EGFR Kinase Inhibitors Research Articles

Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer.

Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies. To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation. Patients with advanced nonsquamous non-small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute-designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting. Plasma ddPCR assay sensitivity, specificity, and TAT. Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRAS G12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically. Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.

ASCT2 (SLC1A5) is an EGFR-associated protein that can be co-targeted by cetuximab to sensitize cancer cells to ROS-induced apoptosis

Therapeutic targeting of ASCT2, a glutamine transporter that plays a major role in glutamine uptake in cancer cells, is challenging because ASCT2 also has a biological role in normal tissues. In this study, we report our novel finding that ASCT2 is physically associated in a molecular complex with epidermal growth factor receptor (EGFR), which is often overexpressed in human head and neck squamous cell carcinoma (HNSCC). Furthermore, we found that ASCT2 can be co-targeted by cetuximab, an EGFR antibody approved for treating metastatic HNSCC. We demonstrated that cetuximab downregulated ASCT2 in an EGFR expression-dependent manner via cetuximab-mediated EGFR endocytosis. Downregulation of ASCT2 by cetuximab led to decreased intracellular uptake of glutamine and subsequently a decreased glutathione level. Cetuximab thereby sensitized HNSCC cells to reactive oxygen species (ROS)-induced apoptosis and, importantly, it is independent of effective inhibition of EGFR downstream signaling by cetuximab. In contrast, knockdown of EGFR by siRNA or inhibition of EGFR kinase with gefitinib, an EGFR kinase inhibitor, failed to sensitize HNSCC cells to ROS-induced apoptosis. Our findings support a novel therapeutic strategy for EGFR-overexpressing and cetuximab-resistant cancers by combining cetuximab with an oxidative therapy.

Abstract 2148: MM-151 elicits broad and unique inhibition of cells harboring EGFR extracellular domain mutations — results of multiscale experiments with genome-edited cell lines

Abstract Mutations in the extracellular domain (ECD) of EGFR have been reported in colorectal (CRC) patients as a mechanism of acquired resistance to the EGFR-directed antibodies cetuximab and panitumumab. Similar to EGFR intracellular domain mutations observed in patients treated with EGFR kinase inhibitors, these EGFR-ECD mutations inhibit the binding of the antibodies while maintaining EGFR signaling activity. MM-151, an investigational agent consisting of three anti-EGFR IgG1 antibodies, has been recently reported to overcome these mutations in over-expression and patient-derived cell line models. Here, we extend these studies to characterize the mechanisms of action that enable MM-151 to provide robust inhibition of EGFR-ECD mutations beyond that achievable by other monoclonal and oligoclonal EGFR antibodies. First, utilizing a cell-free binding assay with recombinant EGFR-ECD mutants, we found that MM-151, but not the Sym004 combination of two antibodies, maintained oligoclonal binding to all mutants. Second, we performed a panel of 2D and 3D in vitro experiments with over-expression and genome-edited (CRISPR-Cas9) cell lines harboring EGFR-ECD mutations to characterize target engagement, cell signaling, proliferation, and the impact of hetero- versus homozygous expression of EGFR wild type or mutant alleles. We observed that the three mechanisms of action for MM-151- antagonism of both low- and high-affinity EGFR ligands, EGFR down-regulation, and immune-effector activity— were preserved in these models. Third, we performed xenograft experiments to evaluate the behavior of MM-151 and found that MM-151 maintained inhibitory activity in an in vivo context. The results of our multiscale experiments identified the impact of combining three antibodies to overcome the high plasticity of the EGFR extracellular domain and thus reveal the potential for clinical evaluation of MM-151 in both EGFR-naïve and EGFR-refractory CRC populations. Citation Format: Hongfang Wang, Shawn P. Carey, Olga Burenkova, Jian Tang, Nastaran Gerami-Moayed, Sabrina Arena, Alberto Bardelli, Rachel Nering, Jeffrey D. Kearns. MM-151 elicits broad and unique inhibition of cells harboring EGFR extracellular domain mutations — results of multiscale experiments with genome-edited cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2148.

Abstract 4559: Sprouty2 differentially regulates signaling and phenotypic responses of glioblastoma cells to DNA damaging agents and receptor kinase inhibitors

Abstract Key to improving glioblastoma patient survival is the exploration of the signaling mechanisms that regulate cellular response to approved and investigational therapeutics, including DNA damaging agents and targeted therapeutics such as EGFR and MET kinase inhibitors. Previous work from our group demonstrated that Sprouty2 (SPRY2) surprisingly acted more as a tumor promoter than tumor suppressor in glioblastoma cell lines and tumor xenografts, with SPRY2 knockdown reducing proliferation of human glioblastoma cells and antagonizing the growth of subcutaneous tumor xenografts in mice. We hypothesized here that these effects may result from a perturbation in the cell cycle and that such effects may augment cell cycle shifts induced by DNA damaging agents or kinase inhibitors. Flow cytometry measurements indicated shifts in DNA content distribution in glioblastoma cell lines consistent with cell cycle arrest in response to SPRY2 knockdown alone, and that these shifts were substantially augmented by carboplatin or to a lesser degree by a combination of EGFR and MET inhibitors, but not by temozolomide. Western blotting indicated that SPRY2 knockdown resulted in increased phosphorylation of Ataxia Telangiectasia Mutated (ATM), a kinase involved in the DNA damage response pathway, at a serine residue (1981) that regulates the dissociation of inactivate ATM homodimers into activated monomers. In response to carboplatin, but not the other therapeutics tested, a robust increase was observed in Checkpoint kinase 1 (CHK1) phosphorylation at serine 345, which has been reported to cause cell cycle arrest. This effect was further enhanced by SPRY2 knockdown, which may help explain why the largest cell cycle perturbations were observed in SPRY2-deficient cells treated with carboplatin. Increased phosphorylation of p38 was also observed in response to carboplatin or EGFR and MET inhibitors. This may have contributed to cell cycle arrest, but may also have cooperated with the inhibition of AKT phosphorylation that occurred only in response to EGFR and MET inhibition to drive cell death, an effect that was also augmented by SPRY2 knockdown. Overall, this work has identified a small network of SPRY2-regulated signaling processes that control phenotypic responses of glioblastoma cells to different clinically relevant therapeutics and provides motivation for further study of the role of SPRY2 in glioblastoma. Citation Format: Nisha G. Sosale, Sally Shin, Matthew J. Lazzara. Sprouty2 differentially regulates signaling and phenotypic responses of glioblastoma cells to DNA damaging agents and receptor kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4559.

Abstract 890: TGFâ upregulation mediates growth retardation in EGFR T790M mutant non-small cell lung cancer

Abstract Introduction: A subset of non-small cell lung cancer is driven by activating mutations in the epidermal growth factor receptor (EGFR). The majority of EGFR-driven lung cancers respond to EGFR kinase inhibitors gefitinib and erlotinib, although the clinical efficacy these inhibitors is limited by the development of drug resistance. A secondary missense mutation encoding EGFR T790M is the most prevalent resistance mechanism observed in patients, accounting for nearly 60% of relapse following gefitinib or erlotinib treatment. Although the T790M mutation enhances EGFR kinase activity, T790M harboring tumors exhibit indolent growth and are associated with favorable prognosis compared to tumors that acquire alternative mechanisms of drug resistance. The mechanism(s) underlying the indolent growth mediated by EGFR T790M are not well characterized. Methods: To identify the factor(s) mediating T790M associated growth retardation, we characterized T790M amplified drug resistant cells. EGFR mutant parental lung cancer cell lines PC9 and H3255 were selected through sequential treatment with gefitinib and the irreversible EGFR inhibitor dacomitinib. The resulting drug resistant cells, PC9DR and H3255DR, harbored amplified EGFR T790M and exhibited marked growth retardation. These T790M amplified models were characterized to identify factors conferring indolent growth. T790M expressing patient tumor samples were also analyzed to validate T790M associated changes. Results: Ectopic overexpression of EGFR T790M induced growth retardation in EGFR inhibitor-naïve cells. Incubation of parental PC9 cells in media conditioned on PC9DR cells was sufficient to slow PC9 growth rates, suggesting that a secreted factor was responsible for the growth retardation observed in the T790M amplified cells. A Luminex assay of PC9DR cells revealed upregulation of transforming growth factor beta 2 (TGFâ2), which was confirmed at the transcript level by qPCR. Treatment of parental PC9 cells with recombinant TGFâ was sufficient to induce slowed growth, while inhibition of TGFâ signaling using a TGFâ receptor inhibitor rescued growth rates in T790M amplified cell lines. Finally, T790M harboring patient tumor samples showed a trend of TGFâ2 transcript upregulation. Conclusions: We have demonstrated that TGFâ2 is upregulated in EGFR T790M amplified lung cancer cells, and is sufficient to elicit T790M associated growth retardation. The correlation between TGFâ expression and indolent growth of T790M expressing tumors identifies TGFâ as a potential biomarker to predict patient prognosis and outcome. Citation Format: Claire E. Repellin, Pinar O. Eser, Marzia Capelletti, Takeshi Shimamura, Dalia Ercan, Chunxiao Xu, Nathanael S. Gray, Kwok-Kin Wong, Pasi A. Jänne. TGFâ upregulation mediates growth retardation in EGFR T790M mutant non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 890.

Kinase inhibitor-responsive genotypes in EGFR mutated lung adenocarcinomas: moving past common point mutations or indels into uncommon kinase domain duplications and rearrangements.

The most frequent epidermal growth factor receptor (EGFR) mutations found by traditional or comprehensive molecular profiling of lung adenocarcinomas include indels of exon 19 (the exon 19 deletion delE746_A750 being the most common) and the exon 21 L858R point mutation. The current approval labels for first line palliative gefitinib 250 mg/day, erlotinib 150 mg/day and afatinib 40 mg/day for advanced lung cancers require the presence of the aforementioned classical/sensitizing EGFR mutations. Other gefitinib, erlotinib and afatinib sensitizing mutations include exon 18 indels, G719X, exon 19 insertions, A763_Y764insFQEA, S768I and L861Q; for which off-label EGFR kinase inhibitor use is generally agreed upon by thoracic oncologists. The main biological mechanism of resistance to approved first line EGFR inhibitors is the selection/acquisition of EGFR-T790M that in itself can be inhibited by osimertinib 80 mg/day, a 3(rd) generation EGFR inhibitor that is bypassed by EGFR-C797X mutations. Another class of de novo inhibitor insensitive mutation includes EGFR exon 20 insertions. More recently, the dichotomy of only point mutations or indels explaining aberrant kinase activation of EGFR plus inhibitor response has been shattered by the discovery of uncommon (<0.5% of all EGFR mutations) genomic events involving exon 18-25 kinase domain duplications (KDD) and rearrangements (EGFR-RAD51 or EGFR-PURB). The latter lead to oncogene addiction, enhanced sensitivity to kinase inhibitors in vitro and clinical responses to approved EGFR inhibitors. The enhanced landscape of EGFR inhibitor-responsive genotypes highlights that comprehensive molecular profiling may be necessary to maximize the identification of all cases that can benefit from precision oncology.

SH3GL1 inhibition reverses multidrug resistance in colorectal cancer cells by downregulation of MDR1/P-glycoprotein via EGFR/ERK/AP-1 pathway.

Multidrug resistance is one of the major reasons colorectal cancer (CRC) chemotherapy-based treatments fail, and novel biologically based therapies are urgently needed. Src homology 3 (SH3)-domain GRB2-like protein 1 (SH3GL1) is a membrane-bound protein which was found to be involved in tumor formation, progression, and metastasis. In this study, immunohistochemistry staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis revealed a high expression of SH3GL1 in human CRC tumor specimens and several CRC cells resistant to chemotherapeutics. Cell Counting Kit-8 (CCK-8) assay showed that transfection of pCDNA3.1(+)-SH3GL1 increased while transfection of SH3GL1 siRNA decreased cell viability in response to 5-fluorouracil (5-FU) treatment (P<0.05). Further studies indicated that transfection of SH3GL1 siRNA significantly downregulated multidrug resistance protein 1 (MDR1)/P-glycoprotein expression (P<0.05), decreased MDR1 promoter activity and activator protein-1 (AP-1) binding activity (P<0.05), and inhibited the activation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling (P<0.05) in CRC cells resistant to chemotherapeutics. Transfection of pCDNA3.1(+)-SH3GL1 caused the opposite effect. Additionally, pre-treatment with either EGFR kinase inhibitor PD153035 or ERK1/2 kinase inhibitor PD98059 in HCT116/5-FU cells partly inhibits P-glycoprotein expression and AP-1 binding activity (P<0.05). In conclusion, we confirmed that inhibition of SH3GL1 reverses multidrug resistance through declining P-glycoprotein expression via the EGFR/ERK/AP-1 pathway.

Design, synthesis and biological evaluation of novel EGFR/HER2 dual inhibitors bearing a oxazolo[4,5-g]quinazolin-2(1H)-one scaffold

For the purpose of developing novel EGFR/HER2 tyrosine kinases inhibitors with high inhibition activity and low toxicity, two novel series of oxazolo[4,5-g]quinazolin-2(1H)-one derivatives as EGFR/HER2 dual inhibitors introducing two electrophiles 2-(2-bromoacetyl)ethyl and 2-(2-chloroacetoxy)ethyl group as side-chain at 1-position respectively and evaluated their EGFR and HER2 inhibition activity and toxicity comparing with Lapatinib. All these compounds were evaluated by EGFR and HER2 kinase inhibition and two anti-proliferation assays in vitro. Most of the designed compounds exhibited moderate to high inhibition activity against EGFR and HER2. Especially, compounds 11o, 11p, 12e and 12f presented high inhibition against EGFR and HER2. Furthermore, compounds 11p and 12f also had well exhibition to excellent anti-proliferation activity against human lung adenocarcinoma cell line (A549) and human breast cancer cell line (SK-Br3), and 12f also exhibited the lowest toxicity against human embryonic lung fibroblast cell line (HELF) cell. Finally, compound 12f presented remarkably higher inhibition efficacy towards tumour growth than Lapatinib in a mouse lewis lung cancer (LLC) xenograft model.

Alteration of red blood cell microrheology by anti-tumor chemotherapy drugs

The aim of this study was to estimate effects of some chemotherapy drugs on the elasticity and deformability of the membrane of a red blood cell (RBC). It was found that incubation of red blood cells (RBCs) with cisplatin or epoetin alpha led to considerable (by 10–17%; p < 0.05) increase in the RBC deformability and that cisplatin could activate tyrosine protein kinases (TPKs). Preincubation of RBCs with a specific inhibitor of EGF-R and Src kinase, lavendustin A, almost completely prevented the cisplatin effect. Tyrosine phosphatase inhibitor, sodium orthovanadate, increased the RBC deformability (p < 0.05). This effect was also abandoned by lavendustin A. To test a hypothesis on the involvement of protein kinases of mature RBCs in control of their membrane elasticity, the cells were incubated with phorbol 12-myristate 13-acetate (PMA) activating protein kinase Cα (PKCα). PMA increased the RBC deformability only moderately (by 8%, p < 0.05) and the effect was canceled by nonselective and selective PKC inhibitors staurosporin and 4-(1-methylindol-3-yl)maleimide hydrochloride. Erythropoietin is known to inhibit the nonselective cation channels of the RBC membrane; however, preincubation of the cells with verapamil did not cancel the increase in their deformability. Hence, this increase in deformability could be a result of the action of tyrosine protein kinases, the more so that this effect was almost completely canceled by lavendustion A. The results suggest that the presence of functionally active protein kinases and phosphatases in the membranes of mature RBC makes them a target for the addressed effects of signal molecules, including some chemotherapy drugs, causing consecutive alterations in the RBC membrane elasticity, microrheological properties, and transport potential.

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.

Abstract P4-14-26: Phase I expansion of S-222611, a reversible inhibitor of EGFR and HER2, in advanced solid tumors, including HER2-positive breast cancer patients with brain metastases

Abstract BACKGROUND S-222611 is an oral, reversible ErbB tyrosine kinase inhibitor of EGFR and HER2 with potent pre-clinical activity. MTD was not reached during the dose-escalation phase, (maximum dose 1600 mg QD). PK and efficacy data supported a daily dose of 800 mg. An expansion cohort of patients has been treated to further explore safety and efficacy. METHODS Subjects with advanced solid tumors expressing EGFR and/or overexpressing HER2 were enrolled. S-222611 800 mg daily was administered until disease progression or unacceptable toxicity. RESULTS 76 patients were included in this phase 1 expansion cohort with a variety of tumor types. Dose reduction was required because of adverse events in 15 patients; the most frequent of which being diarrhea and elevated bilirubin. Two patients discontinued treatment due to drug- related adverse events. Of the 25 patients with HER2-positive metastatic breast cancer (MBC), 4 partial responses were observed, and prolonged stable disease (≥ 6 months) was observed in 3 additional patients. These 25 patients had received prior HER2-directed therapy as shown in Table 1. Table 1. Prior therapies received by patients with HER2-positive MBCPrior therapyn (%)Trastuzumab22 (88)T-DM13 (12)Lapatinib16 (64)Chemotherapy23 (92) Six of these patients had brain metastases, in whom 1 intracranial response and 2 prolonged stable disease (≥ 6 months) were observed (Table 2). Table 2. HER2-positive MBC patients with brain metastases - best overall response to S-222611Pts #HER2 IHCBrain metastasesBest overall response (RECIST 1.1)Patient 13+Target lesionPRPatient 23+Target lesionSD (≥12 M)Patient 33+Target lesionSD (6.0 M)Patient 43+Non-target lesionSD (4.7 M)Patient 53+Non-target lesionSD (3.3 M)Patient 63+Non-target lesionNE The patient showing intracranial response was previously treated with lapatinib and capecitabine after diagnosis of BM. CONCLUSIONS S-222611 was well tolerated at a dose of 800 mg once daily. Anti-tumour activity, including shrinkage of brain metastases, was evident in a heavily pre-treated population of patients with HER2-positive breast cancer. Citation Format: Baird RD, Arkenau H-T, Deva S, Cresti N, Garcia-Corbacho J, Hogarth L, Frenkel E, Kawaguchi K, Arimura A, Donaldson K, Posner J, Sarker D, Jodrell D, Plummer R, Spicer J, Italiano A. Phase I expansion of S-222611, a reversible inhibitor of EGFR and HER2, in advanced solid tumors, including HER2-positive breast cancer patients with brain metastases. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-26.

EPHA2 Blockade Overcomes Acquired Resistance to EGFR Kinase Inhibitors in Lung Cancer.

Despite the success of treating EGFR-mutant lung cancer patients with EGFR tyrosine kinase inhibitors (TKI), all patients eventually acquire resistance to these therapies. Although various resistance mechanisms have been described, there are currently no FDA-approved therapies that target alternative mechanisms to treat lung tumors with acquired resistance to first-line EGFR TKI agents. Here we found that EPHA2 is overexpressed in EGFR TKI-resistant tumor cells. Loss of EPHA2 reduced the viability of erlotinib-resistant tumor cells harboring EGFR(T790M) mutations in vitro and inhibited tumor growth and progression in an inducible EGFR(L858R+T790M)-mutant lung cancer model in vivo. Targeting EPHA2 in erlotinib-resistant cells decreased S6K1-mediated phosphorylation of cell death agonist BAD, resulting in reduced tumor cell proliferation and increased apoptosis. Furthermore, pharmacologic inhibition of EPHA2 by the small-molecule inhibitor ALW-II-41-27 decreased both survival and proliferation of erlotinib-resistant tumor cells and inhibited tumor growth in vivo. ALW-II-41-27 was also effective in decreasing viability of cells with acquired resistance to the third-generation EGFR TKI AZD9291. Collectively, these data define a role for EPHA2 in the maintenance of cell survival of TKI-resistant, EGFR-mutant lung cancer and indicate that EPHA2 may serve as a useful therapeutic target in TKI-resistant tumors.

2D and 3D-QSAR analysis of pyrazole-thiazolinone derivatives as EGFR kinase inhibitors by CoMFA and CoMSIA.

Two and Three-dimensional quantitative structure-activity relationship (2D, 3D-QSAR) study was performed for some pyrazole-thiazolinone derivatives as EGFR kinase inhibitors using the CoMFA, CoMSIA and GA-MLR methods. The utilized data set was split into training and test set based on hierarchical clustering technique. From the five CoMSIA descriptors, electrostatic field presented the highest correlation with the activity. The statistical parameters for the CoMFA (r(2)=0.862, q(2)=0.644) and CoMSIA (r(2)=0.851, q(2)=0.740) were obtained for the training set with the common substructure-based alignment. The obtained parameters indicated the superiority of the CoMSIA model over the CoMFA model. A test set consisted of seven compounds was used to evaluate the proposed models. The results of contour maps which were presented by each method lead to some insights for increasing the inhibition activity of compounds. The 2D-QSAR model was built based on three descriptors selected by genetic algorithm and showed high predictive ability (R(2) train= 0.843, Q(2) LOO=0.787). Molecular docking study was also performed to understand the type interactions presented in binding site of the receptor and ligand. The developed models in parallel with molecular docking can be employed to design and derive novel compounds with the potent EGFR inhibitory activity.

Elaboration of tetra-orthogonally-substituted aromatic scaffolds towards novel EGFR-kinase inhibitors.

Nitration of three regioisomers of bromo-fluorobenzaldehyde proceeds regioselectively, notably with H2SO4/HNO3 at 0 °C. The thereby synthesized tetrasubstituted aromatics, endowed with orthogonal substituents, can be elaborated via Pd-catalysed coupling, reduction and reductive amination reactions. As a test-case, these compounds were converted into EGFR inhibitors related to Gefitinib, whose activity was rationalised by docking studies.

Docking and ADME/T analysis of silibinin as a potential inhibitor of EGFR kinase for ovarian cancer therapy

Epidermal Growth Factor Receptor (EGFR) is mostly deregulated and over expressed in ovarian cancer, which is directly linked with STAT3 activation that leads to the accumulation of anti-apoptotoc events and thus, platinum drug resistance occurs. Regarding this, increasing of platinum drug sensitivity by targeting EGFR receptor along with platinum drugs is one of the major strategies in ovarian cancer treatment. In this context, using molecular simulation studies, the present study described the structural and functional properties of silibinin as a potential inhibitor of EGFR tyrosine kinase, and also its metabolic profile had been investigated by SOM prognosis. According to the results, silibinin have shown the significant binding energy by interacting with important residues in the active site. Again, it also processed medium absorption profile with no Fe accessibility. Furthermore, the study is also useful for further clinical based studies and also for the validation of toxicological and pharmacokinetic study.

Self-optimisation of the final stage in the synthesis of EGFR kinase inhibitor AZD9291 using an automated flow reactor

Self-optimising flow reactors combine online analysis with evolutionary feedback algorithms to rapidly achieve optimum conditions.

Abstract B104: Differentiating somatic and germline variants using targeted next-generation sequencing (NGS) of cell-free plasma DNA (cfDNA)

Abstract Introduction: Genomic analysis of cfDNA is emerging as a powerful tool for noninvasive characterization of advanced cancer. Some cfDNA assays are quantitative, allowing analysis of genomic subpopulations. Here, we sought to use quantitative cfDNA analysis to distinguish somatic and germline variants, gaining insight into cancer biology as well as inherited risk without needing paired germline DNA. To do this, we studied EGFR mutations in cfDNA aiming to distinguish those with somatic T790M, generally acquired after resistance to targeted therapy, from those with germline T790M, a rare allele associated with inherited lung cancer risk. Methods &amp; Results: We first explored an institutional cohort of lung cancer patients undergoing cfDNA genotyping using droplet digital PCR (ddPCR). We identified 64 patients positive for T790M and a concurrent EGFR driver mutation by ddPCR. For the vast majority of cases, the mutant allelic fraction (MAF) of T790M was lower than the MAF of the driver mutation, resulting in a T790M/driver ratio of 0.001-1. For two cases, T790M was the predominant mutation, with T790M/driver ratios of 4.2 and 54.6; MAF of T790M was 49% and 53% for these cases. Under an IRB-approved protocol (NCT01754025), we performed sequencing of DNA extracted from PBMCs which confirmed germline T790M in both. We then submitted cfDNA from 3 patients with lung cancer and germline T790M for blinded NGS using the 68-gene Guardant360 assay. In each, NGS identified over 90 coding and non-coding variants. T790M was identified at a high MAF (56%, 50%, 49%) in the range of other recognized SNPs, while an EGFR driver mutation (L858R or L861Q) was identified at a lower level (23%, 4%, 1%) in the range of coding TP53 mutations. Each case was treated with a third-generation EGFR kinase inhibitor targeting T790M. NGS of post-treatment cfDNA demonstrated the expected response in the EGFR driver mutation (0.2%, 0%, 0%) but minimal change in the high MAF T790M (50%, 49%, 49%), consistent with persistent shed of germline DNA on therapy. To broadly screen for germline T790M carriers, we queried a cohort of 1082 lung cancer patients who had undergone NGS of cfDNA using Guardant360. 74 were positive for EGFR T790M (median MAF 3%, range 0.2%-51%). 58 also harbored a second EGFR driver mutation (median MAF 5%), with 23 also having EGFR amplification (median MAF 16%). Four cases were identified with high MAF T790M in the expected range of SNPs (51%, 49%, 49%, 48%), but no driver EGFR mutations were identified at this high MAF. These 4 cases have been referred for germline testing on a prospective study of germline T790M (NCT01754025). Conclusions: We have identified that quantitative cfDNA analysis with NGS can identify germline mutations through differentiation of high MAF germline variants from lower MAF somatic variants. In this cohort, 4 of 74 cases (5%) positive for T790M using NGS of cfDNA were consistent with a germline mutation. This ability to differentiate germline from somatic variants differentiates NGS of cfDNA from NGS of tumor specimens, where these distinctions are challenging without paired germline DNA. Diagnostic labs performing plasma NGS will need to be vigilant to identify these potential germline alterations, and will need strategies for reporting these to providers and patients as appropriate. Citation Format: Geoffrey R. Oxnard, Adrian G. Sacher, Ryan S. Alden, Nora B. Feeney, Jennifer C. Heng, Rebecca J. Nagy, Richard B. Lanman, Cloud P. Paweletz, Pasi A. Janne. Differentiating somatic and germline variants using targeted next-generation sequencing (NGS) of cell-free plasma DNA (cfDNA). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B104.

Large-Scale Computational Screening Identifies First in Class Multitarget Inhibitor of EGFR Kinase and BRD4

Inhibition of cancer-promoting kinases is an established therapeutic strategy for the treatment of many cancers, although resistance to kinase inhibitors is common. One way to overcome resistance is to target orthogonal cancer-promoting pathways. Bromo and Extra-Terminal (BET) domain proteins, which belong to the family of epigenetic readers, have recently emerged as promising therapeutic targets in multiple cancers. The development of multitarget drugs that inhibit kinase and BET proteins therefore may be a promising strategy to overcome tumor resistance and prolong therapeutic efficacy in the clinic. We developed a general computational screening approach to identify novel dual kinase/bromodomain inhibitors from millions of commercially available small molecules. Our method integrated machine learning using big datasets of kinase inhibitors and structure-based drug design. Here we describe the computational methodology, including validation and characterization of our models and their application and integration into a scalable virtual screening pipeline. We screened over 6 million commercially available compounds and selected 24 for testing in BRD4 and EGFR biochemical assays. We identified several novel BRD4 inhibitors, among them a first in class dual EGFR-BRD4 inhibitor. Our studies suggest that this computational screening approach may be broadly applicable for identifying dual kinase/BET inhibitors with potential for treating various cancers.

Synthesis, characterization and biological activity of tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives as epidermal growth factor receptor inhibitors

Based on the molecular docking studies, which were performed to position Erlotinib and the target compounds into the active site of the epidermal growth factor receptor(EGFR) to determine the probable binding model, a novel series of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives as the novel potential EGFR kinase inhibitors was designed and synthesized. The antitumor activity of all the target compounds against human pulmonary carcinoma cell line A549 has been screened. Of all the target compounds, 4-[2-(1-piperidyl)carbonylmethoxylphenthio]- 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7j) demonstrated the most potent antitumor activity. Several of the target compounds exhibited moderate antitumor activity. The preliminary structure-activity relationships of some target compounds were summarized.

Intermittent high-dose treatment with erlotinib enhances therapeutic efficacy in EGFR-mutant lung cancer.

Treatment with EGFR kinase inhibitors improves progression-free survival of patients with EGFR-mutant lung cancer. However, all patients with initial response will eventually acquire resistance and die from tumor recurrence. We found that intermittent high-dose treatment with erlotinib induced apoptosis more potently and improved tumor shrinkage significantly than the established low doses. In mice carrying EGFR-mutant xenografts intermittent high-dose treatment (200 mg/kg every other day) was tolerable and prolonged progression-free survival and reduced the frequency of acquired resistance. Intermittent EGFR-targeted high-dose schedules induce more profound as well as sustained target inhibition and may afford enhanced therapeutic efficacy.