Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) is inevitable in metastatic EGFR-mutant lung cancers. Here, we modeled disease progression using EGFR-mutant human tumor cell lines. Although five of six models displayed alterations already found in humans, one harbored an unexpected secondary NRAS Q61K mutation; resistant cells were sensitive to concurrent EGFR and MEK inhibition but to neither alone. Prompted by this finding and because RAS/RAF/MEK mutations are known mediators of acquired resistance in other solid tumors (colon cancers, gastrointestinal stromal tumors, and melanomas) responsive to targeted therapies, we analyzed the frequency of secondary KRAS/NRAS/ BRAF/MEK1 gene mutations in the largest collection to date of lung cancers with acquired resistance to EGFR TKIs. No recurrent NRAS, KRAS, or MEK1 mutations were found in 212, 195, or 146 patient samples, respectively, but 2 of 195 (1%) were found to have mutations in BRAF (G469A and V600E). Ectopic expression of mutant NRAS or BRAF in drug-sensitive EGFR-mutant cells conferred resistance to EGFR TKIs that was overcome by addition of a MEK inhibitor. Collectively, these positive and negative results provide deeper insight into mechanisms of acquired resistance to EGFR TKIs in lung cancer and inform ongoing clinical trials designed to overcome resistance. In the context of emerging knowledge about mechanisms of acquired resistance to targeted therapies in various cancers, our data highlight the notion that, even though solid tumors share common signaling cascades, mediators of acquired resistance must be elucidated for each disease separately in the context of treatment.

Purpose We sought to determine the frequency and clinical characteristics of patients with lung cancer harboring NRAS mutations. We used preclinical models to identify targeted therapies likely to be of benefit against NRAS mutant lung cancer cells. Patients and Methods We reviewed clinical data from patients whose lung cancers were identified at 6 institutions or reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) to harbor NRAS mutations. 6 NRAS mutant cell lines were screened for sensitivity against inhibitors of multiple kinases (i.e. EGFR, ALK, MET, IGF-1R, BRAF, PI3K and MEK). Results Among 4562 patients with lung cancers tested, NRAS mutations were present in 30 (0.7%; 95% confidence interval, 0.45% to 0.94%); 28 of these had no other driver mutations. 83% had adenocarcinoma histology with no significant differences in gender. While 95% of patients were former or current smokers, smoking-related G:C>T:A transversions were significantly less frequent in NRAS mutated lung tumors compared to KRAS-mutant NSCLCs (NRAS: 13% (4/30), KRAS: 66% (1772/2733), p<0.00000001). 5 of 6 NRAS mutant cell lines were sensitive to the MEK inhibitors, selumetinib and trametinib, but not to other inhibitors tested. Conclusion NRAS mutations define a distinct subset of lung cancers (~1%) with potential sensitivity to MEK inhibitors. While NRAS mutations are more common in current/former smokers, the types of mutations are not those classically associated with smoking.

Lung cancer in never smokers, which has been partially attributed to household solid fuel use (i.e coal), is etiologically and clinically different from lung cancer attributed to tobacco smoking. To explore the spectrum of driver mutations among lung cancer tissues from never smokers, specifically in a population where high lung cancer rates have been attributed to indoor air pollution from domestic coal use, multiplexed assays were used to detect >40 point mutations, insertions, and deletions (EGFR, KRAS, BRAF, HER2, NRAS, PIK3CA, MEK1, AKT1, and PTEN) among the lung tumors of confirmed never smoking females from Xuanwei, China [32 adenocarcinomas (ADCs), 7 squamous cell carcinomas (SCCs), 1 adenosquamous carcinoma (ADSC)]. EGFR mutations were detected in 35% of tumors. 46% of these involved EGFR exon 18 G719X, while 14% were exon 21 L858R mutations. KRAS mutations, all of which were G12C_34G>T, were observed in 15% of tumors. EGFR and KRAS mutations were mutually exclusive, and no mutations were observed in the other tested genes. Most point mutations were transversions and were also found in tumors from patients who used coal in their homes. Our high mutation frequencies in EGFR exon 18 and KRAS and low mutation frequency in EGFR exon 21 are strikingly divergent from those in other smoking and never smoking populations from Asia. Given that our subjects live in a region where coal is typically burned indoors, our findings provide new insights into the pathogenesis of lung cancer among never smoking females exposed to indoor air pollution from coal.

Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the ␣ 2 ␤ 1 integrin (␣ 2 ␤ 1 ) and the glycoprotein VI (GPVI)/FcR␥ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (␣2-CRP) containing the ␣ 2 ␤ 1 -specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to ␣2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was ␣ 2 ␤ 1 -dependent and GPVI/FcR␥-independent as revealed in experiments with ␣ 2 ␤ 1 -or FcR␥-deficient mouse platelets. We further show that suboptimal activation of other platelet G q -linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to ␣2-CRP. The enhanced ␣ 2 ␤ 1 -mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of ␣ IIb ␤ 3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet G q -linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced ␣ 2 ␤ 1 binding to collagens containing GFOGER sites.Platelets are small anuclear cellular elements that play a central role in hemostasis and contribute to vascular pathology. At sites of intravascular injury, platelets are exposed to multiple prothrombotic factors that promote thrombus formation and trigger firm adhesion of platelets to the subendothelial extracellular matrix. Thrombin and fibrillar collagen are two of the more potent stimuli (1, 2).Thrombin is an essential serine protease in the coagulation cascade that ultimately converts fibrinogen to fibrin. Thrombin also has a direct signaling effect on cells through protease-activated receptors (PARs) 2 , which are G protein-coupled receptors (GPCRs) that are activated by enzymatic cleavage of the N terminus of the receptor to produce a tethered ligand (3). Of the four known PAR isoforms, human platelets express PAR1 and PAR4. In platelets, these receptors show different signaling kinetics; PAR1 is activated at low thrombin concentrations with a quick signal shutoff, whereas, PAR4 is activated at higher thrombin concentrations with a slow signal shut off (4 -6). Thrombin-induced signaling has been shown to modulate ligand affinity of the platelet membrane ␣ IIb ...

Introduction: EGFR mutant lung cancers are highly sensitive to first generation EGFR tyrosine kinase inhibitors (TKIs; gefitinib and erlotinib), but resistance eventually develops. In the majority of patients, such acquired resistance (AR) is mediated by a second-site T790M “gatekeeper” mutation. Second generation TKIs (e.g. afatinib, dacomitinib, neratinib) are more potent but have minimal efficacy as single agents in patients with AR. Notably, second generation TKIs still inhibit the wild type receptor which limits dose escalation, and similar to gefitinib/erlotinib, they still select for T790M-mediated resistance in vitro due to selectivity for drug-sensitive mutations (exon 19 deletions and L858R) compared to T790M. In this study, we characterize the inhibitory properties of CNX-2006, a novel irreversible EGFR TKI developed to inhibit specifically T790M. CNX-2006 is the prototype for CO-1686 which is currently in phase I clinical trials for the treatment of EGFR-mutant lung cancer. Methods and Results: CNX-2006 potency was assessed via multiple methods: i) surrogate kinase assays using immunoblotting of lysates from 293 cells transfected with cDNAs encoding various EGFR mutations and treated with drug, ii) growth inhibition assays using various erlotinib-resistant EGFR mutant cell lines or engineered Ba/F3 cells, and iii) H1975 xenografts expressing EGFR(L858R/T790M). CNX-2006 exhibited specificity and potent in vitro and in vivo activity against T790M. The drug also showed activity against uncommon EGFR mutations including G719S, L861Q, an exon 19 insertion mutant (I744-K745insKIPVAI), and T854A, but not an exon 20 insertion (H773-V774HVdup). In an in vitro resistance model, CNX-2006 significantly inhibited the emergence of resistant cells compared with erlotinib (1/24 vs 11/24, p = 0.0013). Chronic exposure to escalating doses of CNX-2006 failed to select for and/or enhance T790M-mediated resistance using PC-9 or HCC827 cells (both harboring exon 19 deletions), or PC-9/ER and HCC827/ER cells with existing T790M and resistance to erlotinib. In PC-9 and HCC827 cells with acquired resistance to CNX-2006, MET amplification or other known mutations in the RAS/MEK/ERK signaling pathway have not been detected. Conclusions: These results demonstrate that the profile of CNX-2006 is different from first and second generation EGFR TKIs. CNX-2006 selectively targets T790M, shows activity against other common and uncommon EGFR mutations, and does not select for resistance mediated by T790M. Additional studies are ongoing to elucidate mechanisms of acquired resistance to CNX-2006. Citation Format: Kadoaki Ohashi, Kenichi Suda, Jing Sun, Yumei Pan, Annette O. Walter, Alex Dubrovskiy, Robert Tjin, Tetsuya Mitsudomi, William Pao. CNX-2006, a novel irreversible epidermal growth factor receptor (EGFR) inhibitor, selectively inhibits EGFR T790M and fails to induce T790M-mediated resistance in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2101A. doi:10.1158/1538-7445.AM2013-2101A

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EGFR mutant lung cancers are highly sensitive to first generation EGFR tyrosine kinase inhibitors (TKIs; gefitinib and erlotinib), but resistance invariably develops. In the majority of patients, such acquired resistance is mediated by a second-site T790M mutation in EGFR. Second generation EGFR TKIs, such as afatinib, have minimal efficacy in patients with acquired resistance; by contrast, combinations of afatinib with cetuximab or panitumumab - anti-EGFR monoclonal antibodies - are synergistic in pre-clinical models, and afatinib/cetuximab has shown a 30% response rate in a phase Ib trial for patients with acquired resistance. Sym004, currently in phase II trials, is a novel mixture of two anti-EGFR monoclonal antibodies binding non-overlapping epitopes in the extracellular domain III of EGFR. The primary mechanism of action of Sym004 is thought to be EGFR cross-linking, internalization and degradation of the EGFR from the cell surface. To determine if the combination of afatinib and Sym004 shows greater efficacy against TKI-resistant EGFR mutant lung cancer than afatinib combined with individual anti-EGFR monoclonal antibodies, we are investigating mechanisms of sensitivity and resistance to afatinib plus Sym004 in EGFR mutant TKI-resistant lung cancer. Similar to cetuximab, Sym004 has minimal effect on the growth of EGFR mutant cells in 2D culture. However, Sym004 induces growth inhibition of TKI-resistant PC-9/BRc1 cells (EGFR exon 19 deletion/T790M) in soft agar and xenograft models, and afatinib plus Sym004 is more effective at inhibiting growth than either drug alone. In immunoblotting studies, the combination induces greater decrease of total EGFR than either drug alone or afatinib plus cetuximab. Using Alexa Fluor 488-labeled Sym004 and confocal microscopy, the drug induces more efficient internalization and degradation of EGFR than cetuximab in H1975 cells (L858R/T790M). Additional confocal studies will be performed in other TKI-resistant EGFR mutant lung cancer cells. We are also assessing whether addition of Sym004 to TKIs can prevent or delay the acquisition of T790M-mediated resistance in vitro. Finally, we are developing cell lines from PC-9/BRc1and HCC827/R1 (exon 19 deletion/T790M) xenografts that develop resistance to either Sym004, cetuximab, or combinations with afatinib. Collectively, these studies could provide insights into the biology of EGFR mutant lung cancers and preclinical rationale for a trial with the combination treatment of afatinib and Sym004 in patients with EGFR mutant lung cancer and acquired resistance to TKIs. Citation Format: Catherine Meador, Kadoaki Ohashi, Yumei Pan, Elisa de Stanchina, Mikkel W. Pedersen, Ivan Horak, Michael Kragh, William Pao. Mechanisms of sensitivity and resistance to Sym004, a novel anti-EGFR antibody mixture, in EGFR mutant lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2013-2438

6038 Background: In the AA population, previous studies have presented conflicting data on the frequency of EGFR mutations (Reinersman JTO 2011;Leidner JCO 2009), while frequencies of other gene mutations and translocations, including anaplastic lymphoma kinase (ALK), have not been described. Methods: 161 archival FFPE tumor specimens from self reported AA patients with any stage NSCLC from 1997-2010 were collected from 3 sites in Tennessee (132 samples) and one site in Michigan (29 samples). Samples were evaluated for known recurrent driver mutations in EGFR, KRAS, BRAF, NRAS, AKT1, PI3KCA, PTEN, HER-2, MEK1 by standard SNaPshot/sizing assays, and translocations in ALK by FISH. Clinical data was collected on 119 patients. Chi-square was used to compare the frequency of mutations in subgroups and Kaplan-Meier and log rank were used to calculate and compare PFS between groups. Results: 5.0% of tumors had EGFR mutations, 14.9% had KRAS mutations, 0.6% had a BRAF, AKT1, PI3KCA, or HER2 mutation, and 0% had NRAS, PTEN, or MEK1 mutations. Of 35 ‘pan-negative’ non-squamous specimens, 0 had ALK translocations. PFS was the same in those with and without KRAS mutation (p=0.74) and showed a trend towards improvement in those with EGFR mutation (p=0.08). The frequency of EGFR mutations was higher in samples from Detroit versus those from Tennessee (17% vs 2.3%, p<0.01), as was the frequency of adenocarcinoma (62% vs 44%, p<0.05). The frequency of EGFR mutations in never smokers was higher in the samples from Detroit versus Tennessee (83% vs 7.1%, p<0.01). Conclusions: In the largest tumor mutational profiling study of NSCLC from AAs to date, EGFR mutations occurred less frequently than would be expected from a North American population. We noted a regional difference, with fewer EGFR mutations in Tennessee than in Michigan, a finding that may have been the result of more adenocarcinoma samples from Michigan. The rates of other mutations and translocations including ALK were low. While lung cancer tumors should continue to undergo routine molecular testing to prioritize therapy, future comprehensive genotyping efforts should focus on identifying novel driver mutations in this population. Funding: 5RC1CA162260 R01CA060691 R01CA87895.