Epidermal Growth Factor Receptor Kinase Domain Research Articles

113P The analysis of EGFR fusions characteristics in Chinese solid tumor patients

Oncogenic mutations in the epidermal growth factor receptor (EGFR) serve as important predictive biomarkers in non-small cell lung cancer (NSCLC). The constitutive activity and sensitivity to EGFR tyrosine kinase inhibitors (TKIs) in lung cancer with small in-frame deletions in exon 19, mutations in exon 21 in the EGFR tyrosine kinase domain had been conferred. Atypical EGFR aberrations, including EGFR kinase domain duplications (KDD) and EGFR fusions (EGFR-RAD51, EGFR-SEPT14, KIF5B-EGFR) had been identified.

Molecular analysis and systematic profiling of allosteric inhibitor response to clinically significant epidermal growth factor receptor missense mutations in non‐small cell lung cancer

AbstractHuman epidermal growth factor receptor (EGFR) is an attractive anti‐non‐small cell lung cancer (anti‐NSCLC) druggable target, but many traditional adenosine triphosphate (ATP)‐competitive EGFR inhibitors have been clinically observed to eventually establish acquired resistance due to kinase mutations. Allosteric inhibitors represent a new direction of EGFR‐targeted therapy, which have been successfully developed since 2016 to overcome the widely occurring kinase drug‐resistant mutations T790M/C797S [Jia Y, et al. Nature 2016; 534: 129–132]. In this study, a systematic inhibitor response‐to‐mutation profile of 11 reported EGFR allosteric inhibitors against 17 clinically significant EGFR missense mutations in NSCLC was created by integrating computational analyses and kinase assays, from which we were able to identify a number of representative mutation/inhibitor pairs with sensitization and deactivation. It is unraveled that most mutations have only a moderate or modest effect on inhibitor binding, while few mutations located within or nearby the kinase's allosteric site can cause strong sensitization or deactivation to allosteric inhibitors; thus they can influence the inhibitor binding substantially by forming or breaking noncovalent interactions between them. In particular, the gatekeeper mutation T790M and constitutively activated mutation L858R were observed to effectively improve the inhibitory potency of allosteric inhibitors, whereas the gain‐of‐function mutation T854A was revealed to considerably reduce inhibitory activity by breaking a geometrically perfect hydrogen bond. In addition, we also found that the allosteric inhibitor‐addressed conformational change in EGFR kinase domain has only a marginal effect on ATP binding, suggesting that allosteric inhibitors do not use the substrate‐regulatory strategy to suppress kinase activity; instead, they just shift the EGFR conformational equilibrium from active to inactive states by inducing a displacement in kinase's αC‐helix.

Chronicles of EGFR Tyrosine Kinase Inhibitors: Targeting EGFR C797S Containing Triple Mutations.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase widely expressed in many cancers such as non-small cell lung cancer (NSCLC), pancreatic cancer, breast cancer, and head and neck cancer. Mutations such as L858R in exon 21, exon 19 truncation (Del19), exon 20 insertions, and others are responsible for aberrant activation of EGFR in NSCLC. First-generation EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib have clinical benefits for EGFR-sensitive (L858R and Del19) NSCLC patients. However, after 10-12 months of treatment with these inhibitors, a secondary T790M mutation at the gatekeeper position in the kinase domain of EGFR was identified, which limited the clinical benefits. Second-generation EGFR irreversible inhibitors (afatinib and dacomitinib) were developed to overcome this T790M mutation. However, their lack of selectivity toward wild-type EGFR compromised their clinical benefits due to serious adverse events. Recently developed third-generation irreversible EGFR TKIs (osimertinib and lazertinib) are selective toward driving mutations and the T790M mutation, while sparing wild-type EGFR activity. The latest studies have concluded that their efficacy was also compromised by additional acquired mutations, including C797S, the key residue cysteine that forms covalent bonds with irreversible inhibitors. Because second- and third-generation EGFR TKIs are irreversible inhibitors, they are not effective against C797S containing EGFR triple mutations (Del19/T790M/C797S and L858R/T790M/C797S). Therefore, there is an urgent unmet medical need to develop next-generation EGFR TKIs that selectively inhibit EGFR triple mutations via a non-irreversible mechanism.

Quantitative analysis of cell-free DNA by droplet digital PCR reveals the presence of EGFR mutations in non-malignant lung pathologies.

Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) are effectively used in treatment of non-small cell lung cancer (NSCLC). Mutation profile of tyrosine kinase domain of EGFR determines the eligibility of the patients for tyrosine kinase inhibitor (TKI) therapy. Liquid biopsy, which relies on circulating tumor-derived nucleic acids, has emerged as an effective tool in lung cancer management with proven diagnostic, prognostic and predictive applications. We screened 100 subjects, suspected to have lung malignancy, for four hotspot mutations including three activating (G719S, Ex19del E746-A750 and L858R) and one acquired (T790M, de novo) in EGFR gene by droplet digital PCR (ddPCR). While 97 subjects were subsequently confirmed to have lung malignancy based on histo/cytopathological studies, three cases turned out to be non-malignant lung pathologies that were completely cured by antibiotic therapy. Intriguingly, ddPCR revealed the presence of EGFR mutations in these non-malignant subjects. Two cases showed the presence of G719S and T790M mutations respectively and another had compound mutations (T790M and L858R). The detection of EGFR mutations in non-malignant pulmonary conditions opens up a new area of research.

Structural Basis for the Functional Changes by EGFR Exon 20 Insertion Mutations.

Simple SummaryNon-small cell lung cancer (NSCLC) is the most common type of lung cancer that claims the lives of many worldwide. Activating mutations occurring on the epidermal growth factor receptor (EGFR) protein have been associated with the pathogenesis of NSCLC, among which exon 20 insertion mutations play a significant role. The objective of this study is to examine the dynamic structural changes occurring on the EGFR protein as a result of two common EGFR exon 20 insertion mutations, V769insASV and D770insNPG. The study further aims to uncover the mechanisms by which the insertion mutations increase kinase activity. Our results suggest that the insertion mutations stabilize structural elements key to maintaining the active EGFR conformation. Furthermore, the insertions disrupt an interaction essential in stabilizing the inactive conformation, which could drive the kinase from an inactive to an active EGFR state.Activating somatic mutations of the epidermal growth factor receptor (EGFR) are frequently implicated in non-small cell lung cancer (NSCLC). While L858R and exon 19 deletion mutations are most prevalent, exon 20 insertions are often observed in NSCLC. Here, we investigated the structural implications of two common EGFR exon 20 insertions in NSCLC, V769insASV and D770insNPG. The active and inactive conformations of wild-type, D770insNPG and V769insASV EGFRs were probed with molecular dynamics simulations to identify local and global alterations that the mutations exert on the EGFR kinase domain, highlighting mechanisms for increased enzymatic activity. In the active conformation, the mutations increase interactions that stabilize the αC helix that is essential for EGFR activity. Moreover, the key Lys745–Glu762 salt bridge was more conserved in the insertion mutations. The mutants also preserved the state of the structurally critical aspartate–phenylalanine–glycine (DFG)-motif and regulatory spine (R-spine), which were altered in wild-type EGFR. The insertions altered the structure near the ATP-binding pocket, e.g., the P-loop, which may be a factor for the clinically observed tyrosine kinase inhibitor (TKI) insensitivity by the insertion mutants. The inactive state simulations also showed that the insertions disrupt the Ala767–Arg776 interaction that is key for maintaining the “αC-out” inactive conformation, which could consequently fuel the transition from the inactive towards the active EGFR state.

Engineering soluble artificial epidermal growth factor receptor mimics capable of spontaneous in vitro dimerization.

Epidermal growth factor receptor (EGFR) is a clinically validated target for a multitude of human cancers. The receptor is activated upon ligand binding through a critical dimerization step. Dimerization can be replicated in vitro by locally concentrating the receptor kinase domains on the surface of lipid-based vesicles. In this study we investigated the use of coiled coils to induce spontaneous receptor kinase domain dimerization in vitro to form non-membrane-bound artificial receptor mimics in solution. Two engineered forms of EGFR kinase domain fused to coiled coil complementary peptides were designed to self-associate upon mixing. Two fusion protein species (P3-EGFR and P4-EGFR) independently showed the same activity and polymerization profile known to exist with EGFR kinase domains. Upon mixing the two species, coiled coil heterodimers were formed that induced EGFR association to form dimers of the kinase domains. This was accompanied by 11.5-fold increase in the phosphorylation rate indicative of kinase domain activation equivalent to the levels achieved using vesicle localization and mimicking in vivo ligand-induced activation. This study presents a soluble tyrosine kinase receptor mimic capable of spontaneous in vitro activation that can facilitate functional and drug discovery studies for this clinically important receptor class.

Sensitive Detection of Epidermal Growth Factor Receptor T790M using BNAClamp Real-Time PCR

Mutations on Epidermal Growth Factor Receptor (EGFR) cause a variety of cancers including breast and lung cancers. The single mutation T790M on the tyrosine kinase domain of EGFR signifies the response to the cancer drugs gefitinib, which leads to the development of resistance to such a drug. Detecting the mutation thus provides effective therapeutic options for patients who are in need of cancer drug treatments. We sought to develop a facile, rapid detection method for the T790M mutation using Bridged Nucleic Acids (BNA), which has been known to enhance the hybridization affinity of oligonucleotides. Oligonucleotides containing BNA bases, called BNA-clamp and designed to block PCR reaction against wild-type genes were used to discriminate the presence of mutant genes mixed with a large number of wild-type genes. Real-time PCR in conjugation with BNA-clamping allows us to observe different degrees of PCR amplification depending on the ratio of wild-type and mutant genes. In an effort to explore the possibility, several 13-mer oligonucleotide clamps were prepared with various numbers of BNA bases. Tm value analysis suggests that the clamps containing 9 BNA bases (BNA-clamp-9) would be most effective in distinguishing the mutant from wild-type genes, and sensitivity tests using BNA-clamp-9 revealed that the clamp had the ability to detect 0.1% or lower levels of the T790M mutation among wild-type genes. Furthermore, binding structures were analyzed via Molecular Dynamics (MD) simulations, revealing that BNA-clamp-9 distorts the originally constructed BDNA structure. Additionally through umbrella sampling, binding free energies with -60 kJ/mol for the wild-type and -40 kJ/mol for the mutant gene were obtained. This BNA-clamping and real time PCR technology may offer a promising avenue to detect clinically important mutations in the future

Geometrical Features of Epidermal Growth Factor Receptor-Related Dimers Reveal the Mechanisms of Drug Resistance in Lung Cancer Patients

Epidermal growth factor receptor (EGFR) plays an important role in lung cell proliferation. Dimerization of EGFR family members and other receptor tyrosine kinases (RTKs) act as a vital controller for lung cell life cycle signals. Mutations in the kinase domain of EGFR may disorder the signaling networks and lead to cancer. Drug resistance occurs in several generations of EGFR drugs due to genetic mutations, and there is a very less understanding about the mechanism of EGFR-mutated drug resistance. In this work, we investigate the mechanism of wild type EGFR protein and its drug-sensitive and drug-resistant mutations. We performed molecular dynamics (MD) simulation for the 10-ns EGFR-drug mutant complex and investigated the structures’ geometrical properties. With features extracted by alpha shape modeling, different geometrical properties, such as matching rates of atom solid angles at interaction sites and centroid distances between interfacial atoms, were calculated to characterize the binding intensity. Wilcoxon rank sum test was applied to reveal the differences between mutations based on extracted properties. We have developed a framework that explains the drug resistance mechanism based on geometrical properties and binding free energy. Results revealed that drug-sensitive mutants have tighter interactions with corresponding RTK in the complex for all protein-drug systems, while drug-resistant mutants are bound looser. The extracted geometrical properties of the drug mutant complex help understand the drug response mechanism at atomic level.

Toward structure-based drug design against the epidermal growth factor receptor (EGFR)

Most of the available crystal structures of epidermal growth factor receptor (EGFR) kinase domain, bound to drug inhibitors, originated from ligand-based drug design studies. Here, we used variations in 110 crystal structures to assemble eight distinct families highlighting the C-helix orientation in the N-lobe of the EGFR kinase domain. The families shared similar mutational profiles and similarity in the ligand R-groups (chemical composition, geometry, and charge) facing the C-helix, mutation sites, and DFG domain. For structure-based drug design, we recommend a systematic decision-making process for choice of template, guided by appropriate pairwise fitting and clustering before the molecular docking step. Alternatively, the binding site shape/volume can be used to filter and select the compound libraries.

Therapeutic resistance in breast cancer cells can result from deregulated EGFR signaling.

The epidermal growth factor receptor (EGFR) interacts with various downstream molecules including phospholipase C (PLC)/protein kinase C (PKC), Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/GSK-3, Jak/STAT and others. Often these pathways are deregulated in human malignancies such as breast cancer. Various therapeutic approaches to inhibit the activity of EGFR family members including small molecule inhibitors and monoclonal antibodies (MoAb) have been developed. A common problem with cancer treatments is the development of drug-resistance. We examined the effects of a conditionally-activated EGFR (v-Erb-B:ER) on the resistance of breast cancer cells to commonly used chemotherapeutic drugs such as doxorubicin, daunorubicin, paclitaxel, cisplatin and 5-flurouracil as well as ionizing radiation (IR). v-Erb-B is similar to the EGFR-variant EGFRvIII, which is expressed in various cancers including breast, brain, prostate. Both v-Erb-B and EGFRvIII encode the EGFR kinase domain but lack key components present in the extracellular domain of EGFR which normally regulate its activity and ligand-dependence. The v-Erb-B oncogene was ligated to the hormone binding domain of the estrogen receptor (ER) which results in regulation of the activity of the v-Erb-ER construct by addition of either estrogen (E2) or 4-hydroxytamoxifen (4HT) to the culture media. Introduction of the v-Erb-B:ER construct into the MCF-7 breast cancer cell line increased the resistance to the cells to various chemotherapeutic drugs, hormonal-based therapeutics and IR. These results point to the important effects that aberrant expression of EGFR kinase domain can have on therapeutic resistance.

Rational design of EGFR dimerization-disrupting peptides: A new strategy to combat drug resistance in targeted lung cancer therapy

Although a variety of tyrosine kinase inhibitors (TKIs) have been developed to target human epidermal growth factor receptor (EGFR) for lung cancer therapy, many patients treated with first-line small-molecule TKIs are clinically observed to eventually establish drug-resistant mutations T790 M and C797S around kinase active site, which play a primary role in development of acquired drug resistance to first-generation reversible and second-generation irreversible TKIs, respectively. Here, instead of developing small-molecule drugs to directly target the active site, we attempt to derive self-inhibitory peptides (SIPs) from the EGFR:EGFR asymmetric dimerization interface, where is separated from kinase active site and has a relatively low conservation as compared to the active site. It is found that the dimerization is a typical peptide-mediated protein interaction, where the first EGFR N-lobe adopts an N-terminal binding sequence (nBS) to interact with the dimerization interface of second EGFR C-lobe. A core binding sequence (nCBS, 676NQALLRILKE68) is identified in the nBS region as hotspot segment, which is then rationally optimized to generate a number of SIPs. Consequently, three designed SIPs are determined as promising candidates; they have high affinity to EGFR kinase domain, strong competitive potency with native nBS peptide for the dimerization interface, and effective cytotoxicity on human lung cancer cell lines. It is also demonstrated that these peptides are insensitive to drug-resistant EGFR T790 M/C797S mutation at molecular and cellular levels, and exhibit a good selectivity for EGFR over HER2 at molecular level.

Abstract IA10: Modeling sensitivity and resistance to systemic therapies in lung cancer

Abstract Targeted therapies have transformed the landscape for the treatment of metastatic lung cancer. Despite their success, targeted therapies are not curative and acquired resistance is a major impediment to cures for patients treated with these therapies. A paradigm for the success of targeted therapies in lung cancer comes from epidermal growth factor receptor (EGFR) mutant lung cancer where mutations in exons encoding the tyrosine kinase domain of EGFR confer sensitivity to tyrosine kinase inhibitors (TKIs), and several TKIs are currently approved for the first-line treatment of EGFR mutant lung cancer. Moreover, there is heterogeneity in the durability and depth of responses between patients and at different metastatic sites within individual patients. Therefore, there is a need to understand the biology of EGFR mutant tumors and identify factors that affect TKI sensitivity. We developed transgenic mouse models of EGFR mutant lung cancer and have used them to study tumor progression and TKI resistance. When we modeled acquired resistance to the TKI osimertinib as a first-line therapy in transgenic mouse models of EGFRL858R-induced lung adenocarcinoma, we found that it is mediated largely through secondary mutations in EGFR—either C797S or L718V/Q or Kras mutations. We also identified a therapeutic approach to prevent the emergence of secondary mutations in EGFR. To determine whether the presence of co-occurring genetic alterations contributes to a more complex spectrum of resistance mechanisms in vivo, we have developed new mouse models to specifically evaluate the consequences of co-occurring tumor suppressor gene alterations on the progression and TKI sensitivity of EGFR mutant tumors. Collectively, our findings highlight how genetically engineered mouse models of lung cancer, including those with complex genotypes, can be leveraged to study tumor progression and drug resistance in vivo. Citation Format: Katerina A. Politi. Modeling sensitivity and resistance to systemic therapies in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA10.

Stability Analysis at Key Positions of EGFR Related to Non-small Cell Lung Cancer

Background: Mutations in a protein called the Epidermal Growth Factor Receptor (EGFR) can cause Non-Small Cell Lung Cancer (NSCLC), which is the most common form of lung cancer. Many NSCLC cases arise from the L858R mutation, where Leucine (L) is replaced by arginine (R) at the 858th position in the EGFR, and that is also recognized as an exon 21 substitution. Moreover, half of the EKFR-mutated lung cancer patients develop acquired resistance to the first-generation EGFR-TKIs due to another mutation T790M. Objective: In this research work, a novel method is used to investigate the possible reason for the EGFR mutation to takes place in the specific 858th and 790th position, and also, we evaluated the hydrogen bonds to measure the overall stability of different structures. Methods: We performed the molecular dynamics simulation and used Amber tool to achieve our primary objectives and later we use CPPTRAJ to analyze other changes in the hydrogen bonds for different mutational structures of EGFR. Results: First, we investigated the hydrogen bonds in different positions in the EGFR kinase domain and estimated why the first stage mutation (L858R) and resistance mutation (L858R/T790M) take place in the 858th and 790th position respectively. We found the hydrogen bond counts in the 858th and 790th position is lesser than the neighborhood positions and that yields to achieve a least stability in that position. Conclusion: Our method represents an important contribution to molecular dynamics analysis for NSCLC studies. The results obtained from this study provide a useful insight into the NSCLC drug resistance.

Characteristics of EGFR uncommon mutations in Chinese cancer patients.

e13527 Background: Exon 19 deletions and exon 21 L858R substitutions are the most common mutations of epidermal growth factor receptor (EGFR) in cancers, and the remaining other mutations are called uncommon mutations. Recent studies have shown the clinical relevance of EGFR uncommon mutations with tyrosine kinase inhibitors (TKI) therapies and immunotherapies. Therefore, understanding the distribution and characteristics of EGFR uncommon mutations in cancers would provide evidence for future design of trials and drug development. Methods: Next-generation sequencing data were obtained from 3,026 Chinese tumor samples which have been identified with EGFR mutations. Single nucleotide variations (SNV), short and long insertions/deletions (indel), copy number variations and gene rearrangements were assessed. All tests were carried out in a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory in Shanghai, China. Results: EGFR mutations including 32% L858R substitutions, 28% exon 19 deletions, and 40% uncommon mutations were detected in this cohort. EGFR uncommon mutations were most frequently detected in lung cancers, followed by esophageal and gastric cancers. The uncommon mutations of EGFR including 54% SNVs, 30% amplifications, and 9% rare types of mutations such as rearrangement, long indels and complex mutations were detected. The SNVs in exon 18 to 21 which encode the tyrosine kinase domain of EGFR consisted of 16% EGFR mutations. Among them, the mostly frequently SNV was G719X in exon 18 and had 3% EGFR mutations. Mutations in other function domain of EGFR, including extracellular EGF binding domain (0.8%), transmembrane domain (0.03%) and intracellular autophosphorylation domain (0.7%) were also detected. Conclusions: Our data indicated that EGFR uncommon mutations were widely distributed in a variety of cancer types in Chinese patients, mostly in lung cancers. SNVs in the tyrosine kinase domain were the most frequent uncommon mutations. These data will provide clues for future clinical studies.

Correlated Motions and Dynamics in Different Domains of Epidermal Growth Factor Receptor With L858R and T790M Mutations.

Non-small cell lung cancer with an activating epidermal growth factor receptor (EGFR) mutation responds well to targeted drugs. In most cases, drug resistance appears after about a year. Several studies have been conducted on the kinase domain of EGFR to understand the drug resistance mechanism. Since EGFR is a multi-domain protein, mutation in the kinase domain may affect the other domains as well. In this study, we examine the complete structure of the multi-domain EGFR protein and its mutants. We performed molecular dynamics simulations for wildtype EGFR, EGFR with L858R mutation, and EGFR with L858R and T790M mutations. We applied normal mode analysis and complex network analysis to extract the correlated motions in the domains of EGFR. The normal modes are used to construct the dynamic cross-correlation map (DCCM). Simulation results show different patterns of correlated motions in each domain of EGFR mutants compared to the wildtype. In Domains 1 and 3 of the extracellular region, a small number of weak positively correlated motions are extracted. Domains 2 and 4 show large numbers of both positive and negative motions. However, the negatively correlated motions are stronger in mutant structures compared to the wildtype. In Domain 7, some residues showed a positive correlation around the main diagonal. We also identified different communities, nodes and crucial residues in the domains of the structures, which can be important for the function of EGFR. Moreover, hydrogen bond analysis is performed for the stability analysis. The mutant structures have fewer hydrogen bonds compared to the wildtype. Overall, these findings are useful for understanding the dynamics and communications in EGFR domains.

Structural Basis for EGFR Mutant Inhibition by Trisubstituted Imidazole Inhibitors.

Acquired drug resistance in epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer is a persistent challenge in cancer therapy. Previous studies of trisubstituted imidazole inhibitors led to the serendipitous discovery of inhibitors that target the drug resistant EGFR(L858R/T790M/C797S) mutant with nanomolar potencies in a reversible binding mechanism. To dissect the molecular basis for their activity, we determined the binding modes of several trisubstituted imidazole inhibitors in complex with the EGFR kinase domain with X-ray crystallography. These structures reveal that the imidazole core acts as an H-bond acceptor for the catalytic lysine (K745) in the "αC-helix out" inactive state. Selective N-methylation of the H-bond accepting nitrogen ablates inhibitor potency, confirming the role of the K745 H-bond in potent, noncovalent inhibition of the C797S variant. Insights from these studies offer new strategies for developing next generation inhibitors targeting EGFR in non-small-cell lung cancer.

Synthesis, biological evaluation and molecular modeling study of [1,2,4]-Triazolo[4,3-c]quinazolines: New class of EGFR-TK inhibitors

New series of triazolo[4,3-c]quinazolines were designed, synthesized and their structures were elucidated using different spectroscopic techniques. They were evaluated for their in vitro antitumor activity against HepG2, MCF-7, PC-3, HCT-116 and HeLa cancer cell lines using MTT assay. It was found that all compounds showed variable in vitro cytotoxicity. Distinct derivatives exhibited higher inhibitory activity against the tested cell lines with IC50 values ranging from 8.27 to 10.68 µM using DOX standard (IC50 = 4.17–8.87 µM). In vitro epidermal growth factor receptor (EGFR) inhibition assay was performed. Results revealed that compounds 8, 19 and 21 exhibited worthy EGFR inhibitory activity with IC50 values ranging from 0.69 to1.8 µM in comparison to the reference drug Gefitinib (IC50 = 1.74 µM). Further investigation showed that active candidates 8, 19 and 21 caused cell cycle arrest at the G2/M phase, and interestingly, induced cell death by apoptosis of MCF-7 cells cumulatively with 7.14, 17.52 and 24.88%, respectively, compared with DOX as a positive reference (29.09%). Molecular modeling studies, including docking, flexible alignment and surface mapping, were also done to study the interaction mode into the active site of EGFR kinase domain. There was a good agreement between modeling results and biological results. ADMET analysis and parameters of Lipinski’s rule of five were calculated. Pharmacokinetic parameters showed that compound 8 had more expected penetration through blood brain barrier than Gefitinib. The present work displayed new triazoloquinazoline based derivatives with potent cytotoxicity and promising EGFR inhibition activity.

IA31 Genetic Contributors to Tumor Progression and Drug Resistance in EGFR Mutant Lung Cancer

Targeted therapies have transformed the landscape for the diagnosis and treatment of metastatic lung cancer. These tumors are now routinely tested for the presence of mutations or rearrangements in specific oncogenic drivers that, if present, predict sensitivity to targeted therapies directed to the genomic alterations present. Genotype-directed therapies have improved outcomes in specific subsets of patients with metastatic lung cancer. Despite this success, targeted therapies are not curative and acquired resistance is a major impediment to cures for patients treated with these therapies. Moreover, there is heterogeneity in the durability and depth of responses between patients. A paradigm for the success of targeted therapies in lung cancer comes from Epidermal Growth Factor Receptor (EGFR) mutant lung cancer. Mutations in exons encoding the tyrosine kinase domain of EGFR confer sensitivity to tyrosine kinase inhibitors (TKIs), and several are currently approved for the first-line treatment of EGFR mutant lung cancer. Most recently, the third-generation TKI osimertinib was approved and is increasingly being used in the first line. However, we have very limited knowledge of the mechanisms of resistance to osimertinib given its recent adoption in the clinic. Without knowledge about resistance mechanisms, optimal post-osimertinib treatment strategies remain to be defined. We modeled acquired resistance to first-line osimertinib treatment in transgenic mouse models of EGFRL858R-induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR and identified therapeutic strategies to treat these tumors and prevent their emergence. Moreover, since EGFR mutant tumors in patients harbor additional genetic alterations beyond EGFR, many of them in tumor suppressor genes, we tested how the presence of co-occurring genetic alterations in tumor suppressor genes contributes to the progression and osimertinib sensitivity of the tumors in the mouse models of EGFR mutant lung cancer. Collectively, our findings highlight how genetically engineered mouse models of lung cancer, including those with complex genotypes, can be leveraged to study tumor progression and drug resistance in vivo.

Frequent EGFR expression/EGFR amplification and lack of activating mutation in testicular choriocarcinoma

Choriocarcinoma (CC) is the rarest but most aggressive histological component of adult testicular germ cell tumor (TGCT). Although we previously reported a putative role of epidermal growth factor receptor (EGFR) alterations in the progression of CC, little is known about the kinase-activating mutation status of EGFR, which predicts the response to EGFR-tyrosine kinase inhibitors. In this study, we clinicopathologically reviewed a total of 12 cases of mixed TGCTs with CC components. Immunohistochemistry, fluorescence in situ hybridization, and direct sequencing was performed to investigate EGFR expression, EGFR copy number alterations, and functional mutation of EGFR in these CC components, respectively. Four (33%) of 12 cases exhibited predominant CC components (>50%), and all these patients died due to disease within 62 months. Overexpression of EGFR, higher copy number of EGFR, and amplification of EGFR was observed in 12 (100%), 10 (83%), and 9 (75%) of 12 CC components, respectively. None of the cases showed any mutational events in exons 18 to 24, which encode the tyrosine kinase domain of EGFR. These results confirm an important role of EGFR in the tumor aggressiveness of testicular CCs and may suggest its possible innate resistance against conventional anti-EGFR therapies.

Start Selective and Rigidify: The Discovery Path toward a Next Generation of EGFR Tyrosine Kinase Inhibitors.

The epidermal growth factor receptor (EGFR), when carrying an activating mutation like del19 or L858R, acts as an oncogenic driver in a subset of lung tumors. While tumor responses to tyrosine kinase inhibitors (TKIs) are accompanied by marked tumor shrinkage, the response is usually not durable. Most patients relapse within two years of therapy often due to acquisition of an additional mutation in EGFR kinase domain that confers resistance to TKIs. Crucially, oncogenic EGFR harboring both resistance mutations, T790M and C797S, can no longer be inhibited by currently approved EGFR TKIs. Here, we describe the discovery of BI-4020, which is a noncovalent, wild-type EGFR sparing, macrocyclic TKI. BI-4020 potently inhibits the above-described EGFR variants and induces tumor regressions in a cross-resistant EGFRdel19 T790M C797S xenograft model. Key was the identification of a highly selective but moderately potent benzimidazole followed by complete rigidification of the molecule through macrocyclization.