Synergistic Effect of Afatinib with Su11274 in Non-Small Cell Lung Cancer Cells Resistant to Gefitinib or Erlotinib

Epidermal growth factor receptor (EGFR) and c-MET receptors are expressed on many non-small cell lung cancer (NSCLC) cells. Current single agent therapeutic targeting of a mutant EGFR has a high efficacy in the clinic, but is not curative. Here, we investigated the combination of targeting EGFR and c-MET pathways in NSCLC cells resistant to receptor tyrosine kinase inhibitors (TKIs), using RNA interference and inhibition by TKIs. Different NSCLC cell lines with various genomic characteristics (H358, H1650 and H1975) were transfected with EGFR-specific-siRNA, T790M-specific-siRNA, c-MET siRNA or the combination. Subsequently EGFR TKIs (gefitinib, erlotinib or afatinib) or monoclonal antibody cetuximab were combined respectively with the c-MET-specific TKI su11274 in NSCLC cell lines. The cell proliferation, viability, caspase−3/7 activity and apoptotic morphology were monitored by spectrophotometry, fluorimetry and fluorescence microscopy. The combined effect of EGFR TKIs, or cetuximab and su11274, was evaluated using a combination index. The results showed that the cell lines that were relatively resistant to EGFR TKIs, especially the H1975 cell line containing the resistance T790M mutation, were found to be more sensitive to EGFR-specific-siRNA. The combination of EGFR siRNA plus c-MET siRNA enhanced cell growth inhibition, apoptosis induction and inhibition of downstream signaling in EGFR TKI resistant H358, H1650 and H1975 cells, despite the absence of activity of the c-MET siRNA alone. EGFR TKIs or cetuximab plus su11274 were also consistently superior to either agent alone. The strongest biological effect was observed when afatinib, an irreversible pan-HER blocker was combined with su11274, which achieved a synergistic effect in the T790M mutant H1975 cells. In a conclusion, our findings offer preclinical proof of principle for combined inhibition as a promising treatment strategy for NSCLC, especially for patients in whom current EGFR-targeted treatments fail due to the presence of the T790M-EGFR-mutation or high c-MET expression.     

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M

AIM: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR).METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs.RESULTS: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M.CONCLUSION: Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.     

PET probe detecting non-small cell lung cancer susceptible to epidermal growth factor receptor tyrosine kinase inhibitor therapy

Tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR-TKIs) are used as molecular targeted therapy for non-small cell lung cancer (NSCLC) patients. The therapy is applied to the patients having EGFR-primary L858R mutation, but drug tolerance caused by EGFR-secondary mutation is occurred within one and half years. For the non-invasive detection of the EGFR-TKIs treatment positive patients by positron emission tomograpy (PET) imagaing, fluorine-18 labeled thienopyrimidine derivative, [¹⁸F]FTP2 was newly synthesized. EGFR inhibition assay, cell uptake study, and blocking study indicated [¹⁸F]FTP2 binds with high and selective affinity for EGFR with L858R mutation, and not with L858R/T790M dual mutations. On animal PET study using tumor bearing mice, H3255 cells expressing L858R mutated EGFR was more clearly visualized than H1975 cells expressing L858R/T790M dual mutated EGFR. [¹⁸F]FTP2 has potential for detecting NSCLC which is susceptible to EGFR-TKI treatment.     

Curcumin induces EGFR degradation in lung adenocarcinoma and modulates p38 activation in intestine: the versatile adjuvant for gefitinib therapy

Background

Non-small cell lung cancer (NSCLC) patients with L858R or exon 19 deletion mutations in epidermal growth factor receptor (EGFR) have good responses to the tyrosine kinase inhibitor (TKI), gefitinib. However, patients with wild-type EGFR and acquired mutation in EGFR T790M are resistant to gefitinib treatment. Here, we showed that curcumin can improve the efficiency of gefitinib in the resistant NSCLC cells both in vitro and in vivo models.

Methods/Principal Findings

After screening 598 herbal and natural compounds, we found curcumin could inhibit cell proliferation in different gefitinib-resistant NSCLC cell lines; concentration-dependently down-regulate EGFR phosphorylation through promoting EGFR degradation in NSCLC cell lines with wild-type EGFR or T790M EGFR. In addition, the anti-tumor activity of gefitinib was potentiated via curcumin through blocking EGFR activation and inducing apoptosis in gefitinib-resistant NSCLC cell lines; also the combined treatment with curcumin and gefitinib exhibited significant inhibition in the CL1-5, A549 and H1975 xenografts tumor growth in SCID mice through reducing EGFR, c-MET, cyclin D1 expression, and inducing apoptosis activation through caspases-8, 9 and PARP. Interestingly, we observed that the combined treatment group represented better survival rate and less intestinal mucosal damage compare to gefitinib-alone therapy. We showed that curcumin attenuated the gefitinib-induced cell proliferation inhibition and apoptosis through altering p38 mitogen-activated protein kinase (MAPK) activation in intestinal epithelia cell.

Conclusions/Significance

Curcumin potentiates antitumor activity of gefitinib in cell lines and xenograft mice model of NSCLC through inhibition of proliferation, EGFR phosphorylation, and induction EGFR ubiquitination and apoptosis. In addition, curcumin attenuates gefitinib-induced gastrointestinal adverse effects via altering p38 activation. These findings provide a novel treatment strategy that curcumin as an adjuvant to increase the spectrum of the usage of gefitinib and overcome the gefitinib inefficiency in NSCLC patients.     

AZD9291 promotes autophagy and inhibits PI3K/Akt pathway in NSCLC cancer cells

AZD9291, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is highly selective against EGFR T790M-mutant non–small cell lung cancer (NSCLC). On investigating the growth inhibitory effects of AZD9291 on NSCLC and the underlying mechanism, we found that AZD9291 can trigger autophagy-mediated cell death in both A549 and H1975 cells by increasing the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3) and decreasing the expression of p62. In the presence of the autophagy inhibitor chloroquine, the AZD9291-induced increase in LC3 level was further augmented. AZD9291 decreased the levels of phosphoinositide-3 kinase (PI3K), protein kinase B (Akt), and phosphorylated Akt. AZD9291-induced cell death was enhanced by Akt knockdown, and the levels of both EGFR and phosphorylated EGFR were decreased by AZD9291. AZD9291 was also found to significantly suppress the tumor growth in H1975 xenograft nude mice. Thus, AZD9291 was found to induce autophagy, decrease in EGFR levels, and show a strong inhibitory effect on NSCLC both in vitro and in vivo. Furthermore, the PI3K/Akt signaling pathway was found to play a critical role in AZD9291-induced cell death.     

Multiple Analyses of G-Protein Coupled Receptor (GPCR) Expression in the Development of Gefitinib-Resistance in Transforming Non-Small-Cell Lung Cancer

There is increasing evidence that functional crosstalk between GPCRs and EGFR contributes to the progression of colon, lung, breast, ovarian, prostate and head and neck tumors. In this study, we performed multiple analyses of GPCR expression in a gefitinib-resistant non-small cell lung cancer (NSCLC) cell line, H1975, which harbors an L858R/T790M mutation. To determine the expression profile of mRNAs encoding 384 GPCRs in normal human lung fibroblast (NHLF) and H1975 cells, a GPCR-specific microarray analysis was performed. A heat-map of the microarray revealed considerable differences in the expression of GPCRs between NHLF and H1975 cells. From the GPCR expression list, we selected some GPCR agonists/antagonist to investigate whether the respective ligands could affect the growth of H1975 cells. Among them, treatment with either a selective antagonist of adenosine A2a receptors, which were highly expressed in H1975 cell and another gefitinib-resistant NSCLC cells, HCC827GR cells or “small interfering RNA” (siRNA) targeting adenosine A2a receptors produced a significant decrease in cell viability of both H1975 and HCC827GR cells. Among up-regulated GPCRs in H1975 cells, Gs-, Gi- and Gq-coupled GPCRs were expressed almost equally. Among down-regulated GPCRs, Gi-coupled GPCRs were dominantly expressed in H1975 cells. The present results suggest that multilayered crosstalk between GPCRs and EGFR may play an important role in orchestrating downstream signaling molecules that are implicated in the development of gefitinib-resistant NSCLC.     

Epidermal growth factor receptor (EGFR) antibody down-regulates mutant receptors and inhibits tumors expressing EGFR mutations

Activating mutations in the kinase domain of the EGF receptor have been reported in non-small cell lung cancer. The majority of tumors expressing these mutants are sensitive to ATP mimetics that inhibit the EGFR tyrosine kinase. The effect of antibodies that bind to the ectodomain of the receptor is less clear. We report herein the effects and mechanisms of action of the antibody cetuximab in lung cancer cells that naturally express receptor mutations and in ErbB-null 32D hematopoietic cells transfected with mutant EGFR. Treatment with cetuximab down-regulated EGFR levels and inhibited cell growth both in vitro and in vivo. This was associated with inhibition of ligand-independent EGFR signaling. These effects were seen in 32D cells arguing the growth inhibitory action was not because of the blockade of autocrine ligand action. Both antibody-induced EGFR down-regulation and inhibition of growth required receptor dimerization as monovalent Fab fragments only eliminated receptor levels or reduced cell proliferation in the presence of antihuman IgG. Finally, cetuximab inhibited growth of H1975 lung cancer cells and xenografts, which expressed L858R/T790M EGFR and were resistant to EGFR tyrosine kinase inhibitors. These data suggest that cetuximab is an effective therapy against mutant EGFR-expressing cancer cells and thus can be considered in combination with other anti-EGFR molecules.     

Isoliquiritigenin Induces Apoptosis and Inhibits Xenograft Tumor Growth of Human Lung Cancer Cells by Targeting Both Wild Type and L858R/T790M Mutant EGFR

Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR.     

Marsdenia tenacissima extract enhances gefitinib efficacy in non-small cell lung cancer xenografts

PurposeThe stem of Marsdenia tenacissima (Roxb.) Wight et Arn. has long been used as a medicine to treat cancer in China. Our previous in vitro results showed that Marsdenia tenacissima extract (MTE) overcomes gefitinib resistance in non-small cell lung cancer (NSCLC) cells. However, it is unknown whether MTE could enhance gefitinib efficacy in vivo. The present study was intended to investigate the in vivo anti-tumour activity of MTE combined with gefitinib.MethodsHuman NSCLC H460 (K-ras mutation) or H1975 cells (EGFR T790M mutation) were subcutaneously inoculated into nude mice. Tumour volume and body weight were measured regularly. Resected tumours were weighed after the animals were sacrificed. Immunoblotting or immunohistochemistry was used to assess the cellular proliferation and apoptosis in xenograft tumour tissue. Expression of the EGFR downstream pathways and c-Met were measured with western blot analysis to explore possible mechanisms.ResultsMTE (5, 10, 20 g/kg) dose-dependently reduced tumour growth and induced cell apoptosis. MTE suppressed EGFR related signals, and 20 g/kg was the most effective dose. Low-dose MTE (5 g/kg) significantly enhanced gefitinib efficacy in resistant H460 and H1975 xenografts. The combination inhibited tumour proliferation and induced cell apoptosis in both resistant NSCLC xenografts. Constitutive activation of the PI3K/Akt and MEK/ERK pathways is related to EGFR-TKI resistance. Accordingly, phosphorylation of PI3K/Akt/mTOR and ERK1/2 was suppressed after combination treatment. Simultaneously, cross-talked c-Met and EGFR were also prominently lowered in the presence of MTE combined with gefitinib.ConclusionThe present results suggest that the combination of MTE and gefitinib may be a promising therapeutic approach to enhance gefitinib efficacy in resistant NSCLC.     

Licochalcone A inhibits EGFR signalling and translationally suppresses survivin expression in human cancer cells

Dysfunction of epidermal growth factor receptor (EGFR) signalling plays a critical role in the oncogenesis of non–small-cell lung cancer (NSCLC). Here, we reported the natural product, licochalcone A, exhibited a profound anti-tumour efficacy through directly targeting EGFR signalling. Licochalcone A inhibited in vitro cell growth, colony formation and in vivo tumour growth of either wild-type (WT) or activating mutation EGFR-expressed NSCLC cells. Licochalcone A bound with L858R single-site mutation, exon 19 deletion, L858R/T790M mutation and WT EGFR ex vivo, and impaired EGFR kinase activity both in vitro and in NSCLC cells. The in silico docking study further indicated that licochalcone A interacted with both WT and mutant EGFRs. Moreover, licochalcone A induced apoptosis and decreased survivin protein robustly in NSCLC cells. Mechanistically, we found that treatment with licochalcone A translationally suppressed survivin through inhibiting EGFR downstream kinases ERK1/2 and Akt. Depletion of the translation initiation complex by eIF4E knockdown effectively inhibited survivin expression. In contrast, knockdown of 4E-BP1 showed the opposite effect and dramatically enhanced survivin protein level. Overall, our data indicate that targeting survivin might be an alternative strategy to sensitize EGFR-targeted therapy.     

Structure based designing of triazolopyrimidone-based reversible inhibitors for kinases involved in NSCLC

Secondary acquired mutant EGFR (L858R-T790M) overexpressed NSCLC forms one of the prevalent form of resistant NSCLC. Another subset of resistant NSCLC includes amplified cMET in mutant EGFR derived tumours. Thus, in continuation to our previous work on these two major targets of resistant NSCLC, i.e., EGFR (L858R-T790M) and cMET, we are hereby reporting reversible inhibitors of these kinases. Out of 11 lead molecules reported in our previous study, we selected triazolo-pyrimidone (BAS 09867482) scaffold for further development of small molecule dual and reversible inhibitors. Analogues of lead with different substituents on the side ring were sketched and docked in both the target kinases, followed by molecular dynamic simulations. Analogues maintaining hydrophobic interaction with M790 in secondary acquired mutant EGFR (L858R-T790M) were selected and duly synthesized. In vitro biochemical evaluation of these molecules against EGFR (L858R-T790M) and cMET kinase, along with EGFR (L858R) kinase disclosed that three molecules were having significant dual kinase inhibitory potential with IC₅₀ values well below 100 nM. Further, in vitro anti-proliferative assay against three cell lines (A549, A431 and H460) was performed. Out of all, two compounds were having significant potency against these cell lines.     

Exosomes transmit T790M mutation‐induced resistance in EGFR‐mutant NSCLC by activating PI3K/AKT signalling pathway

Emerging evidence has shown that exosomes derived from drug‐resistant tumour cells are able to horizontally transmit drug‐resistant phenotype to sensitive cells. However, whether exosomes shed by EGFR T790M‐mutant–resistant NSCLC cells could transfer drug resistance to sensitive cells has not been investigated. We isolated exosomes from the conditioned medium (CM) of T790M‐mutant NSCLC cell line H1975 and sensitive cell line PC9. The role and mechanism of exosomes in regulating gefitinib resistance was investigated both in vitro and in vivo. Exosome‐derived miRNA expression profiles from PC9 and H1975 were analysed by small RNA sequencing and confirmed by qRT‐PCR. We found that exosomes shed by H1975 could transfer gefitinib resistance to PC9 both in vitro and in vivo through activating PI3K/AKT signalling pathway. Small RNA sequencing and RT‐PCR confirmed that miR‐3648 and miR‐522‐3p were the two most differentially expressed miRNAs and functional study showed that up‐regulation of miR‐522‐3p could induce gefitinib resistance in PC9 cell. The findings of our study reveal an important mechanism of acquired resistance to EGFR‐TKIs in NSCLC.     

Discovery of selective EGFR modulator to inhibit L858R/T790M double mutants bearing a N-9-Diphenyl-9H-purin-2-amine scaffold

Based upon the modeling binding mode of marketed AZD9291 with T790M, a series of N-9-Diphenyl-9H-purin-2-amine derivatives were designed and synthesized with the purpose to overcome the drug resistance resulted from T790M/L858R double mutations. The most potent compound 23a showed excellent enzyme inhibitory activities and selectivity with nanomolar IC₅₀ values for both the single T790M and double T790M/L858R mutant EGFRs, and was more than 8-fold selective for wild type EGFR. Compound 23a displayed strong antiproliferative activity against the H1975 non-small cell lung cancer (NSCLC) cells bearing T790M/L858R. And it was less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety index.     

Dihydromyricetin suppresses tumor growth via downregulation of the EGFR/Akt/survivin signaling pathway

Deregulation of epidermal growth factor receptor (EGFR) signaling is frequently observed in non-small cell lung cancer (NSCLC). The present study aimed to determine the impact of dihydromyricetin (DHM) on NSCLC, a natural compound extracted from Ampelopsis grossedentata with various pharmacological activities. Results of the present study demonstrated that DHM may act as a promising antitumor agent for NSCLC therapy, inhibiting the growth of cancer cells in vitro and in vivo. Mechanistically, results of the present study demonstrated that exposure to DHM downregulated the activity of wild-type (WT) and mutant EGFRs (mutations, exon 19 deletion, and L858R/T790M mutation). Moreover, western blot analysis indicated that DHM induced cell apoptosis via suppression of the antiapoptotic protein, survivin. Results of the present study further demonstrated that depletion or activation of EGFR/Akt signaling may regulate survivin expression though modulating ubiquitination. Collectively, these results suggested that DHM may act as a potential EGFR inhibitor, and may provide a novel choice of treatment strategy for patients with NSCLC.     

A systematic profile of clinical inhibitors responsive to EGFR somatic amino acid mutations in lung cancer: implication for the molecular mechanism of drug resistance and sensitivity

Human epidermal growth factor receptor (EGFR) has become a well-established target for the treatment of patients with non-small cell lung cancer (NSCLC). However, a large number of somatic mutations in such protein have been observed to cause drug resistance or sensitivity during pathological progression, limiting the application of reversible EGFR tyrosine kinase inhibitor therapy in NSCLC. In the current work, we describe an integration of in silico analysis and in vitro assay to profile six representative EGFR inhibitors against a panel of 71 observed somatic mutations in EGFR tyrosine kinase domain. In the procedure, the changes in interaction free energy of inhibitors with EGFR upon various mutations were calculated one by one using a rigorous computational scheme, which was pre-optimized based on a set of structure-solved, affinity-known samples to improve its performance in characterizing the EGFR-inhibitor system. This method was later demonstrated to be effective in inferring drug response to the classical L858R and G719S mutations that confer constitutive activation for the EGFR kinase. It is found that the Staurosporine, a natural product isolated from the bacterium Streptomyces staurosporeus, exhibits selective inhibitory activity on the T790M and T790M/L858R mutants. This finding was subsequently solidified by in vitro kinase assay experiment; the inhibitory IC₅₀ values of Staurosporine against wild-type, T790M and T790M/L858R mutant EGFR were measured to be 937, 12 and 3 nM, respectively.     

Discovery of 5-(methylthio)pyrimidine derivatives as L858R/T790M mutant selective epidermal growth factor receptor (EGFR) inhibitors

To overcome the drug-resistance of first generation EGFR inhibitors and the nonselective toxicities of second generation inhibitors among NSCLC patients, a series of 5-(methylthio)pyrimidine derivatives were discovered as novel EGFR inhibitors, which harbored not only potent enzymatic and antiproliferative activities against EGFR^L858R/T790M mutants, but good selectivity over wide-type form of the receptor. This goal was achieved by employing structure-based drug design and traditional optimization strategies, based on WZ4002 and CO1686. These derivatives inhibited the enzymatic activity of EGFR^L858R/T790M mutants with IC₅₀ values in subnanomolar ranges, while exhibiting hundreds of fold less potency on EGFR^WT. These compounds also strongly inhibited the proliferation of H1975 non-small cell lung cancer cells bearing EGFR^L858R/T790M, while being significantly less toxic to A431 human epithelial carcinoma cells with overexpressed EGFR^WT. The EGFR kinase inhibitory and antiproliferative activities were further validated by Western blot analysis for activation of EGFR and the downstream signaling in cancer cells.     

Design,synthesis and evaluation of the osimertinib analogue (C-005) as potent EGFR inhibitor against NSCLC

Osimertinib has been approved as a first-line treatment for non-small-cell lung cancer (NSCLC) patients whose tumor carries EGFR activation and / or resistant mutations. To mitigate Osimertinib’s toxicity caused by AZ5104, the N-demethylation metabolite of Osimertinib, we designed and synthesized a series of Osimertinib analogs with different headpieces. In vitro and in vivo analysis rendered a potential clinical candidate C-005 which had pyrrolo-pyridine headpiece. Biochemically, C-005 and its main human hepatocyte metabolite showed over 30 fold selectivity of L858R/T790M mutant EGFR over WT EGFR. Such selectivity profile was retained at cellular level. In general, C-005 is 2-14 fold more selective than Osimertinib in a panel of WT EGFR cancer cell lines. Furthermore, C-005 demonstrated robust antitumor efficacy and good tolerability in NCI-H1975, PC-9 and HCC827 xenograft mouse models, making it a potential candidate for human test in clinical.     

BEBT-109, a pan-mutant-selective EGFR inhibitor with potent antitumor activity in EGFR-mutant non-small cell lung cancer

EGFR mutation-positive NSCLC tumors are highly heterogeneous, therefore, exploring an agent simultaneously targeting multiple EGFR mutations may be valuable for clinical practice. Compared with osimertinib, BEBT-109 shows more sensitive and extensive antitumor activity in EGFR mutant NSCLC, while sparing wild-type EGFR cell lines. Meanwhile, unlike the metabolite of osimertinib AZ5104, the main metabolites of BEBT-109 are found lacking in activity against wild-type EGFR cell lines. Preclinical and clinical studies demonstrate a unique pharmacokinetic profiles of BEBT-109 with rapid absorption and quick in vivo clearance without accumulation, which are conducive to minimizing the off-target toxicity of the covalent irreversible EGFR inhibitor. Oral administration of BEBT-109 induces tumor regression in EGFR exon 20 insertion xenografts, and even tumor disappearance in PC-9, HCC827 and H1975 xenograft models. Furthermore, in clinical trials, the objective responses were observed in NSCLC patients with EGFR T790M mutation in the first and second dosing cohorts. These findings demonstrate that BEBT-109, a potent pan-mutant-selective EGFR inhibitor with improved pharmacokinetic properties, might offer a promising new option for the treatment of multiple mutant-EGFR-driven NSCLC.