Discovery of a novel third-generation EGFR inhibitor and identification of a potential combination strategy to overcome resistance

Abstract

Background: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond to first-generation EGFR inhibitors; however, the efficacy of these drugs is limited by acquired resistance driven by the EGFR ^T790M mutation. The discovery of third-generation EGFR inhibitors overcoming EGFR ^T790M and their new resistance mechanisms have attracted much attention.

Methods: We examined the antitumor activities and potential resistance mechanism of a novel EGFR third-generation inhibitor in vitro and in vivo using ELISA, SRB assay, immunoblotting, flow cytometric analysis, kinase array, qRT-PCR and tumor xenograft models. The clinical effect on a patient was evaluated by computed tomography scan.

Results: We identified compound ASK120067 as a novel inhibitor of EGFR ^T790M, with selectivity over EGFR ^WT. ASK120067 exhibited potent anti-proliferation activity in tumor cells harboring EGFR ^T790M (NCI-H1975) and sensitizing mutations (PC-9 and HCC827) while showed moderate or weak inhibition in cells expressing EGFR ^WT. Oral administration of ASK120067 induced tumor regression in NSCLC xenograft models and in a PDX model harboring EGFR ^T790M. The treatment of one patient with advanced EGFR T790M-positive NSCLC was described as proof of principle. Moreover, we found that hyperphosphorylation of Ack1 and the subsequent activation of antiapoptotic signaling via the AKT pathway contributed to ASK120067 resistance. Concomitant targeting of EGFR and Ack1 effectively overrode the acquired resistance of ASK120067 both in vitro and in vivo.

Conclusions: Our results idenfity ASK120067 as a promising third-generation EGFR inhibitor and reveal for the first time that Ack1 activation as a novel resistance mechanism to EGFR inhibitors that guide to potential combination strategy.

Keywords: Ack1; Drug resistance; EGFR T790M; Non-small cell lung cancer (NSCLC); Small-molecule inhibitor.

Fig. 1

Chemical structure, binding mode and target inhibition of compound ASK120067. a Chemical structure of ASK120067. b Structure modeling of ASK120067 binding to EGFR ^T790M. The 2-aminopyrimidine core of ASK120067 forms two hydrogen bonds to the hinge residue Met793, and the acrylamide group forms a covalent bond with the conserved C797 residue in the ATP-binding pocket. The C5-Cl substitution points to the gatekeeper Met790 residue. The 2,4-disubstituted pyrimidine scaffold adapts a U-shaped configuration. The amine moiety faces an open space in the solvent exposure area. c Inhibition of ASK120067 on EGFR kinases determined by ELISA assay. IC₅₀ values are shown as the mean ± SD from at least three independent experiments. d Kinase profiling of ASK120067 at a concentration of 100 nM against 258 human kinases. The graphic was generated by BioMed X GmbH and Merck KgaA, with slight modifications. Circles indicate detected kinases, and the circle size indicates the strength of inhibition

Fig. 2

Effect of ASK120067 on cancer cells harboring mutant or wild-type EGFR. a and b ASK120067 or osimertinib inhibited the phosphorylation of EGFR at Tyrosine residue 1068 and its downstream signaling proteins AKT and ERK in NCI-H1975 cells (EGFR ^L858R/T790M) a while showing less activity against the activation of EGFR and its downstream signaling in A431 cells expressing EGFR ^WTb. c Apoptosis of NCI-H1975 cells was evaluated by flow cytometry after treatment with increasing concentrations of ASK120067 for 24 to 72 h. Data are plotted as the mean ± SEM, and significance of differences was determined by Student’s t test (^∗p < 0.05, ^∗∗p < 0.01). d The expression levels of cleaved PARP and cleaved caspase-3 in NCI-H1975 cells after ASK120067 treatment were determined by Western blot analysis

Fig. 3

ASK120067 exerts in vivo antitumor activity against EGFR-mutant tumor xenograft models, and proof-of-concept clinical studies validate ASK120067 as an EGFR ^T790M inhibitor. a Antitumor activity of ASK120067 in the NCI-H1975 lung cancer xenograft model following 21 days of daily treatment with ASK120067 at doses of 1, 5, and 10 mg/kg/qd, with osimertinib (10 mg/kg/qd) as a positive control. b The phosphorylation of EGFR and AKT in tumor tissues of the NCI-H1975 xenograft model were evaluated by IHC staining after 21 days of treatment with ASK120067 or vehicle control and are presented by quantitative analysis. c Antitumor efficacy of ASK120067 in a PC-9 lung cancer xenograft model. d Antitumor activity of ASK120067 in the A431 epidermoid carcinoma xenograft model. e Antitumor efficacy of ASK120067 in PDX models harboring the EGFR ^L858R/T790M mutation. f The expression of phosphorylated EGFR and Ki-67 in PDX xenograft tumor tissues was evaluated by IHC staining and is presented by quantitative analysis. Data are presented as the mean ± SEM, and the significance of differences was determined by Student’s t test (^∗p < 0.05, ^∗∗p < 0.01). g Computed tomography scans of the chest from a patient before and after treatment with 40 mg ASK120067 in a phase I trial: images from a 74-year-old Chinese female diagnosed with stage IV EGFR-mutant (exon 19del) NSCLC in August 2016. The patient was previously treated with the first-line therapy icotinib for 16 months and achieved a partial response before eventually developing disease progression. See the main text for details

Fig. 4

Activation of Ack1 is sufficient to cause resistance to ASK120067, and drug combinations suppress proliferation and induce apoptosis of ASK120067-resistant cells. a Immunoblot analysis of total Ack1 and phosphorylated Ack1 (p-Ack1) levels in parental NCI-H1975 and 67R cells. b The expression of p-Ack1 in NCI-H1975 xenograft tumors and 67R xenograft tumors was compared by immunoblotting. c The p-Ack1 protein in AZDR and NCI-H1975 was detected by immunoblotting. d Correlation analysis of Ack1 expression and relapse-free survival (RFS) of 204 lung adenocarcinoma cancer patients (GSE22138) is presented as a Kaplan-Meier plot. e The antiproliferation effects of ASK120067 on NCI-H1975 cells ectopically expressing negative control vector or Ack1 were assessed using an SRB assay. f Knockdown of Ack1 expression in 67R effectively enhanced the antiproliferation potency of ASK120067. g ASK120067 in combination with AIM-100 caused a significantly higher growth-inhibition rate in ASK120067-resistant cells than that with ASK120067 treatment alone. h AIM-100 synergistically enhanced the apoptosis-inducing activity of ASK120067 in 67R cells. Cells were treated with ASK120067, AIM-100 alone or both drugs in combination for 48 h, and apoptosis was assessed using flow cytometry. i and j Combination ASK120067 with dasatinib i or bosutinib j partially restored the growth-inhibition sensitivity of ASK120067-resistant cells to ASK120067 treatment. k Combination ASK120067 with AIM-100, dasatinib or bosutinib synergistically inhibited the growth of AZDR cells. Data are plotted as the mean ± SD, and significance of differences was evaluated by Student’s t test (^∗p < 0.05, ^∗∗p < 0.01)

Fig. 5

Activation of antiapoptotic signaling through the Ack1/AKT pathway contributes to ASK120067 resistance. a The levels of AKT phosphorylation (p-AKT) in NCI-H1975 and 67R cells were determined by immunoblotting analysis. b The inhibitory activity of ASK120067 on p-AKT expression in NCI-H1975 cells and 67R cells was compared. c Knockdown of Ack1 expression using short hairpin RNA (shRNA) decreased the levels of phosphorylated AKT in 67R cells. d The mRNA and protein levels of proapoptotic protein BIM in NCI-H1975 and 67R cells were determined by real-time PCR (left panel) and Western blot analysis (right panel), respectively. e The effect of ASK120067 on BIM expression in NCI-H1975 and 67R cells was examined. f Knockdown of Ack1 expression in 67R cells increased the expression of BIM by decreasing the phosphorylation of AKT. g to i, the combination of ASK120067 with Ack1 inhibitors synergistically suppressed AKT activation g and induced the transcription h and protein expression of BIM i

Fig. 6

Combination therapy with ASK120067 and Ack1 inhibitors showed synergistic in vivo antitumor effects in the 67R xenograft model. a Growth of 67R xenograft tumors following daily treatment with 5 mg/kg ASK120067, 25 mg/kg dasatinib or a combination of ASK120067 and dasatinib for 21 days. b The expression levels of p-Ack1 and p-AKT in 67R xenograft tumors were assessed after 21 days of treatment. c Cell apoptosis in 67R xenograft tumors was tested by the TUNEL assay after 21 days of treatment. Significance of differences was determined by Student’s t test (^∗p < 0.05, ^∗∗p < 0.01). d Proposed mechanism for the efficacy of the combination strategy in ASK120067-resistant NCI-H1975 cells