EGFR exon 20 insertions (Ex20Ins) account for 4% to 10% of EGFR activating mutations in non-small cell lung cancer (NSCLC). EGFR Ex20Ins tumors are generally unresponsive to first- and ...second-generation EGFR inhibitors, and current standard of care for NSCLC patients with EGFR Ex20Ins is conventional cytotoxic chemotherapy. Therefore, the development of an EGFR TKI that can more effectively target NSCLC with EGFR Ex20Ins mutations represents a major advance for this patient subset. Osimertinib is a third-generation EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M; however, the activity of osimertinib in EGFR Ex20Ins NSCLC has yet to be fully assessed. Using CRISPR-Cas 9 engineered cell lines carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%), and a series of patient-derived xenografts, we have characterized osimertinib and AZ5104 (a circulating metabolite of osimertinib) activities against NSCLC harboring Ex20Ins. We report that osimertinib and AZ5104 inhibit signaling pathways and cellular growth in Ex20Ins mutant cell lines
and demonstrate sustained tumor growth inhibition of EGFR-mutant tumor xenograft harboring the most prevalent Ex20Ins
The antitumor activity of osimertinib and AZ5104 in NSCLC harboring EGFR Ex20Ins is further described herein using a series of patient-derived xenograft models. Together these data support clinical testing of osimertinib in patients with EGFR Ex20Ins NSCLC.
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(or
) aberrations, including both amplification and mutations, have been classified as oncogenic drivers that contribute to 2% to 6% of lung adenocarcinomas.
amplification is also an important ...mechanism for acquired resistance to EGFR tyrosine kinase inhibitors (TKI). However, due to limited preclinical studies and clinical trials, currently there is still no available standard of care for lung cancer patients with
aberrations. To fulfill the clinical need for targeting
in patients with non-small cell lung cancer (NSCLC), we performed a comprehensive preclinical study to evaluate the efficacy of a third-generation TKI, osimertinib (AZD9291).
Three genetically modified mouse models (GEMM) mimicking individual
alterations in NSCLC were generated, and osimertinib was tested for its efficacy against these
aberrations
Osimertinib treatment showed robust efficacy in HER2
overexpression and
models, but not in
exon 20 insertion tumors. Interestingly, we further identified that combined treatment with osimertinib and the BET inhibitor JQ1 significantly increased the response rate in
-mutant NSCLC, whereas JQ1 single treatment did not show efficacy.
Overall, our data indicated robust antitumor efficacy of osimertinib against multiple
aberrations in lung cancer, either as a single agent or in combination with JQ1. Our study provides a strong rationale for future clinical trials using osimertinib either alone or in combination with epigenetic drugs to target aberrant
in patients with NSCLC.
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Epidermal growth factor receptor (EGFR) inhibitors have been used clinically in the treatment of non-small-cell lung cancer (NSCLC) patients harboring sensitizing (or activating) mutations for a ...number of years. Despite encouraging clinical efficacy with these agents, in many patients resistance develops leading to disease progression. In most cases, this resistance is in the form of the T790M mutation. In addition, EGFR wild type receptor inhibition inherent with these agents can lead to dose limiting toxicities of rash and diarrhea. We describe herein the evolution of an early, mutant selective lead to the clinical candidate AZD9291, an irreversible inhibitor of both EGFR sensitizing (EGFRm+) and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition in preclinical models, the clinical candidate was administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC and early efficacy has been observed, accompanied by an encouraging safety profile.
Tyrosine kinase inhibitors (TKI) targeting mutant EGFR in non-small cell lung cancer (NSCLC) have been successful to control cancer growth, but acquired resistance inevitably occurs, including ...mutations directly on EGFR, for example, T790M and C797S. Strategies to prevent such acquired mutations by reducing mutant-EGFR expression have met limited success. Here, we propose a new model of mutant-EGFR trafficking and demonstrate that clathrin inhibition induces rapid degradation across a large panel of endogenous mutant-EGFR (Ex19del, L858R, and Ex20Ins). This panel included mutant-EGFR (T790M) resistant to the first- and second-generation EGFR inhibitors and to the third-generation TKI osimertinib and occurs through both mutational (C797S) and nonmutational EGFR mechanisms. Clathrin-mediated endocytosis inhibition of mutant EGFR induced a macropinocytosis-dependent lysosomal pathway associated with a loss of mutant-EGFR-dependent signaling (pAKT, pERK). Moreover, induction of this macropinocytic pathway led to robust apoptosis-dependent death across all mutant-EGFR cell lines tested, including those resistant to TKIs. We, therefore, propose a novel strategy to target mutant-EGFR refractory to approved existing TKI treatments in NSCLC and where new treatment strategies remain a key area of unmet need.
These findings extend our mechanistic understanding of NSCLC mutant EGFR trafficking biology, the role that trafficking may play in resistance of mutant EGFR to tyrosine kinase inhibitors, and provide new therapeutic and biological insights to tackle this fundamental issue and improve benefit to patients.
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Resistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct ...development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition.
Osimertinib, a mutant-specific third-generation EGFR tyrosine kinase inhibitor, is emerging as the preferred first-line therapy for
-mutant lung cancer, yet resistance inevitably develops in ...patients. We modeled acquired resistance to osimertinib in transgenic mouse models of
-induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in
-either C797S or L718V/Q. Analysis of circulating free DNA data from patients revealed that L718Q/V mutations almost always occur in the context of an L858R driver mutation. Therapeutic testing in mice revealed that both erlotinib and afatinib caused regression of osimertinib-resistant C797S-containing tumors, whereas only afatinib was effective on L718Q mutant tumors. Combination first-line osimertinib plus erlotinib treatment prevented the emergence of secondary mutations in
. These findings highlight how knowledge of the specific characteristics of resistance mutations is important for determining potential subsequent treatment approaches and suggest strategies to overcome or prevent osimertinib resistance
. SIGNIFICANCE: This study provides insight into the biological and molecular properties of osimertinib resistance
mutations and evaluates therapeutic strategies to overcome resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/2017/F1.large.jpg.
Osimertinib is an oral, third-generation, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI-sensitizing and
T790M-resistance ...mutations with lower activity against wild-type EGFR and has demonstrated efficacy in non-small cell lung cancer (NSCLC) CNS metastases. The sensitizing mutations, the in-frame deletions in exon 19 and the L858R point mutation in exon 21, represent between 80% and 90% of all EGFR mutations. The remaining 10% to 20% are referred to as uncommon activating mutations and are a diverse group of mutations in exons 18 to 21 within the kinase domain of the EGFR gene. Excluding those found as insertion mutations in exon 20, the uncommon mutations involving codons G719, S768, and L861 are the most prevalent.Although the efficacy of EGFR-TKIs for the common EGFR mutations is well established, much less is known about rare EGFR mutations, such as exon 20 insertions, G719X, L861Q, S768I, as most of the data consist of single case reports or small case series.Using available patient-derived xenografts (PDX) and cell lines derived from two of these PDXs that harbor the G719X mutation, we have evaluated
and
the preclinical activity of osimertinib. We report osimertinib inhibits signaling pathways and cellular growth in G719X-mutant cell lines
and demonstrate sustained tumor growth inhibition of PDX harboring the G719X mutation alone or in combination with L861Q and S768I.Together, these data support clinical testing of osimertinib in patients with uncommon EGFR NSCLC.
The epidermal growth factor receptor (EGFR) harboring activating mutations is a clinically validated target in non-small-cell lung cancer, and a number of inhibitors of the EGFR tyrosine kinase ...domain, including osimertinib, have been approved for clinical use. Resistance to these therapies has emerged due to a variety of molecular events including the C797S mutation which renders third-generation C797-targeting covalent EGFR inhibitors considerably less potent against the target due to the loss of the key covalent-bond-forming residue. We describe the medicinal chemistry optimization of a biochemically potent but modestly cell-active, reversible EGFR inhibitor starting point with sub-optimal physicochemical properties. These studies culminated in the identification of compound 12 that showed improved cell potency, oral exposure, and in vivo activity in clinically relevant EGFR-mutant-driven disease models, including an Exon19 deletion/T790M/C797S triple-mutant mouse xenograft model.
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.
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.
A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with ...gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.
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