Osimertinib + Savolitinib to Overcome Acquired MET-Mediated Resistance in Epidermal Growth Factor Receptor-Mutated, MET-Amplified Non-Small Cell Lung Cancer: TATTON.

MET-inhibitor and EGFR tyrosine kinase inhibitor (EGFR-TKI) combination therapy could overcome acquired MET-mediated osimertinib resistance. We present the final phase Ib TATTON (NCT02143466) analysis (Part B, n = 138/Part D, n = 42) assessing oral savolitinib 600 mg/300 mg once daily (q.d.) + osimertinib 80 mg q.d. in patients with MET-amplified, EGFR-mutated (EGFRm) advanced non-small cell lung cancer (NSCLC) and progression on prior EGFR-TKI. An acceptable safety profile was observed. In Parts B and D, respectively, objective response rates were 33% to 67% and 62%, and median progression-free survival (PFS) was 5.5 to 11.1 months and 9.0 months. Increased antitumor activity may occur with MET copy number ≥10. EGFRm circulating tumor DNA clearance on treatment predicted longer PFS in patients with detectable baseline ctDNA, while acquired resistance mechanisms to osimertinib + savolitinib were mediated by MET, EGFR, or KRAS alterations. SIGNIFICANCE: The savolitinib + osimertinib combination represents a promising therapy in patients with MET-amplified/overexpressed, EGFRm advanced NSCLC with disease progression on a prior EGFR-TKI. Acquired resistance mechanisms to this combination include those via MET, EGFR, and KRAS. On-treatment ctDNA dynamics can predict clinical outcomes and may provide an opportunity to inform earlier decision-making. This article is highlighted in the In This Issue feature, p. 1.

ERK5 is required for VEGF-mediated survival and tubular morphogenesis of primary human microvascular endothelial cells.

Extracellular signal-regulated kinase 5 (ERK5) is activated in response to environmental stress and growth factors. Gene ablation of Erk5 in mice is embryonically lethal as a result of disruption of cardiovascular development and vascular integrity. We investigated vascular endothelial growth factor (VEGF)-mediated ERK5 activation in primary human dermal microvascular endothelial cells (HDMECs) undergoing proliferation on a gelatin matrix, and tubular morphogenesis within a collagen gel matrix. VEGF induced sustained ERK5 activation on both matrices. However, manipulation of ERK5 activity by siRNA-mediated gene silencing disrupted tubular morphogenesis without impacting proliferation. Overexpression of constitutively active MEK5 and ERK5 stimulated tubular morphogenesis in the absence of VEGF. Analysis of intracellular signalling revealed that ERK5 regulated AKT phosphorylation. On a collagen gel, ERK5 regulated VEGF-mediated phosphorylation of the pro-apoptotic protein BAD and increased expression of the anti-apoptotic protein BCL2, resulting in decreased caspase-3 activity and apoptosis suppression. Our findings suggest that ERK5 is required for AKT phosphorylation and cell survival and is crucial for endothelial cell differentiation in response to VEGF.

Structure-activity relationship of a series of non peptidic RGD integrin antagonists targeting α5ß1: part 1.

Potent antagonists of the integrin α(5)ß(1), which are RGD mimetics built from tyrosine are described. This letter describes the optimization of in vitro potency obtained by variation of two parts of the molecule, the basic group and the linker between the basic group and the phenyl central core.

Structure-activity relationship of a series of non peptidic RGD integrin antagonists targeting α5ß1: part 2.

Potent antagonists of the integrin α(5)ß(1), which are RGD mimetics built from tyrosine are described. This paper describes the optimization of in vitro potency obtained by variation of two parts of the molecule, the central aromatic core and the amide moiety.

Potent and Selective Inhibitors of the Epidermal Growth Factor Receptor to Overcome C797S-Mediated Resistance.

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.

Preclinical Comparison of the Blood-brain barrier Permeability of Osimertinib with Other EGFR TKIs.

PURPOSE: Osimertinib is a potent and selective EGFR tyrosine kinase inhibitor (EGFR-TKI) of both sensitizing and T790M resistance mutations. To treat metastatic brain disease, blood-brain barrier (BBB) permeability is considered desirable for increasing clinical efficacy. EXPERIMENTAL DESIGN: We examined the level of brain penetration for 16 irreversible and reversible EGFR-TKIs using multiple in vitro and in vivo BBB preclinical models. RESULTS: In vitro osimertinib was the weakest substrate for human BBB efflux transporters (efflux ratio 3.2). In vivo rat free brain to free plasma ratios (Kpuu) show osimertinib has the most BBB penetrance (0.21), compared with the other TKIs (Kpuu ≤ 0.12). PET imaging in Cynomolgus macaques demonstrated osimertinib was the only TKI among those tested to achieve significant brain penetrance (C max %ID 1.5, brain/blood Kp 2.6). Desorption electrospray ionization mass spectroscopy images of brains from mouse PC9 macrometastases models showed osimertinib readily distributes across both healthy brain and tumor tissue. Comparison of osimertinib with the poorly BBB penetrant afatinib in a mouse PC9 model of subclinical brain metastases showed only osimertinib has a significant effect on rate of brain tumor growth. CONCLUSIONS: These preclinical studies indicate that osimertinib can achieve significant exposure in the brain compared with the other EGFR-TKIs tested and supports the ongoing clinical evaluation of osimertinib for the treatment of EGFR-mutant brain metastasis. This work also demonstrates the link between low in vitro transporter efflux ratios and increased brain penetrance in vivo supporting the use of in vitro transporter assays as an early screen in drug discovery.

Osimertinib, an Irreversible Next-Generation EGFR Tyrosine Kinase Inhibitor, Exerts Antitumor Activity in Various Preclinical NSCLC Models Harboring the Uncommon EGFR Mutations G719X or L861Q or S768I.

Osimertinib is an oral, third-generation, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI-sensitizing and EGFR 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 in vitro and in vivo the preclinical activity of osimertinib. We report osimertinib inhibits signaling pathways and cellular growth in G719X-mutant cell lines in vitro 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.

Drug Sensitivity and Allele Specificity of First-Line Osimertinib Resistance EGFR Mutations.

Osimertinib, a mutant-specific third-generation EGFR tyrosine kinase inhibitor, is emerging as the preferred first-line therapy for EGFR-mutant lung cancer, yet resistance inevitably develops in patients. We modeled acquired resistance to osimertinib in transgenic mouse models of EGFRL858R -induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR-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 EGFR. 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 in vivo. SIGNIFICANCE: This study provides insight into the biological and molecular properties of osimertinib resistance EGFR mutations and evaluates therapeutic strategies to overcome resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/2017/F1.large.jpg.

ERK5 and the regulation of endothelial cell function.

ERK5 (extracellular-signal-regulated kinase 5), also termed BMK1 [big MAPK1 (mitogen-activated protein kinase 1)], is the most recently discovered member of the MAPK family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling, regulating hypoxia, tumour angiogenesis and cell migration. This review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function.

Reactivation of Mutant-EGFR Degradation through Clathrin Inhibition Overcomes Resistance to EGFR Tyrosine Kinase Inhibitors.

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.Significance: 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. Cancer Res; 78(12); 3267-79. ©2018 AACR.

Targeting HER2 Aberrations in Non-Small Cell Lung Cancer with Osimertinib.

Purpose:HER2 (or ERBB2) aberrations, including both amplification and mutations, have been classified as oncogenic drivers that contribute to 2% to 6% of lung adenocarcinomas. HER2 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 HER2 aberrations. To fulfill the clinical need for targeting HER2 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).Experimental Design: Three genetically modified mouse models (GEMM) mimicking individual HER2 alterations in NSCLC were generated, and osimertinib was tested for its efficacy against these HER2 aberrations in vivoResults: Osimertinib treatment showed robust efficacy in HER2wt overexpression and EGFR del19/HER2 models, but not in HER2 exon 20 insertion tumors. Interestingly, we further identified that combined treatment with osimertinib and the BET inhibitor JQ1 significantly increased the response rate in HER2-mutant NSCLC, whereas JQ1 single treatment did not show efficacy.Conclusions: Overall, our data indicated robust antitumor efficacy of osimertinib against multiple HER2 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 HER2 in patients with NSCLC. Clin Cancer Res; 24(11); 2594-604. ©2018 AACRSee related commentary by Cappuzzo and Landi, p. 2470.

Antitumor Activity of Osimertinib, an Irreversible Mutant-Selective EGFR Tyrosine Kinase Inhibitor, in NSCLC Harboring EGFR Exon 20 Insertions.

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 in vitro and demonstrate sustained tumor growth inhibition of EGFR-mutant tumor xenograft harboring the most prevalent Ex20Ins in vivo 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. Mol Cancer Ther; 17(5); 885-96. ©2018 AACR.

Current Status and Future Perspectives on Neoadjuvant Therapy in Lung Cancer.

This Review Article provides a multi-stakeholder view on the current status of neoadjuvant therapy in lung cancer. Given the success of oncogene-targeted therapy and immunotherapy for patients with advanced lung cancer, there is a renewed interest in studying these agents in earlier disease settings with the opportunity to have an even greater impact on patient outcomes. There are unique opportunities and challenges with the neoadjuvant approach to drug development. To achieve more rapid knowledge turns, study designs, endpoints, and definitions of pathologic response should be standardized and harmonized. Continued dialogue with all stakeholders will be critical to design and test novel induction strategies, which could expedite drug development for patients with early lung cancer who are at high risk for metastatic recurrence.

The structure-guided discovery of osimertinib: the first U.S. FDA approved mutant selective inhibitor of EGFR T790M.

The winners of the Malcolm Campbell Memorial Prize for 2017 discuss the structure-guided discovery of Osimertinib and the difficulties associated with discovering a new drug.

Utilization of Structure-Based Design to Identify Novel, Irreversible Inhibitors of EGFR Harboring the T790M Mutation.

A novel series of covalent inhibitors of EGFR (epidermal growth factor receptor) kinase was discovered through a combination of subset screening and structure-based design. These compounds preferentially inhibit mutant forms of EGFR (activating mutant and T790M mutant) over wild-type EGFR in cellular assays measuring EGFR autophosphorylation and proliferation, suggesting an improved therapeutic index in non-small cell lung cancer patients would be achievable relative to established EGFR inhibitors. We describe our design approaches, resulting in the identification of the lead compound 5, and our efforts to develop an understanding of the structure-activity relationships within this series. In addition, strategies to overcome challenges around metabolic stability and aqueous solubility are discussed. Despite limitations in its physical properties, 5 is orally bioavailable in mice and demonstrates pronounced antitumor activity in in vivo models of mutant EGFR-driven cancers.

Optimizing the sequence of anti-EGFR-targeted therapy in EGFR-mutant lung cancer.

Metastatic EGFR-mutant lung cancers are sensitive to the first- and second-generation EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib, and afatinib, but resistance develops. Acquired resistance to gefitinib or erlotinib occurs most commonly (>50%) via the emergence of a second-site EGFR mutation, T790M. Two strategies to overcome T790M-mediated resistance are dual inhibition of EGFR with afatinib plus the anti-EGFR antibody cetuximab (A+C), or mutant-specific EGFR inhibition with AZD9291. A+C and AZD9291 are now also being tested as first-line therapies, but whether these therapies will extend progression-free survival or induce more aggressive forms of resistance in this setting remains unknown. We modeled resistance to multiple generations of anti-EGFR therapies preclinically to understand the effects of sequential treatment with anti-EGFR agents on drug resistance and determine the optimal order of treatment. Using a panel of erlotinib/afatinib-resistant cells, including a novel patient-derived cell line (VP-2), we found that AZD9291 was more potent than A+C at inhibiting cell growth and EGFR signaling in this setting. Four of four xenograft-derived A+C-resistant cell lines displayed in vitro and in vivo sensitivity to AZD9291, but four of four AZD9291-resistant cell lines demonstrated cross-resistance to A+C. Addition of cetuximab to AZD9291 did not confer additive benefit in any preclinical disease setting. This work, emphasizing a mechanistic understanding of the effects of therapies on tumor evolution, provides a framework for future clinical trials testing different treatment sequences. This paradigm is applicable to other tumor types in which multiple generations of inhibitors are now available.

Acquired Resistance to the Mutant-Selective EGFR Inhibitor AZD9291 Is Associated with Increased Dependence on RAS Signaling in Preclinical Models.

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.

Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor.

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.

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

UNLABELLED: 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.

Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR).

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.