Molecular Mechanism Of The EGFR Inhibitor 244-MPT Overcomes Gefitinib Resistance In Non-small Cell Lung Cancer Cells

Cancer has become a major public health problem in both developed and developing countries. It is the second leading cause of death in the United States and is expected to go beyond heart disease to become the first cause of death in the next few years. In 2012,1800,000 lung cancer cases were diagnosed, which count up 13% of all newly diagnosed cancer patients. Based on 2015 updated database, lung cancer will be the most common cause of cancer death in both men and women. In China, the morbidity and mortality of lung cancer are expected to leading in all malignancy, and showed a trend of rising every year. Non-small cell lung cancer (NSCLC) is the major type of lung cancer (87%) and the overall 5-year survival rate for NSCLC is onlyl8.2%. At present, the treatment of lung cancer is still the preferred surgical resection, radiation and chemotherapy or targeted therapies are the strategies for patients who lost surgical opportunity. The epidermal growth factor receptor (EGFR) reportedly plays a critical role in NSCLC, with about 40-80% of NSCLC patients exhibiting elevated EGFR expression. In NSCLC, mutations are frequently observed in the EGFR. Classical-activating mutations include deletions in exon 19 and a point mutation of L858R in exon 21, which constitute about 90% of all EGFR-activating mutations. EGFR activating mutations changed the balance between active and unactive conformation. Because of the crucial role of EGFR in tumorigenesis, several EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, have been developed as effective clinical therapies for patients with activating mutations. The magnesium-ATP-binding pocket of the intracellular tyrosine kinase domain can be blocked by TKIs, which means that the ligand-induced receptor auto-phosphorylation function is obstructed by the binding of a TKI to the tyrosine kinase domain. This binding disrupts tyrosine-kinase activity, thereby inhibiting intracellular downstream signaling.Unfortunately, acquired resistance to the TKIs often manifests within 6 to 12 months of treatment. Reports indicate that TKIs resistance are associated with MET amplification, HER2 amplification, K-Ras mutations and PTEN loss. Meanwhile, approximately one-half of the cases of the emergence of a secondary mutation of EGFR, T790M, which leads to a substitution of methionine for threonine at position 790 (T790M) in the kinase domain. Research results have shown that Thr 790 is a gatekeeper residue, which is a key factor of TKIs binding in the EGFR ATP binding pocket. The T790M mutation causes resistance to TKIs by increasing the affinity of EGFR for ATP. Although resistance to TKI treatment in NSCLC has increased clinically, the treatment strategies to successfully overcome TKI resistance are still limited. Therefore, finding a drug that can inhibit the double mutant EGFR(L858R/T790M) to overcome gefitinib resistance in NSCLC treatment is greatly needed.By using computational methods to screen for small molecule inhibitors in the ZINC natural compound database, we identified 244-MPT (2-(4-(4-methoxyphenoxy)-1H-pyrazol-3-yl)-5-(p-tolylmethoxy)phenol, a potential EGFR inhibitor that could be used in NSCLC therapy. We found that this compound could suppress gefitinib-sensitive and -resistant NSCLC cell growth by inhibiting EGFR activity and its downstream signaling pathways in vitro and ex vivo. Moreover, we observed that 244-MPT could strongly suppress gefitinib-resistant NSCLC tumor growth in both mouse xenograft model and patient-derived xenograft (PDX) model.Chapter I 244-MPT inhibits anchorage-independent growth and proliferation of both gefitinib-sensitive and-resistant NSCLC cells.Methods1. By using computational methods to screen for small molecule inhibitor against EGFR.2. The MTS assay was used to determine whether 244-MPT exerted any cytotoxic effects against normal lung cells.3. The soft agar assay was performed to examine the effects of 244-MPT on gefitinib-sensitive or-resistant NSCLC cell lines.4. To examine the effects of 244-MPT on gefitinib-sensitive or -resistant NSCLC cell lines by cell proliferation assay.Results1.244-MPT is a potential wildtype and mutant (L858R/T790M) EGFR inhibitorFrom the computational screening result, we found that 244-MPT got a high score which can bind well with wildtype and mutant (L858R/T790M) EGFR as a novel compound. Thus,244-MPT was selected for further study.2.244-MPT has no cytotoxicity against on normal lung cellsThe MTS result showed that 244-MPT had no cytotoxicity against MRC-5 cells at concentrations less than 40μM.3.244-MPT inhibits anchorage-independent growth of both gefitinib-sensitive and -resistant NSCLC cellsSoft agar assay results revealed that gefitinib and 244-MPT strongly inhibited gefitinib-sensitive HCC827 colony formation. Notably,244-MPT also markedly suppressed H1975 gefitinib-resistant colony formation dose-dependently, whereas gefitinib was ineffective.4.244-MPT inhibits cell proliferation of both gefitinib-sensitive and-resistant NSCLC cellsMTS results showed that 244-MPT markedly suppresses cell proliferation of gefitinib-sensitive or -resistant NSCLC cell lines dose-dependently, whereas no effect of gefitinib was observed on gefitinib-resistant NSCLC cell line.Chapter Ⅱ 244-MPT binds and inhibits both wildtype and mutant EGFR activities in vitro and ex vivo.Methods1. To better understand the mechanisms and the activity of 244-MPT, we conducted an in silico docking study and an energy minimization and molecular dynamics (MD) simulation.2. To further validate these simulations, we conducted ATP competition assays with 244-MPT-conjugated SepharoseTM 4B beads.3. Experiment of ex vivo binding assay between 244-MPT-conjuated SepharoseTM 4B beads and cell lysate overexpressing exogenous EGFR.4. EGFR in vitro kinase assays were conducted to determine the inhibiting efficiency of 244-MPTResults1.244-MPT binds EGFR at the ATP-binding pocketThe computational binding models showed that several hydrogen bonds were formed between 244-MPT and the EGFR ATP pocket in either the wildtype or mutant protein. And after 5ns MD, results showed that 244-MPT still formed some hydrogen bonding, hydrophobic and other interactions with double mutant EGFR (L858R/T790M).2.244-MPT binds EGFR at the ATP-binding pocket, compete with ATPAn ATP competition assay further showed that either wildtype or mutant EGFR was pulled down by 244-MPT-conjugated SepharoseTM 4B beads but not SepharoseTM 4B beads alone.3.244-MPT binds EGFR at the ATP-binding pocket ex vivoWe observed ex vivo binding between 244-MPT and EGFR in HEK-293T cells overexpressing exogenous wildtype or mutant EGFR.244-MPT could strongly bind with wildtype, the L858R single mutant EGFR or the L858R/T790M double mutant EGFR.4.244-MPT inhibits both wildtype and mutant EGFR activities in vitroThe in vitro kinase assay showed 244-MPT strongly inhibited wildtype EGFR kinase activity, whereas 244-MPT also substantially inhibited the kinase activity of the EGFR L858R/T790M double mutant while gefitinib was ineffective.Chapter III 244-MPT attenuates EGFR signaling in both gefitinib-sensitive and -resistance NSCLC cells.Methods1. To examine the effects of 244-MPT on EGFR signaling pathway in gefitinib-sensitive and -resistant NSCLC cell lines by Western blot.2. To establish NL-20 cells stably overexpressing wildtype or mutant EGFR.3. To examined the effect of 244-MPT on NL-20 cells stably overexpressing wildtype or mutant EGFR by Western blot.Results1. Western blot results showed that 244-MPT strongly suppressed phosphorylation of EGFR, Akt and ERK1/2 in both gefitinib-sensitive and -resistant NSCLC cells.2. We observed NL20 cells could stably overexpress wildtype or mutant EGFR.3. Western blot indicated that 244-MPT strongly attenuated phosphorylation of EGFR, Akt and ERK1/2 in EGFR overexpressing system.Chapter IV 244-MPT induces apoptosis in gefitinib-resistant NSCLC cells.Methods1. To examine whether 244-MPT could induce apoptosis in H1975 gefitinib-resistant NSCLC cells.2. To examine the effects of 244-MPT on apoptosis signaling pathway in gefitinib-resistance NSCLC cell lines by Western blot.3. By using TUNEL assay to confirm 244-MPT could induce apoptosis in H1975 gefitinib-resistant NSCLC cells.Results1.244-MPT could induce apoptosis in H1975 gefitinib-resistant NSCLC cell lineData showed that treatment with 244-MPT caused significant apoptosis in H1975 cell line.2.244-MPT has effect on apoptosis signaling pathway in H1975 gefitinib-resistant NSCLC cell lineTreatment of H1975 cells with 244-MPT induced the cleavage of caspase-3 and PARP, which are markers of apoptosis, increased the expression of the pro-apoptotic protein, Bax, and reduced the expression of the anti-apoptotic protein, Bel-2.3.244-MPT could induce apoptosis in H1975 gefitinib-resistant NSCLC cellsThe TUNEL assay provided results showed that treatment with 244-MPT caused substantially more apoptosis.Chapter V 244-MPT suppresses tumor growth of gefitinib-resistant NSCLC cells in a xenograft mouse model.Methods1. To determine the chemotherapeutic effect of 244-MPT in vivo, we used an athymic nude xenograft mouse model of gefitinib-resistant H1975 cells.2. Immunohistochemical analysis of 244-MPT treated H1975 xenograft tumors, to confirm the effect of 244-MPT was associated with its inhibition of EGFR or not.Results1.244-MPT suppresses tumor growth of gefitinib-resistance xenograftsXenograft mouse model results showed that 244-MPT suppressed H1975 xenograft tumor growth, whereas gefitinib was ineffective in reducing tumor size. Thsese results indicated that 244-MPT effectively overcome gefitinib-resistant xenograft growth in mice.2.244-MPT suppressing tumor growth of gefitinib-reisistant xenografts was associated with its inhibition of EGFRResults showed that Ki-67 expression and phosphorylation of EGFR, Akt and ERK1/2 were each significantly suppressed in the 244-MPT treated-groups compared with the vehicle-or gefitinib-treated group. These results clearly indicated that 244-MPT exerts a substantial chemotherapeutic effect to overcome gefitinib-resistant xenograft growth in mice acting mainly through the suppression of EGFR activation.Chapter VI 244-MPT suppresses tumor growth in a gefitinib-resistant NSCLC PDX model.Methods1. To create a NSCLC PDX model that mimicked a patient who was sensitive to gefitinib, then induce the model to a gefitinib-resistant NSCLC PDX model.2. To determine the chemotherapeutic effect of 244-MPT in a NSCLC gefitinib-resistant NSCLC PDX model.3. Immunohistochemical analysis of 244-MPT treated PDX tumors was conducted to evaluate the expression level of phosphorylated EGFR, ERK1/2, and Ki-67.Results1. The method to creat gefitinib-resistant NSCLC PDX model workedThe NSCLC gefitinib-resistant NSCLC PDX model was established by exposure to stepwise increasing doses of gefitinib over time. Thses results indicated that created a model that mimicked a patient who was sensitive to gefitinib, treated with gefitinib for a certain period, and finally followed by the emergence of gefitinib-resistance finally worked.2.244-MPT suppresses tumor growth in a NSCLC gefitinib-resistant NSCLC PDX modelIn the NSCLC gefitinib-resistant NSCLC PDX model, results showed that 244-MPT suppressed NSCLC gefitinib-resistant NSCLC PDX tumor growth with no toxicity, whereas gefitinib had no effect.3.244-MPT suppressing tumor growth of gefitinib-reisistant NSCLC PDX model was associated with its inhibition of EGFRImmunohistochemical analysis showed that Ki-67 and phosphorylation of EGFR and ERK1/2 were significantly suppressed in the 244-MPT treated-groups compared with the vehicle- or gefitinib-treated group.Conclusions1. Several different approaches, supercomputer simulation, in vitro, ex vivo and in vivo, all confirmed that 244-MPT binds and inhibits both wildtype and mutant EGFR activities by competing ATP in EGFR binding pocket, and attenuated primary downstream pathways of EGFR signaling. All these results indicated that 244-MPT overcomes gefitinib-resistant NSCLC cell growth by suppressing EGFR.2.244-MPT markedly inhibites NSCLC tumor growth of both in vivo xenograft study and PDX model.244-MPT exerts a substantial chemotherapeutic effect against gefitinib -resistant NSCLC without affecting body weight of mice, all these data indicated that 244-MPT might be a very promising novel drug candidate tobe evaluated in clinical trials.