Multiple Epidermal Growth Factor Receptor Research Articles

Optimization of Potent, Efficacious, Selective and Blood-Brain Barrier Penetrating Inhibitors Targeting EGFR Exon20 Insertion Mutations.

Herein, we report the optimization of a series of epidermal growth factor receptor (EGFR) Exon20 insertion (Ex20Ins) inhibitors using structure-based drug design (SBDD), leading to the discovery of compound 28, a potent and wild type selective molecule, which demonstrates efficacy in multiple EGFR Ex20Ins xenograft models and blood-brain barrier penetration in preclinical species. Building on our earlier discovery of an in vivo probe, SBDD was used to design a novel bicyclic core with a lower molecular weight to facilitate blood-brain barrier penetration. Further optimization including strategic linker replacement and diversification of the ring system interacting with the c-helix enabled photolytic and metabolic stability improvements. Together with refinement of molecular properties important for achieving high brain exposure, including molecular weight, H-bonding, and polarity, 28 was identified.

Uncovering the Molecular Basis for the Better Gefitinib Sensitivity of EGFR with Complex Mutations over Single Rare Mutation: Insights from Molecular Simulations.

Epidermal growth factor receptor (EGFR) is an intensively focused target for anti-tumor compounds used in non-small cell lung cancer (NSCLC) therapy. Compared to the classical activating mutations, there are still many uncommon EGFR mutations associated with poorer responses to EGFR inhibitors. A detailed understanding of the molecular basis for multiple EGFR mutants exhibiting diverse responses to inhibitors is of critical importance for related drug development. Herein, we explored the molecular determinants contributing to the distinct responses of EGFR with a single rare mutation (G719S) or combined mutations (G719S/L858R and G719S/l861Q) to Gefitinib (IRE). Our results indicated that interactions, formed within the tetrad of residues S768 (in the αC-helix), D770 (in the αC-β4 loop), Y827 (in the αE-helix), and R831 (in the catalytic loop), play an important role in the stability of αC-helix and the maintenance of K745–E762 salt bridge in the absence of IRE, which are weakened in the EGFRG719S system and enhanced in the EGFRG719S/L858R system upon IRE binding. Besides, the introduced hydrogen bonds by the co-occurring mutation partner also contribute to the stability of αC-helix. The work done for inhibitor dissociation suggests that IRE exhibits a stronger binding affinity to EGFRG719S/L858R mutant. Together, these findings provide a deeper understanding of minor mutations, which is essential for drug development targeting EGFR with less common mutations.

Abstract 5940: Inhibition of β-catenin/CBP signaling alters EGFR fucosylation status in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy in the world with oral squamous cell carcinomas (OSCC) accounting for the majority of HNSCC cases. A major driver of OSCC is the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) with 12 N-glycosylation sites. Fucosylated N-linked glycans on EGFR are associated with survival e.g., they suppress receptor dimerization and signaling. High levels of fucosylated glycan epitopes have been observed in OSCC, where invasive regions lose expression of linkage-specific fucosylated epitopes, suggesting that fucosylated glycans are involved in the suppression of cell growth and invasion. Previously, it was shown that inhibition of the interaction between nuclear β-catenin and CREB-binding protein (CBP) in human OSCC cells and in mouse tumor xenografts with a small molecule inhibitor ICG-001, interfered with OSCC proliferation and aggressive features in cellular, zebrafish, and murine models. E7386, a novel β-catenin/CBP modulator displays activity profile that closely overlaps with that of ICG-001 and exhibits ~50 - 100-fold lower EC50 values. Treatment with ICG-001 and E7386, increased expression of two glycosyltransferases, FUT2 and FUT3 coincident with decreased EGFR abundance that was accompanied by higher fucosylation of EGFR and upregulated expression of E-cadherin and junctional β-catenin. Further, genomic analyses showed a positive correlation between the ICG-001 and E7386 treatments and EGFR inhibition, suggesting that higher expression of antennary fucosyltransferase genes suppresses EGFR signaling. We now show using nLC-MS/MS analyses that EGFR from metastatic HSC-3 cells had low levels of fucosylated N-glycans, while EGFR from indolent CAL27 cells displayed higher levels of fucosylation at multiple EGFR N-linked glycosylation sites, and that these changes were statistically significant. In-depth characterization of multiply-fucosylated N-glycans via tandem mass spectrometry of EGFR glycopeptides revealed new insights into the identity of fucosylated glycan epitopes. Collectively, these results suggest that the β-catenin/CBP axis promotes EGFR signaling through downregulation of fucosyltransferase expression and activity. We conclude that inhibition of β-catenin/CBP signaling with a novel small molecule E7386 may serve as a therapeutic approach to downregulate EGFR pro-tumorigenic activity in HNSCC patients. Citation Format: Kevin B. Chandler, Khalid Alamoud, Bac-Cuc Nguyen, Vanessa L. Stahl, Takashi Owa, Kenichi Nomoto, Stefano Monti, Maria A. Kukuruzinska, Catherine E. Costello. Inhibition of β-catenin/CBP signaling alters EGFR fucosylation status in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5940.

Abstract 5659: AFM24, a bispecific EGFR/CD16A innate cell engager with the potential to overcome resistance to current targeted treatments for EGFR-positive malignancies

Abstract The epidermal growth factor receptor (EGFR) is a prime target for cancer therapy across many solid tumor types, including CRC, NSCLC, breast, esophageal cancer, and SCCHN. Binding of EGFR by its ligand EGF induces cell proliferation, a process which is uncontrolled or constitutively active in many cancers. The development of anti-EGFR therapies such as monoclonal antibodies and tyrosine kinase inhibitors enabled clinicians to treat specific patient populations. Nevertheless, prognoses for patients remain unfavorable due to occurrence of intrinsic or acquired resistance via mutations of EGFR and/or downstream signaling factors such as KRAS and BRAF which are drivers of tumorigenesis. Inhibitors that target specific KRAS mutations, such as the G12C mutation (i.e. 14% of the most frequently observed in NSCLC and up to 5% in CRC), have shown early promising efficacy. While such drugs hold promise for specific mutations, there continues to be a need for new treatments with more differentiated and novel modes of action that utilize the power of the innate immune system, such as antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). Affimed has engineered the tetravalent bispecific EGFR- and CD16A-binding antibody AFM24 to engage and redirect innate immune cells such as NK cells and macrophages to EGFR+ tumor cells. In contrast to currently available EGFR-targeted therapies (e.g. cetuximab), this innate cell engager has been designed to exhibit a significantly reduced inhibition of EGFR signaling aiming for an improved safety profile, i.e. avoiding skin and gastro-intestinal toxicities. In vitro, AFM24 demonstrated high affinity binding to immune cells regardless of CD16A variants and potent killing of multiple EGFR+ tumor cell lines, carrying relevant KRAS or BRAF mutations via a different mode of action, namely ADCC and ADCP, rather than EGFR signaling inhibition. It was shown that AFM24's activity is independent of both, the EGFR cell surface expression level and the cellular origin of the respective cell lines. Cynomolgus toxicity studies revealed a very good safety profile, and no toxicity was observed after repeated administration (q7x28d, in total 5 infusions) of AFM24 up to 75 mg/kg. In contrast, EGFR-targeting mAbs (e.g. cetuximab) exhibited severe toxicities particularly in skin in toxicology studies when investigated with a similar dosing schedule. Due to its potent activation of innate immunity and its beneficial safety/tolerability profile, AFM24 has the potential to become a novel treatment option for patients suffering from EGFR-expressing cancers and to overcome limitations of available EGFR-targeted therapies such as resistance and/or associated toxicities. AFM24 has cleared the IND and clinical investigations are planned in patients with cancers known to express EGFR. Citation Format: Uwe Reusch, Michael Damrat, Susanne Wingert, Stefan H.J. Knackmuss, Thomas Mueller, Ivica Fucek, Ute Schniegler-Mattox, Kristina Ellwanger, Torsten Haneke, Andras Strassz, Wolfgang Fischer, Erich Rajkovic, Michael Tesar. AFM24, a bispecific EGFR/CD16A innate cell engager with the potential to overcome resistance to current targeted treatments for EGFR-positive malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5659.

Response to tyrosine kinase inhibitors in lung adenocarcinoma with the rare epidermal growth factor receptor mutation S768I and G724S: A case report and literature review

Mutations in the epidermal growth factor receptor (EGFR) are drivers of a subset of lung cancers. In recent years, the treatment of non‐small cell lung cancer (NSCLC), especially with EGFR inhibitors, has made rapid progress, and the median progression‐free survival (PFS) of patients with EGFR gene‐sensitive mutations has been significantly prolonged. However, the response effect of some uncommon EGFR mutations to tyrosine kinase inhibitors (TKIs) remains unclear.Here, we present a patient with multiple EGFR mutations that highlights tumor heterogeneity leading to a mixed molecular response to targeted drugs and emphasizes the complexity of EGFR‐driven lung cancer. He received chemotherapy and molecular‐targeted treatment including icotinib, afatinib, osimertinib and afatinib + osimertinib.In conclusion, patients with lung adenocarcinoma harboring the EGFR S768I and G724S mutations appear less sensitive to icotinib than patients with sensitive EGFR. However, the patient in our report benefited from treatment with afatinib. Here, we hope to provide information for the treatment of rare and compound mutations in patients.

EGFR: An essential receptor tyrosine kinase-regulator of cancer stem cells.

The Epidermal Growth Factor Receptor (EGFR) is frequently expressed at elevated levels in different forms of cancer and expression often correlates positively with cancer progression and poor prognosis. Different mutant forms of this protein also contribute to cancer heterogeneity. A constitutively active form of EGFR, EGFRvIII is one of the most important variants. EGFR is responsible for the maintenance and functions of cancer stem cells (CSCs), including stemness, metabolism, immunomodulatory-activity, dormancy and therapy-resistance. EGFR regulates these pathways through several signaling cascades, and often cooperates with other RTKs to exert further control. Inhibitors of EGFR have been extensively studied and display some anticancer efficacy. However, CSCs can also acquire resistance to EGFR inhibitors making effective therapy even more difficult. To ameliorate this limitation of EGFR inhibitors when used as single agents, it may be of value to simultaneously combine multiple EGFR inhibitors or use EGFR inhibitors with regulators of other important cancer phenotype regulating molecules, such as STAT3, or involved in important processes such as DNA repair. These combinatorial approaches require further experimental confirmation, but if successful would expand and improve therapeutic outcomes employing EGFR inhibitors as one arm of the therapy.

Abstract 4228: Integrated drug high-throughput screening and quantitative phosphoproteomics rationalizes combination therapy for mutant KRAS pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly recalcitrant malignancy for which better therapies are urgently needed. KRAS is the most frequently altered gene in PDAC and it orchestrates a number of effector pathways that ultimately drives PDAC growth and proliferation. The recent development of allele-specific inhibitors of oncogenic KRAS G12C, such as ARS-1620, provides an unprecedented opportunity to explore direct targeting of oncogenic KRAS signaling in PDAC. Nearly 3% PDAC patient harbor KRAS G12C, corresponding to ~1,600 new cases of PDAC/year in the US alone for which currently there are no robust targeted therapies. Even if the inhibitor effectively block KRAS function, compensatory mechanism will probably be triggered to overcome the KRAS-mutant PDAC.To better position the direct mutant KRAS inhibitor, we have developed a platform that includes: a high-throughput screen anchored with ARS-1620 to identify companion inhibitor to further dial down the oncogenic KRAS signaling, quantitative mass spectrometric method to map out the adaptive signaling mechanisms of this combination therapy, and bioinformatics pipeline to reveal and rank targetable kinases that drive the adaptive resistance mechanism. To do so, we will apply our platform on a panel of KRAS G12C primary PDAC models. We will profile the proteomic/phosphoproteomic changes of cells treated with combination therapy to identify adaptive signaling pathways that could be potentially targeted.High-throughput chemical-genomics screening identified receptor tyrosine kinases (RTKs) to be synergistic to the KRAS inhibitor, ARS-1620. In particular, multiple epidermal growth factor receptor (EGFR) inhibitors restrict growth of patient-derived PDAC, XWR200, when combined with ARS-1620. When profiling the proteomic/phosphoproteomics changes induced by the combination therapy, we quantified nearly 9600 protein groups and 44,000 phosphopeptides with 36,000 class 1 phosphopeptides having quantitation in all groups (FDR < 1%). We interrogated the Drug Signature Database (DSigDB) using the ~2,400 significantly-altered proteins (FDR < 0.1%) and found the protein gene set to be highly correlated to genes downregulated by topoisomerase inhibition. To identify adaptive signaling pathways, we performed kinase-substrate enrichment analysis (KSEA) on the phosphoproteomic dataset containing ~8,000 significantly altered phosphopeptides (FDR < 1%). KSEA identified hyperactivation of several G2/M checkpoint kinases such as Aurora kinases A and B. This integrative platform has revealed synergistic drug combinations with ARS-1620 and the phosphoproteomics has prescribed possibly the third drug of the combination therapy. Note: This abstract was not presented at the meeting. Citation Format: Joseph Capri, Thuc Le, Caius Radu, Timothy Donahue. Integrated drug high-throughput screening and quantitative phosphoproteomics rationalizes combination therapy for mutant KRAS pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4228.

Downregulating CD26/DPPIV by apigenin modulates the interplay between Akt and Snail/Slug signaling to restrain metastasis of lung cancer with multiple EGFR statuses

BackgroundMetastasis rather than the primary cancer determines the survival of cancer patients. Activation of Akt plays a critical role in the epithelial-to-mesenchymal transition (EMT), the initial step in lung cancer metastasis. Apigenin (API), a flavonoid with a potent Akt-inhibitory effect, shows oncostatic activities in various cancers. However, the effects of API on metastasis of non-small cell lung cancer (NSCLC) remain unclear.MethodsNSCLC cell lines with different epidermal growth factor receptor (EGFR) statuses and in vivo orthotopic bioluminescent xenograft model were employed to determine antitumor activity of API. Western blot and genetic knockdown by shRNA or genetic overexpression by DNA plasmids were performed to explore the underlying mechanisms. The Cancer Genome Atlas (TCGA) database was used to investigate the prognosis of API-targeted genes.ResultsAPI was demonstrated to inhibit the migration/invasion of NSCLC cells harboring different EGFR statuses via suppressing the Snail/Slug-mediated EMT. Mechanistic investigations showed that CD26/dipeptidyl peptidase IV (DPPIV) was downregulated by API following suppressive interplay of Akt and Snail/Slug signaling to modulate the EMT and the invasive ability of NSCLC cells. CD26 expression was positively correlated with the invasive abilities of NSCLC cells and a worse prognosis of lung cancer patients. Furthermore, we observed that patients with CD26high/Akthigh tumors had the shortest recurrence-free survival times. In vivo, API drastically reduced the growth and metastasis of A549 xenografts through targeting CD26.ConclusionsCD26 may be a useful biomarker for predicting NSCLC progression. API effectively suppressed lung cancer progression by targeting the CD26-Akt-Snail/Slug signaling pathway.

Abstract 1836: Induction of secreted soluble decoy EGFR variants by splicing interference overcomes drug resistance in human non-small cell lung cancer

Abstract Activation of the epidermal growth factor receptor (EGFR) is involved in the oncogenesis of multiple cancers, particularly lung cancer. Current treatments are limited by the appearance of TKI resistance from secondary somatic mutations in the EGFR gene, such as the T790M mutation (>50% of biopsies) in the EGFR kinase domain following erlotinib or gefitinib treatment. As these tumors are still EGFR-dependent, EGFR remains a prime therapeutic target in NSCLC. We have recently described multiple natural soluble decoy isoforms for multiple RTKs, including EGFR, which depend on the differential inclusion of functional domains by alternative splicing. We employ antisense oligo nucleotide (ASO) to reprogram EGFR pre-mRNA processing in order to increase expression of mRNA variants that encode for dominant-negative, soluble decoys EGFR (sdEGFR), at the expense of the oncogenic full-length EGFR receptor. These alternative splicing isoforms are generated via intronic polyadenylation (IPA) of pre-mRNA in a U1-snRNP (U1)-dependent manner. More specifically, we are able to induce the sdEGFR variants by using specific ASOs, designed to block the upstream U1 binding site and thus activate the appropriate IPA sites. Here, we show that our novel strategy effectively induces the expression of potent natural inhibitors of EGFR signaling in treatment-refractory lung cancer models in vitro and in vivo, leading to suppression of downstream pathways and tumor growth inhibition. The ASO-induced natural sdEGFR compounds function in a dominant-negative manner and induce dramatic cell death in NSCLC cells harboring multiple activating and resistance EGFR mutations, and thus provides a novel alternative strategy for treatment of refractory NSCLC, to overcome resistance mediated by the EGFR secondary mutations (including T790M, C797S and others). Citation Format: Jeong Eun Park, Lee Spraggon, Elisa de Stanchina, Luca Cartegni. Induction of secreted soluble decoy EGFR variants by splicing interference overcomes drug resistance in human non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1836.

Mechanism of Allosteric Coupling into and through the Plasma Membrane by EGFR

Epidermal growth factor receptor (EGFR) interacts through its extracellular domain with seven different growth factors. These factors induce different structures within the cytoplasmic juxtamembrane (JM) segment of the dimeric receptor and propagate different growth factor-dependent signals to the cell interior. How this process occurs is unknown. Here we apply diverse experimental and computational tools to show that growth factor identity is encoded by the EGFR transmembrane (TM) helix into discrete helix dimer populations that differ in both cross-location and cross-angle. Helix dimers with smaller cross-angles at multiple cross locations are decoded to induce an EGF-type coiled coil in the adjacent JM, whereas helix dimers with larger cross-angles at fewer cross locations induce the TGF-α-type coiled coil. We propose an updated model for how conformational coupling across multiple EGFR domains results in growth factor-specific information transfer, and demonstrate that this model applies to both EGFR and the related receptor ErbB2.

Hybrid Capture-Based Comprehensive Genomic Profiling Identifies Lung Cancer Patients with Well-Characterized Sensitizing Epidermal Growth Factor Receptor Point Mutations That Were Not Detected by Standard of Care Testing.

In our recent study, of cases positive for epidermal growth factor receptor (EGFR) exon 19 deletions using comprehensive genomic profiling (CGP), 17/77 (22%) patients with prior standard of care (SOC) EGFR testing results available were previously negative for exon 19 deletion. Our aim was to compare the detection rates of CGP versus SOC testing for well-characterized sensitizing EGFR point mutations (pm) in our 6,832-patient cohort. DNA was extracted from 40 microns of formalin-fixed paraffin-embedded sections from 6,832 consecutive cases of non-small cell lung cancer (NSCLC) of various histologies (2012-2015). CGP was performed using a hybrid capture, adaptor ligation-based next-generation sequencing assay to a mean coverage depth of 576×. Genomic alterations (pm, small indels, copy number changes and rearrangements) involving EGFR were recorded for each case and compared with prior testing results if available. Overall, there were 482 instances of EGFR exon 21 L858R (359) and L861Q (20), exon 18 G719X (73) and exon 20 S768I (30) pm, of which 103 unique cases had prior EGFR testing results that were available for review. Of these 103 cases, CGP identified 22 patients (21%) with sensitizing EGFR pm that were not detected by SOC testing, including 9/75 (12%) patients with L858R, 4/7 (57%) patients with L861Q, 8/20 (40%) patients with G719X, and 4/7 (57%) patients with S768I pm (some patients had multiple EGFR pm). In cases with available clinical data, benefit from small molecule inhibitor therapy was observed. CGP, even when applied to low tumor purity clinical-grade specimens, can detect well-known EGFR pm in NSCLC patients that would otherwise not be detected by SOC testing. Taken together with EGFR exon 19 deletions, over 20% of patients who are positive for EGFR-activating mutations using CGP are previously negative by SOC EGFR mutation testing, suggesting that thousands of such patients per year in the U.S. alone could experience improved clinical outcomes when hybrid capture-based CGP is used to inform therapeutic decisions. This study points out that genomic profiling, as based on hybrid capture next-generation sequencing, can identify lung cancer patients with point mutation in epidermal growth factor receptor (EGFR) missed by standard molecular testing who can likely benefit from anti-EGFR targeted therapy. Beyond the specific findings regarding false-negative point mutation testing for EGFR, this study highlights the need for oncologists and pathologists to be cognizant of the performance characteristics of testing deployed and the importance of clinical intuition in questioning the results of laboratory testing.

Uncommon <em>EGFR</em> mutations in cytological specimens of 1,874 newly diagnosed Indonesian lung cancer patients

PurposeWe aimed to evaluate the distribution of individual epidermal growth factor receptor (EGFR) mutation subtypes found in routine cytological specimens.Patients and methodsA retrospective audit was performed on EGFR testing results of 1,874 consecutive cytological samples of newly diagnosed or treatment-naïve Indonesian lung cancer patients (years 2015–2016). Testing was performed by ISO15189 accredited central laboratory.ResultsOverall test failure rate was 5.1%, with the highest failure (7.1%) observed in pleural effusion and lowest (1.6%) in needle aspiration samples. EGFR mutation frequency was 44.4%. Tyrosine kinase inhibitor (TKI)-sensitive common EGFR mutations (ins/dels exon 19, L858R) and uncommon mutations (G719X, T790M, L861Q) contributed 57.1% and 29%, respectively. Approximately 13.9% of mutation-positive patients carried a mixture of common and uncommon mutations. Women had higher EGFR mutation rate (52.9%) vs men (39.1%; p<0.05). In contrast, uncommon mutations conferring either TKI responsive (G719X, L861Q) or TKI resistance (T790M, exon 20 insertions) were consistently more frequent in men than in women (67.3% vs 32.7% or 69.4% vs 30.6%; p<0.05). Up to 10% EGFR mutation–positive patients had baseline single mutation T790M, exon 20 insertion, or in coexistence with TKI-sensitive mutations. Up to 9% patients had complex or multiple EGFR mutations, whereby 48.7% patients harbored TKI-resistant mutations. One patient presented third-generation TKI-resistant mutation L792F simultaneously with T790M.ConclusionRoutine diagnostic cytological techniques yielded similar success rate to detect EGFR mutations. Uncommon EGFR mutations were frequent events in Indonesian lung cancer patients.

Abstract A028: Biomarker analysis in a phase 1 study of the antibody-drug conjugate ABBV-221 in patients with solid tumors likely to overexpress the epidermal growth factor receptor (EGFR)

Abstract Background: Aberrant EGFR plays a vital role in the oncogenesis of multiple tumor types. ABBV-221 is a 2nd-generation antibody-drug conjugate (ADC) targeting EGFR, based on the 1st-generation ADC depatuxizumab mafodotin (depatux-m, formerly ABT-414). Depatux-m demonstrates efficacy in glioblastoma (GBM) patients (pts) with EGFR amplification in ongoing studies. ABBV-221 is an affinity matured monoclonal antibody against EGFR linked to the microtubule toxin monomethylauristatin E. ABBV-221 has higher affinity for EGFR than depatux-m, potentially allowing it to target a broader range of tumor types. To understand the population most suitable for ABBV-221, molecular assays characterizing EGFR pathway status, including EGFR protein, EGFR mRNA, and EGFR ligand mRNA expression, were evaluated from available tumor tissue of enrolled patients. The results of each assay were correlated with patient outcome to determine which marker may be best associated with prognosis. Methods: In this phase 1, multicenter study of ABBV-221, eligible pts have solid tumors that are likely to overexpress EGFR. EGFR protein expression in tumor tissue samples was analyzed with immunohistochemistry (IHC) using the Dako pharmDx IHC assay. EGFR and mRNA expression from the EGFR ligands epiregulin (EREG) and amphiregulin (AREG) were measured using next-generation RNA sequencing (RNAseq). Results: As of 29 March 2017, 42 pts were treated (13 colon, 5 head &amp; neck [H&amp;N] cancer, 5 non-small cell lung cancer, 5 GBM, 2 breast, 12 other). Ten dose escalation cohorts have been completed with the last cleared dose 4.5 mg/kg per cycle. The most common adverse events were infusion reactions in 19/42 pts (45%) and fatigue in 17/42 pts (41%). Sixteen pts (38%) had stable disease (SD), including 4 pts who remained on study longer than 6 months. All markers are expressed in a wide range across enrolled pts. The pts with response had high EGFR and EGFR ligand. One pt with head and neck cancer who received 5 cycles of ABBV-221 had a partial response and elevated levels of both EGFR (3-fold higher than median for all pts) and EGFR ligand (8-fold higher AREG and 19-fold higher EREG than median for all pts). One colorectal cancer pt with durable SD &amp;gt;6 months had high levels of ligand (EREG) but not receptor, and one H&amp;N pt with SD &amp;gt;6 months had high levels of EGFR receptor but not ligand. Conclusions: Multiple EGFR pathway status biomarkers were evaluated and correlated with clinical outcomes observed in this phase 1 trial of ABBV-221. Patients who observed clinical benefit had high levels of EGFR pathway markers. The duration of SD in pts with refractory solid tumors is encouraging. Clinical trial information: NCT02365662 Citation Format: Emiliano Calvo, James M. Cleary, Victor Moreno, Maryella Gifford, Lisa Roberts-Rapp, Peter J. Ansell, Ho-Jin Lee, Beibei Hu, David Barch, Christopher Ocampo, Anthony W. Tolcher. Biomarker analysis in a phase 1 study of the antibody-drug conjugate ABBV-221 in patients with solid tumors likely to overexpress the epidermal growth factor receptor (EGFR) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A028.

Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer

Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms. The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover, phospho-Akt levels are increased in most clinical specimens obtained from EGFR-mutant non-small-cell lung cancer patients with acquired EGFR tyrosine kinase inhibitor resistance. Our findings provide a rationale for clinical trials testing Akt and EGFR inhibitor co-treatment in patients with elevated phospho-Akt levels to therapeutically combat the heterogeneity of EGFR tyrosine kinase inhibitor resistance mechanisms.

The activation of SRC family kinases and focal adhesion kinase with the loss of the amplified, mutated EGFR gene contributes to the resistance to afatinib, erlotinib and osimertinib in human lung cancer cells.

Second- and third-generation inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase activity (EGFR-TKIs) are improving the treatment of patients with non-small cell lung cancer. Here we established two sublines (BR1-8 and BR2-3) resistant to a second-generation inhibitor, afatinib, from the human lung cancer cell line HCC827 that harbors a mutation that activates the tyrosine kinase activity of EGFR. These afatinib-resistant sublines were resistant to first-generation EGFR-TKIs, gefitinib and erlotinib, and a third-generation EGFR-TKI, osimertinib. These resistant sublines showed markedly reduced levels of multiple EGFR family proteins, including the activated mutant EGFR, and complete loss of EGFR amplification as compared with their parental HCC827 cells harboring amplification of EGFR gene. Treatment with the multikinase inhibitor dasatinib or transfection with a SRC small interfering RNA inhibited cell survival and AKT phosphorylation in drug-resistant sublines to a greater extent compared with HCC827 cells. Further, the migration of drug-resistant cells was greater compared with that of HCC827 cells and was inhibited by dasatinib or an FAK inhibitor. These findings indicate that compensatory activation of SRC family kinases (SFKs) and FAK supports the survival and migration of afatinib-resistant cells when the expression of multiple EGFR family proteins was mostly abrogated. Combinations of potent drugs that target SFKs and FAK may overcome the resistance of lung cancer cells to second-generation TKIs.

Multiplex Ultrasensitive Genotyping of Patients with Non-Small Cell Lung Cancer for Epidermal Growth Factor Receptor (EGFR) Mutations by Means of Picodroplet Digital PCR

Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~0.01% for each mutation. All 33 EGFR-activating mutations detected in formalin-fixed paraffin-embedded (FFPE) tissue samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation (T790M; including an ultra-low-level mutation, <0.1%) was detected in the same reaction. Regression analysis of the duplex assay and multiplex assay showed a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for an exon 19 deletion, and 0.9959 for T790M. Using ddPCR, we designed a multiplex ultrasensitive genotyping platform for 3 common EGFR mutations. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations concurrently with an ultra-rare pretreatment mutation (T790M).

Current Status and Perspectives Regarding the Therapeutic Potential of Targeting EGFR Pathway by Curcumin in Lung Cancer.

Lung cancer is among the leading causes of cancer-related-death. Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. More than 70% of NSCLC patients have locally advanced or metastatic disease in diagnosis stage, which are then being treated with platinum-based chemotherapy or epidermal-growthfactor- receptor (EGFR) inhibitors. Several molecules which target multiple ErbB receptors and EGFR have been developed, including gefitinib and erlotinib. Identification of novel agents with less toxicity is warranted. Several interesting data have been reported about the antitumor activity of curcumin in several tumors, including lung, breast and colorectal cancers. In particular, a recent phase I trial evaluated the activity of curcumin in combination with FOLFOX chemotherapy in patients with inoperable colorectal cancer. They showed that curcumin added benefit in subsets of patients when administered with FOLFOX and was well-tolerated chemotherapy adjunct. Another ongoing trial is now investigating the beneficial effects of curcumin plus gefitinib or erlotinib for EGFRmutant NSCLC. Improved understanding of molecular mechanisms behind resistance to EGFR tyrosine kinase inhibitors suggests the importance of a genotype-guided approach to therapy and inhibition of parallel and downstream pathways, using agents which target heat-shock-protein-90, poly (ADP-ribose) polymerase and PI3K/mTOR pathway. The aim of the current review is to give an overview of the possible molecular mechanisms of curcumin in the preclinical and clinical investigations in solid tumors, with particular emphasis on its combination with other chemotherapeutic agents in lung cancers.

Molecular Targeting of Growth Factor Receptor Signaling in Radiation Oncology.

Ionizing radiation has been shown to activate and interact with multiple growth factor receptor pathways that can influence tumor response to therapy. Among these receptor interactions, the epidermal growth factor receptor (EGFR) has been the most extensively studied with mature clinical applications during the last decade. The combination of radiation and EGFR-targeting agents using either monoclonal antibody (mAb) or small-molecule tyrosine kinase inhibitor (TKI) offers a promising approach to improve tumor control compared to radiation alone. Several underlying mechanisms have been identified that contribute to improved anti-tumor capacity after combined treatment. These include effects on cell cycle distribution, apoptosis, tumor cell repopulation, DNA damage/repair, and impact on tumor vasculature. However, as with virtually all cancer drugs, patients who initially respond to EGFR-targeted agents may eventually develop resistance and manifest cancer progression. Several potential mechanisms of resistance have been identified including mutations in EGFR and downstream signaling molecules, and activation of alternative member-bound tyrosine kinase receptors that bypass the inhibition of EGFR signaling. Several strategies to overcome the resistance are currently being explored in preclinical and clinical models, including agents that target the EGFR T790M resistance mutation or target multiple EGFR family members, as well as agents that target other receptor tyrosine kinase and downstream signaling sites. In this chapter, we focus primarily on the interaction of radiation with anti-EGFR therapies to summarize this promising approach and highlight newly developing opportunities.

Epidermal growth factor receptor, KRAS and BRAF gene mutations and their correlation with clinicopathological characteristics in non-small-cell lung cancer

Objective To investigate the epidermal growth factor receptor (EGFR), KRAS and BRAF mutations and their correlation with clinicopathological characteristics in non-small-cell lung cancer (NSCLC). Methods The mutations of exon 18, exon 19, exon 20 and exon 21 of the EGFR, codon 12, codon 13 of the KRAS and codon 600 of the BRAF gene in 143 cases of NSCLC were detected by gene sequencing. The relationship between the mutations and clinicopathological features was analyzed by SPSS 16.0. Results EGFR mutation was detected in 57 cases (39.9 %), including 2 mutations in exon 18, 25 in exon 19, 3 in exon 20, 24 in exon 21 and 3 multiple point mutations. KRAS mutation was found in 25 cases (17.5 %), including 23 in codon 12 and 2 in codon 13. BRAF V600E mutation was detected only in 2 cases (1.4 %). No patient harboring multiple EGFR, KRAS and BRAF mutations was found. EGFR mutation rate was related to gender, smoking history, histological types, differentiation and tumor size (P 0.05). There was no association between KRAS mutation and clinicopathological features including gender, smoking history, histological types, differentiation, tumor size, lymph node metastasis and pTNM stage (P> 0.05). Conclusions The frequency of EGFR mutation in NSCLC is high, and usually occurs in female, non-smokers, smaller tumors, better differentiation and adenocarcinomas. The frequency of KRAS mutation is not associated with the clinicolpathological features. The frequency of BRAF mutation is very low, and EGFR, KRAS and BRAF gene mutations do not occur at the same time.These results contribute to the target therapy of NSCLC. Key words: Carcinoma, non-small-cell lung; Epidermal growth factor receptor; KRAS; BRAF; Clinicopathological characteristics

Abstract 768: A kinome-wide siRNA screen identifies modifiers of sensitivity to the EGFR T790M-targeted tyrosine kinase inhibitor (TKI), AZD9291, in EGFR mutant lung adenocarcinoma

Abstract New epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) that are targeted against EGFR T790M, such as AZD9291, are among the most promising agents for the treatment of EGFR mutant lung adenocarcinomas, as they can overcome a key mechanism of acquired resistance to first or second generation EGFR-TKIs. Emerging clinical data indicate that these drugs achieve partial responses in ∼60% of patients and demonstrate variable duration of benefit (Yang J et al. Ann Oncol 2014;25 (suppl 4): iv149). However, most responding patients are expected to develop eventually progressive disease. Mechanisms of resistance for this class of mutant specific EGFR TKIs are only beginning to be elucidated. To identify modifiers of sensitivity to AZD9291 in EGFR mutant lung adenocarcinoma, we performed a kinome-wide short-interfering (si) RNA screen using clonal PC-9/BRc1 (EGFR exon19 deletion/T790M) cells and siRNA triplicates directed against 714 phylogenetically-related kinases (GE Dharmacon). The cells were derived from PC-9 cells chronically exposed to afatinib (Chmielecki J et al. Sci Transl Med. 2011;3:90ra59). The screen was performed in the absence and presence of AZD9291 (5 nM). Among the top 20 hits, siRNAs against the serine threonine kinases, MAPK1 (ERK2) and BRAF, sensitized cells to AZD9291. Both genes encode products involved in the RAS-MAP kinase signaling pathway. Confirming the significance of these hits, combinations of AZD9291 and the MEK inhibitor, selumetinib, were more effective at inhibiting cell growth than AZD9291 alone. Consistent with these findings, we found that ERK1/2 is readily dephosphorylated upon exposure to AZD929, however, ERK1/2 becomes re-phosphorylated after continuous exposure to AZD9291 for 96 hours. These data suggest that reactivation of ERK signaling may provide an early escape mechanism mediating drug resistance. ERK reactivation after AZD9291 treatment and stronger growth inhibition by AZD9291 + selumetinib combination therapy were also seen in multiple other EGFR mutant lines. The SRC kinase is known to activate RAS-MAP kinase pathway signaling, and pharmacologic or genetic ablation of SRC partially inhibited ERK reactivation, suggesting that SRC is involved in this escape mechanism. In conclusion, through a kinome wide siRNA screen, we identified that gene products in the MAP kinase signaling pathway modify sensitivity to AZD9291. Such sensitivity may be associated with ERK re-phosphorylation within 96h of drug treatment. Collectively, these data suggest rational drug combinations that could be used to forestall resistance to AZD9291. Additional hits from the screen are currently under investigation. This study is supported by AstraZeneca Oncology Innovative Medicines, National Institutes of Health (NIH) NCI grants R01-CA121210, P01-CA129243, U54-CA143798, and the Uehara Memorial Foundation. Citation Format: Eiki Ichihara, Joshua A. Bauer, Pengcheng Lu, Fei Ye, Darren Cross, William Pao, Christine M. Lovly. A kinome-wide siRNA screen identifies modifiers of sensitivity to the EGFR T790M-targeted tyrosine kinase inhibitor (TKI), AZD9291, in EGFR mutant lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 768. doi:10.1158/1538-7445.AM2015-768