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. 2014 Sep;4(9):1046-61.
doi: 10.1158/2159-8290.CD-14-0337. Epub 2014 Jun 3.

AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer

Darren A E Cross  1 , Susan E Ashton  2 , Serban Ghiorghiu  2 , Cath Eberlein  2 , Caroline A Nebhan  3 , Paula J Spitzler  3 , Jonathon P Orme  4 , M Raymond V Finlay  2 , Richard A Ward  2 , Martine J Mellor  2 , Gareth Hughes  2 , Amar Rahi  2 , Vivien N Jacobs  2 , Monica Red Brewer  3 , Eiki Ichihara  3 , Jing Sun  3 , Hailing Jin  3 , Peter Ballard  2 , Katherine Al-Kadhimi  2 , Rachel Rowlinson  2 , Teresa Klinowska  2 , Graham H P Richmond  2 , Mireille Cantarini  2 , Dong-Wan Kim  5 , Malcolm R Ranson  6 , William Pao  7
Affiliations

AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer

Darren A E Cross et al. Cancer Discov. 2014 Sep.

Abstract

First-generation EGFR tyrosine kinase inhibitors (EGFR TKI) provide significant clinical benefit in patients with advanced EGFR-mutant (EGFRm(+)) non-small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent, and selective third-generation irreversible inhibitor of both EGFRm(+) sensitizing and T790M resistance mutants that spares wild-type EGFR. This mono-anilino-pyrimidine compound is structurally distinct from other third-generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Preclinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm(+) and EGFRm(+)/T790M(+) mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR-mutant tumor xenograft and transgenic models. The treatment of 2 patients with advanced EGFRm(+) T790M(+) NSCLC is described as proof of principle.

Significance: We report the development of a novel structurally distinct third-generation EGFR TKI, AZD9291, that irreversibly and selectively targets both sensitizing and resistant T790M(+) mutant EGFR while harboring less activity toward wild-type EGFR. AZD9291 is showing promising responses in a phase I trial even at the first-dose level, with first published clinical proof-of-principle validation being presented.

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Figures

Figure 1
Figure 1
AZD9291 binding mode and structure. A, Structural model showing the covalent mode of binding of AZD9291 to EGFR T790M via Cys-797. Shows pyrimidine core forming two hydrogen bonds to the hinge region (Met-793), orientation of the indole group adjacent to the gatekeeper residue, the amine moiety positioned in the solvent channel and the covalent bond formed to Cys-797 via the acrylamide group of AZD9291. B, Chemical structure of AZD9291.
Figure 2
Figure 2
Effect of AZD9291 on EGFR phosphorylation in vitro. A, In comparison to early generation TKIs, AZD9291 inhibits EGFR phosphorylation across cell lines harboring sensitising (PC-9, H3255, H1650) or T790M resistance (H1975, PC-9VanR) mutations, whilst having less activity against wild-type EGFR phosphorylation (LOVO, A431, H2073). Apparent geomean IC50 (nM) values quantified in cell extracts after 2 h compound treatment using a phospho-EGFR ELISA from at least two separate experiments (expressed with 95% confidence intervals where n>3, or individual IC50 values where n=2). B, AZD9291 inhibits EGFR phosphorylation and downstream signaling pathways across representative mutant EGFR lines (PC-9, H1975, H1650, H3255), whilst having less activity against EGFR phosphorylation in the LOVO wild-type EGFR cell line compared to early generation TKIs, after 6 h treatment. The data is representative of at least two separate experiments.
Figure 3
Figure 3
Additional characteristics of AZD9291 in vitro. A, AZD9291 demonstrates greater inhibition of viability against mutant EGFR cell lines compared to wild-type, as assessed using a Sytox Green live/dead assay measured after 3 days treatment. The data represents the geomean IC50 nM value from at least two separate experiments (expressed with 95% confidence intervals where n>3). B, Sensitivity of isogenic pairs of EGFR mutant drug-sensitive and –resistant lung cancer cell lines (PC-9, ex19del; PC-9/BRc1, ex19del/T790M; HCC827, ex19del; HCC827/ER1, ex19del/T790M; HCC827/ER2, ex19del/METamplification; H1650, ex19del/PTEN loss; HCC4006, ex19del; HCC4006/ER, EMT (epithelial mesenchymal transition); H3255, L858R; H3255/XLR, L858R/T790M; H1975, L858R/T790M; 11-18, L858R; 11-18/ER, L858R/NRAS) to AZD9291, erlotinib, and afatinib. IC50s (μM) were based on data obtained from growth inhibition assays. C, T790M mutation was detected in multiple independent populations of PC-9 cells with acquired resistance to gefitinib or afatinib, but not in populations resistant to AZD9291.
Figure 4
Figure 4
In vivo anti-tumor efficacy of AZD9291 in subcutaneous xenograft models of EGFR-TKI sensitising and T790M resistant lung cancer. A, PC-9 (ex19del) xenograft following 14 days of daily treatment (n=6 or 8 animals depending on treatment group). B, H1975 (L858R/T790M) xenograft following 14 days of daily treatment (n=6 or 8 animals depending on treatment group). C, PC-9 following chronic daily oral dosing of 5mg/kg AZD9291 (n=8) or 6.25 mg/kg gefitinib (n=11). D, H1975 following chronic daily oral dosing of 1, 5 or 25 mg/kg AZD9291 (n=10 or 12 animals depending on dose group). Data are plotted as mean standard error.
Figure 5
Figure 5
AZD9291 induces significant and sustained tumor regression in transgenic models of EGFR-TKI sensitising (C/L858R) and T790M resistant (C/L+T) lung cancer. A, Percent change in radiographic tumor volume from baseline by treatment for individual lung tumor-bearing C/L858R (top) and C/L+T (middle, bottom) animals with vehicle, afatinib (7.5 mg/kg/day), or AZD9291 (5 mg/kg/day). B and C, Representative MRI images and H&E staining (original magnification, ×40) of lungs from tumor-bearing animals (B, C/L858R and C, C/L+T) pre and post treatment with vehicle, afatinib, or AZD9291 for 1 week. H – heart; L – liver; arrow denotes tumor.
Figure 6
Figure 6
AZD9291 inhibits EGFR phosphorylation and downstream signallng in murine models of EGFR T790M resistant lung cancer. A, Subcutaneous H1975 (L858R/T790M) xenografts, treated with a single 5 mg/kg dose of AZD9291 for the indicated times, were examined for phospho-EGFR, -ERK, -S6, and -PRAS40 status by immunohistochemistry. Representative images were taken from scans at 20× magnification and then size adjusted to fill the screen. B, Lungs from representative transgenic mice treated with control, AZD9291, or afatinib were harvested 6 hours after dose administration. Formalin-fixed paraffin-embedded sections were stained with the indicated antibodies. Representative images were taken from Aperio scans at 100× magnification. C. Lungs were harvested from either untreated tumor-bearing transgenic mice (as confirmed by MRI) (control) or from tumor-bearing mice 8 hours after a single treatment with AZD9291 5 mg/kg. Corresponding lysates from individual animals were immunoblotted with the indicated antibodies. Anti-SP-C (surfactant protein C) antibody was used as a surrogate marker for tumor burden, as tumors express the protein.
Figure 7
Figure 7
Proof of concept clinical studies validating AZD9291 as a mutant-selective EGFR kinase T790M inhibitor. A, Preliminary pharmacokinetic profile showing mean (+ SD) total plasma levels of AZD9291, AZ5104 and AZ7550 versus time from cohort of 6 advanced NSCLC patients. After a single dose of AZD9291 mesylate salt, followed by a 7 day washout and then 8 days of once daily 20 mg oral dosing in AURA Phase 1 study (NCT01802632). B and C, Serial computed tomography scans of the chest from patients before and after treatment with AZD9291 in a phase I trial. B, Images from a 57-year old Korean female patient diagnosed with Stage IV non-small cell lung cancer in May 2011. See main text for details. C, Images from a 57-year old British female never smoker diagnosed with Stage IV lung adenocarcinoma in December 2010. The patient was previously treated with first-line gefitinib for 14 months, achieving a partial response before eventually developing progressive disease. See main text for details.
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