculating the nuclear diffusion factor, which represents the ratio between the total area of the halo plus nucleus and that of the nucleus. Data are expressed as relative nuclear diffusion factor, calculated by subtracting the nuclear diffusion factor of control cells from that of treated cells.

Data from three independent experiments were used for evaluation of dose-response curves. IC₅₀ values, expressed as mean of three independent determinations (±SD), were calculated by fitting the experimental data with a hyperbolic function and constraining Y_max to 100 (GraphPad Prism 4.00; GraphPad Software). Nonlinear regression R² values were >0.80 (Fig. 1C) or >0.95 (Fig. 2A).

Statistical significance of differences among data was estimated, unless differently stated, by two-tailed Student's t test.

UPR1024, a 1,5-disubstituted hydantoin (Fig. 1A), is a new compound previously characterized as an inhibitor of EGFR autophosphorylation in A431 cells (13). We analyzed the effect of UPR1024 on the level of EGFR autophosphorylation in the A549 lung cancer cell line by Western blotting. UPR1024 inhibited the EGF-induced EGFR autophosphorylation in a dose-dependent manner with an IC₅₀ of 19.58 ± 6.02 μmol/L (Fig. 1B and C).

To test whether UPR1024 induced reversible or irreversible inhibition, cells were treated with the inhibitor (50 μmol/L) for 1 h and then washed free of drug. After 8 h, EGF was added for 5 min and EGFR autophosphorylation was determined. UPR1024 inhibition was completely reversible because phosphorylation was recovered after drug removal.

We tested the antiproliferative effect of UPR1024 on a panel of NSCLC cell lines previously characterized in our laboratory for sensitivity to gefitinib.

After 72 h of treatment with 20 μmol/L UPR1024, all the cell lines tested showed a significant inhibition of cell proliferation (>40%; Fig. 1D) despite a very different sensitivity to gefitinib as indicated in the caption of the figure. UPR1024 inhibited A549 colony formation in a dose-dependent manner as assessed by clonogenic assay with an IC₅₀ of 20.17 ± 1.81 μmol/L (Fig. 2A). As indicated in Fig. 2B, drug exposure up to 24 h, followed by recovery time, did not significantly affect colony formation ability. However, a significant growth inhibitory activity was retained after 48-h exposure to 40 μmol/L, suggesting that long exposure to the drug is required for the appearance of the antiproliferative effect.

UPR1024 was cytostatic up to 10 μmol/L; however, at higher doses, a cytotoxic effect was detectable with appearance of floating dead cells and Annexin V–FITC/propidium iodide–positive cells (Fig. 2C). This result is in agreement with that observed by Chang (21) on the same cell line treated with similar concentrations of ZD1839 (gefitinib). To determine the effect of UPR1024 on A549 cell cycle distribution, cells treated with 20 and 40 μmol/L for 24 and 48 h were analyzed by flow cytometry. As shown in Fig. 2D, UPR1024 caused an increase in the proportion of cells in S phase with a decrease in G₁ phase. By contrast to gefitinib that induced a G₁ arrest in NSCLC cell lines (21–24), UPR1024 induced S arrest of the cell cycle, suggesting a different or additive target of action. This result has also been confirmed by BrdUrd incorporation experiments (not shown). An apoptotic sub-G₁ peak was detected, confirming that growth inhibition was associated with the activation of apoptotic pathways.

To investigate the involvement of apoptotic cell death in UPR1024-induced cytotoxicity, several known markers of apoptosis were evaluated.

As shown in Fig. 3A, the activation of caspase-3 was negligible until 6 h of treatment becoming activated after 16 h at 40 μmol/L. The maximal activity was detectable after 48 h for both concentrations of drug tested.

We then investigated the cleavage of caspase-3, caspase-8, and caspase-9 by Western blotting to define the apoptotic pathways involved (Fig. 3B). We showed that UPR1024 treatment was associated with the activation of all three caspases. These proapoptotic proteins were strongly cleaved from their inactive precursor forms into active fragments. In line with these observations, cytochrome c was detected in the cytoplasm (Fig. 3C) concomitant with a marked decrease in mitochondrial membrane potential (data not shown) as determined by MitoCapture assay kit.

Because the Fas signaling pathway is involved in gefitinib-mediated apoptosis (21, 25), we monitored Fas plasma membrane expression in our system. UPR1024 induced an increase in Fas protein expression on the cell surface (data not shown).

As expected, the caspase-8 inhibitor z-IETD completely abolished cleavage of caspase-8 but did not prevent the cleavage of caspase-9 (Fig. 3D), indicating that caspase-9 activation was not a mere consequence of caspase-8 activity.

Taken together, these results indicated that intrinsic and extrinsic pathways are both involved in UPR1024-triggered apoptotic death in A549 cells.

Taking into account the activation of the apoptotic intrinsic pathway induced by UPR1024 and considering that the α,β-unsaturated carbonyl fragment is featured by different cytotoxic agents that undergo interactions with DNA, we investigated the ability of the molecule to induce DNA damage. Fast halo assay was used to assess DNA damage in whole cells as previously reported (20). Control and UPR1024-treated cells were embedded in melted agarose spread onto microscope slides and lysed with a hypotonic solution at pH 13, which induced a radial diffusion from the nucleus of DNA fragments. Nuclei from A549 cells treated with 20 μmol/L UPR1024 for 3 h (Fig. 4A) showed a marked radial expansion (halo) of DNA fragments compared with nuclei from control cells. Quantitation of this effect, expressed as relative nuclear diffusion factor, is shown in Fig. 4B. Treatments with UPR1024 induced a concentration and time-dependent increase in the DNA damage, which reached a plateau at 16 h. Nuclear damage detected in cells treated with UPR1024 was not secondary to the onset of apoptosis as shown by the lack of caspase-3 activation until 16 h of treatment (see Fig. 3A).

As shown in Fig. 4C, UPR1024 induced DNA damage after 4 h of treatment that was completely repaired soon after withdrawal of the drug, suggesting a reversible effect on DNA.

As shown by Western blot analysis, UPR1024 treatment induced p53 accumulation in A549 cells expressing wild-type p53 protein (Fig. 5A and B). The increase in the p53 level was evident after 16 h of treatment but not at earlier time points. p21^WAF1 protein level was detectable at 24 h and strongly increased after 48 h of treatment.

To determine whether the presence of p53 affects the biological effects of UPR1024, we used Calu-1 cells (a NSCLC cell line lacking p53) stably transfected with an ecdysone-inducible p53 expression vector. In this model, treatment with the inducer Ponasterone A caused a dose-dependent expression of p53 protein (14).

Calu-1 clones treated with 2.5 μmol/L Ponasterone A and 20 μmol/L UPR1024 showed higher levels of p53 in comparison with cells treated with Ponasterone A alone (Fig. 5C). Furthermore, additional Western blot analysis showed an Mdm2 down-regulation simultaneously with an increase in p53 in A549 treated with UPR1024 (data not shown), suggesting that Mdm2 down-regulation might be responsible for p53 accumulation.

p53 expression leads to p21^WAF1 up-regulation, and higher levels of the protein were observed in the presence of UPR1024.

Calu-1 clones were then analyzed for growth capability in the absence or in the presence of Ponasterone A and UPR1024 for 72 h.

As shown in Fig. 5D, in absence of Ponasterone A stimulation, cell number was significantly reduced only at 20 μmol/L UPR1024 treatment. Upon addition of the inducer (Ponasterone A), the number of Calu-1 cells reexpressing p53 treated with UPR1024 was reduced at lower concentration of the drug (1 μmol/L). It should be noted that the expression of p53 alone induced by 2.5 μmol/L Ponasterone A in the absence of UPR1024 significantly reduced cell proliferation (20% versus control).

Taking into account that UPR1024 has a double target, we did an experiment designed to test whether UPR1024 treatment can overcome gefitinib resistance in SKLU-1 cells. As shown in Fig. 6A, gefitinib almost completely inhibited EGFR autophosphorylation at 1 μmol/L without affecting cell proliferation (Fig. 6B), indicating that resistance mechanisms were activated to bypass EGFR inhibition. When UPR1024 was added to gefitinib, a significant inhibition in colony formation was observed (Fig. 6B), suggesting that UPR1024 is able to reduce proliferation even after the EGFR kinase activity was fully inhibited by gefitinib.

Two main strategies for therapeutic targeting of EGFR have been developed: small-molecule inhibitors of the tyrosine kinase domain, such as gefitinib and erlotinib, and monoclonal antibodies, such as cetuximab, that are directed against the extracellular domain of EGFR and that inhibit phosphorylation and activation and stimulate internalization (26). In the case of gefitinib, survival improvement was found to be confined to certain patient subgroups (27). Investigations are in progress to identify molecular predictive markers of gefitinib responsiveness, such as EGFR mutations (7, 28, 29), EGFR gene copy number (30), and CA repeat polymorphism (31).

Tumor cells can develop several mechanisms that may result in resistance to gefitinib and erlotinib. Studies have revealed that additional oncogenic changes downstream of the EGFR (e.g., changes in the phosphatidylinositol 3′ kinase Akt pathway or K-ras) could also result in resistance to gefitinib and erlotinib (8). For this reason, new strategies for the simultaneous inhibition of multiple molecular targets are being pursued. One of these entails the combination of different molecular targets inhibitors (32).

The design and synthesis of new tyrosine kinase inhibitors is another rational approach to treat acquired resistance to erlotinib or gefitinib (33). The clinically available EGFR tyrosine kinase inhibitors belong to the chemical class of 4-aniloquinazolines, whose lack of structural diversity may lead to the appearance of cross-resistance and points to the need for novel molecules (34).

In a previous study, we examined the effect of a series of 1,5-disubstituted hydantoins on the inhibition of EGFR kinase activity and on cell growth. The results showed that UPR1024 is able to inhibit EGFR autophosphorylation and cell proliferation of A431 tumor cell line overexpressing EGFR protein (13).

In the present study, we confirmed our previous observations on a different cell model (NSCLC cell lines) showing that UPR1024 is capable of inhibiting EGF-induced EGFR autophosphorylation and cell proliferation leading to a preferential accumulation of cells in the S phase of the cell cycle. Moreover, new data on the biological effects of the molecule under investigation are presented, showing that UPR1024 has a second target at the DNA level.

Treatment with gefitinib induced in A549 cancer cell line a G₁ arrest associated with up-regulation of p27 without any changes in p21^WAF1 and p53 proteins (21, 25). Here, we show that in contrast to gefitinib, UPR1024 induced an S-phase growth arrest with up-regulation of p21^WAF1 and p53. No p27 increase was observed in the same conditions. Moreover, we found that treatment with UPR1024 (at concentration of >10 μmol/L) for >24 h resulted in an apoptotic cell death as shown by externalization of phosphatidylserine, release of cytochrome c, appearance of DNA fragmentation, and activation of caspase-3, caspase-8, and caspase-9.

Hence, we show the involvement of both the extrinsic and intrinsic pathways in the UPR1024-mediated apoptosis, whereas activation of extrinsic apoptotic pathway depending on Fas seems to play a major role in the gefitinib-induced apoptosis (21, 25). Interestingly, increased Fas expression has been correlated with p53 translocation to the nucleus (25); however, modulation of p53 level in response to gefitinib has not been observed.

Treatment with UPR1024 induced a concentration-dependent and time-dependent increase in the DNA damage with a concomitant accumulation of p53 and p21^WAF1. It is known that DNA damage induces arrest in the cell cycle progression acting mainly in the G₁ phase, depending on the activation of the p53 and subsequently up-regulation of p21^WAF1 (35). However, consistent with our results, treatment of the immortalized breast cell line MCF10A with topoisomerase I inhibitor SN38 (36) and treatment of human ovarian carcinoma cells with a new platinum (IV) complex (LA-12; ref. 37) induced an accumulation in S phase associated with the increased expression of p53 and p21^WAF1.

The role of p53 in the biological response to UPR1024 was evaluated in experiments involving reintroduction of p53 into a p53-negative lung cancer cell line (Calu-1).

Calu-1 cells, induced to express p53 treated with increased concentration of UPR1024, exhibited a more pronounced decrease in proliferation compared with cells not expressing p53. The drug was effective also in p53-negative cell lines (Calu-1, H1299) suggesting that p53 is not required for the inhibitory effect. However, there was a significant increase in sensitivity to UPR1024 in p53-positive cell lines.

These data indicate that, apart from anti-EGFR tyrosine kinase activity, UPR1024 induces additional cytotoxic DNA damage, a mechanism of action that may result in a significant antiproliferative and proapoptotic activity in refractory tumors. This conclusion is strongly supported by the observation that, in a cell model in which gefitinib is unable to reduce cell proliferation although autophosphorylation of the receptor is inhibited, the concomitant addition of UPR 1024 markedly reduced the capability to form colonies.

In particular, the α,β-unsaturated carbonyl substructure of UPR1024 may be partially responsible for its antiproliferative activity toward A549 cells. The characteristic reactivity of the α,β-unsaturated carbonyl moiety toward bionucleophiles may be responsible for the observed DNA damage effect and S-phase cell cycle arrest.

In a previous study, the exo-cyclic, conjugated double bond (the O=C-C=CHPh moiety) was shown to be an essential structural requirement for the biological activity of UPR1024 (13). Compounds containing the α,β-unsaturated carbonyl system can form cyclic adducts with DNA bases (38, 39) or can act as Michael acceptors in the reaction with -SH or -NH groups of bionucleophiles (40, 41). In addition to covalent bond formation, α,β-unsaturated carbonyl compounds can deplete cellular glutathione and promote the generation of reactive oxygen species (42). Therefore, the α,β-unsaturated carbonyl structure is an important feature for the cytotoxic antitumor activity of a wide range of natural products, such as helenalin (43), tenulin (44), and acronycine derivatives (45).

Although a clear characterization of its mechanism of action at the molecular level is required for further development of this class of compounds, the present data show that UPR1024 may be considered a new combi-molecule and could be inserted in a novel tumor strategy termed combi-targeting associating two modes of action within a single molecule. This approach had been successfully exploited by Jean-Claude's group for the anilinoquinazoline derivatives carrying a chloroethylamino (ZR2002; ref. 46), a triazene (RB24; ref. 47), or a nitrosourea (JDA58; ref. 48) fragment, all combining EGFR inhibition and DNA damage. The cooperative effect on double targets might enable broader antitumor activity and improve efficacy. Further investigations on UPR1024 or its derivatives should lead to the design of compounds circumventing resistance to classic tyrosine kinase inhibitors in the treatment of lung cancer.

We thank Prof. Prisco Mirandola for helpful discussion.