Small-molecule Epidermal Growth Factor Receptor Research Articles

CSN6 controls the proliferation and metastasis of glioblastoma by CHIP-mediated degradation of EGFR.

CSN6, a critical subunit of the constitutive photomorphogenesis 9 (COP9) signalosome (CSN), has received attention as a regulator of the degradation of cancer-related proteins such as p53, c-myc and c-Jun, through the ubiquitin-proteasome system, suggesting its importance in cancerogenesis. However, the biological functions and molecular mechanisms of CSN6 in glioblastoma (GBM) remain poorly understood. Here, we report that GBM tumors overexpressed CSN6 compared with normal brain tissues and that CSN6 promoted GBM cell proliferation, migration, invasion and tumorigenesis. Erlotinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, was used to reveal that the proliferative and metastatic effects of CSN6 on GBM cells were EGFR dependent. We also found that CSN6 positively regulated EGFR stability via reduced levels of EGFR ubiquitination, thereby elevating steady expression of EGFR. In addition, this study is the first description of a novel role for the CSN6-interacting E3 ligase, CHIP (carboxyl terminus of heat-shock protein 70-interacting protein), regulating EGFR ubiquitination in cancer cells. We showed that CSN6 associated with CHIP and led to CHIP destabilization by increasing CHIP self-ubiquitination. Moreover, CSN6 decreased CHIP expression and increased EGFR expression in the tumor samples. Deregulation of this axis promoted GBM cell's proliferation and metastasis. Thus, our study provides insights into the applicability of using the CSN6-CHIP-EGFR axis as a potential therapeutic target in cancer.

Effects of epidermal growth factor receptor kinase inhibition on radiation response in canine osteosarcoma cells.

BackgroundRadiation therapy is a palliative treatment modality for canine osteosarcoma, with transient improvement in analgesia observed in many cases. However there is room for improvement in outcome for these patients. It is possible that the addition of sensitizing agents may increase tumor response to radiation therapy and prolong quality of life. Epidermal growth factor receptor (EGFR) expression has been documented in canine osteosarcoma and higher EGFR levels have been correlated to a worse prognosis. However, effects of EGFR inhibition on radiation responsiveness in canine osteosarcoma have not been previously characterized. This study examined the effects of the small molecule EGFR inhibitor erlotinib on canine osteosarcoma radiation responses, target and downstream protein expression in vitro. Additionally, to assess the potential impact of treatment on tumor angiogenesis, vascular endothelial growth factor (VEGF) levels in conditioned media were measured.ResultsErlotinib as a single agent reduced clonogenic survival in two canine osteosarcoma cell lines and enhanced the impact of radiation in one out of three cell lines investigated. In cell viability assays, erlotinib enhanced radiation effects and demonstrated single agent effects. Erlotinib did not alter total levels of EGFR, nor inhibit downstream protein kinase B (PKB/Akt) activation. On the contrary, erlotinib treatment increased phosphorylated Akt in these osteosarcoma cell lines. VEGF levels in conditioned media increased after erlotinib treatment as a single agent and in combination with radiation in two out of three cell lines investigated. However, VEGF levels decreased with erlotinib treatment in the third cell line.ConclusionsErlotinib treatment promoted modest enhancement of radiation effects in canine osteosarcoma cells, and possessed activity as a single agent in some cell lines, indicating a potential role for EGFR inhibition in the treatment of a subset of osteosarcoma patients. The relative radioresistance of osteosarcoma cells does not appear to be related to EGFR signalling exclusively. Angiogenic responses to radiation and kinase inhibitors are similarly likely to be multifactorial and require further investigation.

EGFR Inhibition Blocks Palmitic Acid-induced inflammation in cardiomyocytes and Prevents Hyperlipidemia-induced Cardiac Injury in Mice.

Obesity is often associated with increased risk of cardiovascular diseases. Previous studies suggest that epidermal growth factor receptor (EGFR) antagonism may be effective for the treatment of angiotensin II-induced cardiac hypertrophy and diabetic cardiomyopathy. This study was performed to demonstrate if EGFR plays a role in the pathogenesis of hyperlipidemia/obesity-related cardiac injuries. The in vivo studies using both wild type (WT) and apolipoprotein E (ApoE) knockout mice fed with high fat diet (HFD) showed the beneficial effects of small-molecule EGFR inhibitors, AG1478 and 542, against obesity-induced myocardial injury. Administration of AG1478 and 542 significantly reduced myocardial inflammation, fibrosis, apoptosis, and dysfunction in both two obese mouse models. In vitro, EGFR signaling was blocked by either siRNA silencing or small-molecule EGFR inhibitors in palmitic acid (PA)-stimulated cardiomyocytes. EGFR inhibition attenuated PA-induced inflammatory response and apoptosis in H9C2 cells. Furthermore, we found that PA-induced EGFR activation was mediated by the upstream TLR4 and c-Src. This study has confirmed the detrimental effect of EGFR activation in the pathogenesis of obesity-induced cardiac inflammatory injuries in experimental mice, and has demonstrated the TLR4/c-Src-mediated mechanisms for PA-induced EGFR activation. Our data suggest that EGFR may be a therapeutic target for obesity-related cardiovascular diseases.

Challenges and Perspectives on the Development of Small-Molecule EGFR Inhibitors against T790M-Mediated Resistance in Non-Small-Cell Lung Cancer.

Because of the development of drug-resistance mutations, particularly the "gatekeeper" threonine(790)-to-methionine(790) (T790M) mutation in the ATP-binding pocket of the epidermal growth factor receptor (EGFR), the current generation of EGFR tyrosine kinase inhibitors lost their clinical efficacy. Recently, a large number of small-molecule inhibitors with striking inhibitory potency against EGFR mutants with the T790M change have been identified. In particular, the inhibitors rociletinib and osimertinib, which can selectively target both sensitizing mutations and the T790M resistance while sparing the wild-type (WT) form of the receptor, have been designated as breakthrough therapies in the treatment of mutant non-small-cell lung cancer (NSCLC) by the U.S. FDA in 2014. We hope that this review on the small-molecule EGFR T790M inhibitors, along with their discovery strategies, will assist in the design of future T790M-containing EGFR inhibitors with high levels of selectivity over WT EGFR, broad kinase selectivity, and desirable physicochemical properties.

Abstract 3747: Identification of NSCLC biomarkers underlying quantifiable drug-induced clonal fitness

Abstract Lung cancer accounts for nearly 20% of all cancer-related deaths worldwide. Subsets of non-small-cell lung cancer (NSCLC; >80% of all lung cancers) harboring activating epidermal growth factor receptor (EGFR) mutations are highly sensitive to erlotinib, a small-molecule EGFR tyrosine kinase inhibitor. However, clinical responses to erlotinib are variable and limited in duration as all tumors eventually relapse. Clonal heterogeneity is thought to be a major cause of relapse, but it has been difficult to quantify in drug-sensitive cancer cell populations. To overcome this obstacle, we quantify clonal fitness via live-cell imaging as a drug-induced proliferation (DIP) rate that incorporates cell division, death, and quiescence into a single metric. This fitness rate varies across cell types from positive (division prevails) to negative (death prevails) and, unlike traditional IC50 measurements, can effectively discriminate intricate degrees of drug sensitivity. Moreover, in monoclonal sublines isolated naïve-to-drug from NSCLC parental cell lines, erlotinib-treated DIP rates calculated in vitro predict the long-term growth rates (250+ days) of in vivo tumor xenografts. Using global proteomics across a panel of monoclonal sublines, we have characterized networks of biomarkers that act as molecular determinants of phenotypic drug fitness. We have identified unsuspected molecular correlates in the EGFR and AKT pathways and are currently investigating whether they can act as lead targets for small molecule inhibition in combination therapies. Our data indicate that a multi-scale systems approach integrating molecular subtyping with quantifiable clonal fitness can yield insights into clonal heterogeneity and lead to novel strategies to overcome tumor relapse. Note: This abstract was not presented at the meeting. Citation Format: Katherine L. Jameson, Peter L. Frick, Darren R. Tyson, Thomas E. Yankeelov, Vito Quaranta. Identification of NSCLC biomarkers underlying quantifiable drug-induced clonal fitness. [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 3747. doi:10.1158/1538-7445.AM2015-3747

Chemical Proteomics Uncovers EPHA2 as a Mechanism of Acquired Resistance to Small Molecule EGFR Kinase Inhibition.

Tyrosine kinase inhibitors (TKIs) have become an important therapeutic option for treating several forms of cancer. Gefitinib, an inhibitor of the epidermal growth factor receptor (EGFR), is in clinical use for treating non-small cell lung cancer (NSCLC) harboring activating EGFR mutations. However, despite high initial response rates, many patients develop resistance to gefitinib. The molecular mechanisms of TKI resistance often remain unclear. Here, we describe a chemical proteomic approach comprising kinase affinity purification (kinobeads) and quantitative mass spectrometry for the identification of kinase inhibitor resistance mechanisms in cancer cells. We identified the previously described amplification of MET and found EPHA2 to be more than 10-fold overexpressed (p < 0.001) in gefitinib-resistant HCC827 cells suggesting a potential role in developing resistance. siRNA-mediated EPHA2 knock-down or treating cells with the multikinase inhibitor dasatinib restored sensitivity to gefitinib. Of all dasatinib targets, EPHA2 exhibited the most drastic effect (p < 0.001). In addition, EPHA2 knockdown or ephrin-A1 treatment of resistant cells decreased FAK phosphorylation and cell migration. These findings confirm EPHA2 as an actionable drug target, provide a rational basis for drug combination approaches, and indicate that chemical proteomics is broadly applicable for the discovery of kinase inhibitor resistance.

Gefitinib: a review of its use in adults with advanced non-small cell lung cancer.

Gefitinib (Iressa®) is a selective small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR TKI) indicated for the treatment of adults with locally advanced or metastatic non-small cell lung cancer (NSCLC) with activating mutations of EGFR tyrosine kinase. Large phase III or IV clinical trials in patients with locally advanced or metastatic NSCLC showed that gefitinib as first- or subsequent-line treatment significantly prolonged progression-free survival (PFS) and improved objective response rates and/or health-related quality of life parameters in patients with activating EGFR mutations and in clinically selected patients (e.g., Asian patients or never-smokers) who are more likely to harbour these mutations. Overall survival did not increase significantly with gefitinib, although post-study treatments may have had a confounding effect on this outcome. Gefitinib was generally well tolerated in these studies, with mild or moderate skin reactions, gastrointestinal disturbances and elevations in liver enzymes among the most common adverse reactions in gefitinib recipients; interstitial lung disease has also been reported in <6% of gefitinib recipients. Compared with chemotherapy, gefitinib as first- or subsequent-line therapy provided similar or greater PFS benefit and was generally associated with fewer haematological adverse events, neurotoxicity, asthenic disorders, as well as grade ≥3 adverse events. Although the position of gefitinib with respect to other EGFR TKIs is not definitively established, current evidence indicates that gefitinib monotherapy is an effective and generally well-tolerated first- or subsequent-line treatment option for patients with NSCLC and activating EGFR mutations who have not received an EGFR TKI previously.

Tyrosine Kinase Inhibitors for EGFR Gene Mutation-Positive Non-SmallCell Lung Cancers: An Update for Recent Advances in Therapeutics

The presence of activating gene mutations in the epidermal growth factor receptor (EGFR) of non-small cell lung cancer (NSCLC) patients is predictive (improved progression-free survival and improved response rate) when treated with small molecule tyrosine kinase inhibitors (TKIs) such as gefitinib, erlotinib and afatinib. The two most common mutations that account for greater than 85% of all EGFR gene mutations, are in-frame deletions in exon 19 (LREA deletions) and point mutations in exon 21 (L858R). Exon 18 mutations occur much less frequently at about 4% of all EGFR gene mutations. Together, deletion19 and L858R gene mutations are present in about 10% of Caucasian patients and 20–40% of Asian patients with NSCLC. T790M gene mutation at exon 20 is associated with acquired resistance to EGFR TKIs. Early studies showed that activating EGFR gene mutations are most common in patients with adenocarcinoma histology, women, never smokers and those of Asian ethnicity. A recent multi-center phase III trial suggested that frontline EGFR TKI therapy with afatinib is associated with improved progressionfree survival compared to chemotherapy regardless of race. Moreover, guidelines suggest EGFR testing should be conducted in all patients with lung adenocarcinoma or mixed lung cancers with an adenocarcinoma component), regardless of characteristics such as smoking status, gender, or race. The success of targeted therapies in NSCLC patients has changed the treatment paradigm in metastatic NSCLC. However, despite a durable response of greater than a year, resistance to EGFR TKIs inevitably occurs. This mini-review describes the clinically significant EGFR gene mutations and the efficacy of small molecule EGFR TKIs as targeted therapies for these gene mutations. Therapeutic strategies to overcome resistance, including selected emerging and novel therapies are discussed.

Abstract 1184: Modeling mechanisms of resistance of epidermal growth factor receptor (EGFR) mutations to targeted drugs through patient-derived xenografts (PDX) of non-small cell lung cancer (NSCLC)

Abstract Background: Resistance to small molecule EGFR tyrosine kinase inhibitors (TKIs) is seen in some NSCLC patients with activating EGFR mutations. We evaluated in vivo PDX models for their utility in studying EGFR-targeted drug resistance mechanisms. Methods: Surgically resected early stage NSCLC tumors were implanted into non-obese diabetic severe combined immune deficient (NOD-SCID) mice. Tumors were passaged and expanded in new mouse hosts once the humane endpoint of 1.5 cm maximum diameter was reached. EGFR TKI treatment was initiated at an average tumor volume of 150mm3. Treatments included daily oral gavage with first and second generation EGFR TKIs, and with weekly intraperitoneally administered cetuximab. Results: Of the 55 NSCLC tumors with EGFR activating mutations, only 6 engrafted (11%) and could be propagated beyond the first passage, and 4 have been studied for their responsiveness to EGFR-targeted agents. Model 148, developed from a patient who received pre-operative erlotinib, showed intrinsic pan-resistance to all EGFR-targeted therapies despite having an L858R mutation. The corresponding patient did not respond to erlotinib, relapsed after surgery and did not receive additional TKI therapy. Model 137, with an exon19 E746-A750 deletion, recapitulated the patient's response to gefitinib at relapse; this model was sensitive to first and second generation EGFR TKIs. Model 192 also has the exon19 E746-A750 deletion, however it did not recapitulate the patient's observed sensitivity to erlotinib. Selection for a MET amplified population during engraftment may be the cause for the disparate drug sensitivities. Model 164 has a double exon19 L747-T751 deletion and T790M EGFR mutation. Neither patient nor xenograft responded to erlotinib; the xenograft responded to cetuximab. Resistance developed over time to a second generation EGFR TKI; this resistant phenotype was not stable as each subsequent passage of the ‘resistant’ tumor exhibited the same initial response pattern. Two potential mechanisms for this transient sensitivity are currently being investigated: epigenetic mechanisms and intratumoural mutational heterogeneity. Conclusions: PDX models may provide important insight into biomarkers and mechanisms of resistance to targeted therapies, and provide a means to test novel treatment strategies to improve future treatment efficacies. Citation Format: Erin L. Stewart, Celine Mascaux, Shingo Shakashita, Devang Panchal, Dennis Wang, Ming Li, Nhu-An Pham, Natasha Leighl, Geoffrey Liu, Frances A. Shepherd, Ming-Sound Tsao. Modeling mechanisms of resistance of epidermal growth factor receptor (EGFR) mutations to targeted drugs through patient-derived xenografts (PDX) of non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1184. doi:10.1158/1538-7445.AM2014-1184

Toward precision medicine with next-generation EGFR inhibitors in non-small-cell lung cancer.

The use of genomics to discover novel targets and biomarkers has placed the field of oncology at the forefront of precision medicine. First-generation epidermal growth factor receptor (EGFR) inhibitors have transformed the therapeutic landscape of EGFR mutant non-small-cell lung carcinoma through the genetic stratification of tumors from patients with this disease. Somatic EGFR mutations in lung adenocarcinoma are now well established as predictive biomarkers of response and resistance to small-molecule EGFR inhibitors. Despite early patient benefit, primary resistance and subsequent tumor progression to first-generation EGFR inhibitors are seen in 10%–30% of patients with EGFR mutant non-small-cell lung carcinoma. Acquired drug resistance is also inevitable, with patients developing disease progression after only 10–13 months of antitumor therapy. This review details strategies pursued in circumventing T790M-mediated drug resistance to EGFR inhibitors, which is the most common mechanism of acquired resistance, and focuses on the clinical development of second-generation EGFR inhibitors, exemplified by afatinib (BIBW2992). We discuss the rationale, mechanism of action, clinical efficacy, and toxicity profile of afatinib, including the LUX-Lung studies. We also discuss the emergence of third-generation irreversible mutant-selective inhibitors of EGFR and envision the future management of EGFR mutant lung adenocarcinoma.

Dual EGFR inhibition in combination with anti-VEGF treatment in colorectal cancer.

Preclinical studies demonstrate that epidermal growth factor receptor (EGFR) signals through both kinase-dependent and independent pathways and that combining a small-molecule EGFR inhibitor, EGFR antibody, and/or anti-angiogenic agent is synergistic. We conducted a dose-escalation, phase I study combining erlotinib, cetuximab, and bevacizumab. The subset of patients with metastatic colorectal cancer was analyzed for safety and antitumor activity. Forty-one patients with heavily pretreated metastatic colorectal cancer received treatment on a range of dose levels. The most common treatment-related grade ≥2 adverse events were rash (68%), hypomagnesemia (37%), and fatigue (15%). Thirty of 34 patients (88%) treated at the full FDA-approved doses of all three drugs tolerated treatment without drug-related dose-limiting effects. Eleven patients (27%) achieved stable disease (SD) ≥6 months and three (7%) achieved a partial response (PR) (total SD>6 months/PR= 14 (34%)). Of the 14 patients with SD≥6 months/PR, eight (57%) had received prior sequential bevacizumab and cetuximab, two (5%) had received bevacizumab and cetuximab concurrently, and four (29%) had received prior bevacizumab but not cetuximab or erlotinib (though three had received prior panitumumab). The combination of bevacizumab, cetuximab, and erlotinib was well tolerated and demonstrated antitumor activity in heavily pretreated patients with metastatic colorectal cancer.

Sensitivity of human granulosa cell tumor cells to epidermal growth factor receptor inhibition

Epidermal growth factor receptor (EGFR) is implicated in the progression of many human cancers, but its significance in ovarian granulosa cell tumor (GCT) pathobiology remains poorly understood. We assessed the EGFR gene copy number, surveyed the mRNA and protein expression patterns of EGFR in 90 adult GCTs, and assessed the in vitro sensitivity of GCT cells to EGFR inhibition. Low-level amplification of EGFR gene was observed in five GCTs and high-level amplification in one sample. EGFR mRNA was robustly expressed in GCTs. Most tumors expressed both unphosphorylated and phosphorylated EGFR protein, but the protein expression did not correlate with clinical parameters, including the risk of recurrence. Small-molecule EGFR inhibitors reduced the EGF-induced activation of EGFR and its downstream signaling molecules at nanomolar doses, but cell viability was reduced, and caspase-3/7 was activated in GCT cells only at micromolar doses. Based on the present results, EGFR is active and abundantly expressed in the majority of GCTs, but probably has only minor contribution to GCT cell growth. Given the high doses of EGFR inhibitors required to reduce GCT cell viability in vitro, they are not likely to be effective for GCT treatment as single agents; they should rather be tested as part of combination therapies for these malignancies.

Recovery from paraplegia with administration of erlotinib in a patient with lung adenocarcinoma.

Recovery from paraplegia with administration of erlotinib in a patient with lung adenocarcinoma.

Abstract C111: In vitro characterization of TAS-121, a novel, highly potent, and mutant-specific EGFR-TKI.

Abstract Background: The active mutation of epidermal growth factor receptor (EGFR), L858R or deletion in exon19, are present in patients with NSCLC and responsive to EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib. Over time, most tumors develop resistance to these EGFR inhibitors by the acquiring of drug-resistant mutations including gatekeeper T790M mutation (more than 50% cases). Severe skin rash and diarrhea caused by wild-type EGFR (wtEGFR) inhibition limit EGFR TKI treatment. Therefore targeting mutant EGFR (mtEGFR) but not wtEGFR represents an attractive therapeutic strategy. In this report, we identify a small molecule EGFR inhibitor, TAS-121, that targets mtEGFR but not wtEGFR and its biological characterization in vitro. Methods: In vitro enzyme inhibition activity of TAS-121 for EGFR and its mutants was determined by using purified EGFRs and peptide substrates. Cellular phosphorylation of EGFR was assayed by immunoblot analysis. For growth inhibition assay, cells were treated with TAS-121 for 3 days, and living cells were determined by using CellTiter GloTM which measures cellular ATP. Apoptosis assay was conducted by using Caspase-GloTM and Cell Counting Kit. Results: TAS-121 was found to be a more potent mutant selective EGFR-TKI based on bioavailability characteristics compared to our proto-type compound, TAS-2913. TAS-121 inhibits kinase activity of EGFR mutants harboring T790M in a sub-nano molar range and much more potently than that of wtEGFR. TAS-121 inhibits auto-phosphorylation of EGFR and intercellular signaling pathways in NCI-H1975 (EGFR T790M / L858R) and HCC827 (EGFR del E746-A750). In a cell proliferation assay, TAS-121 also suppresses growth of NCI-H1975 and HCC827 more potently than that of a normal cell which grows in an EGF-dependent manner. Therefore, the wtEGFR / mtEGFR inhibition ratio of TAS-121 appears to be higher than those of other EGFR TKIs. Furthermore, TAS-121 induces apoptosis in NCI-H1975 but erlotinib could not. Conclusion: These results suggest that TAS-121, a potent mutant-selective EGFR inhibitor, may represent a new therapeutic strategy in the treatment of NSCLC resistant to currently available EGFR/TKIs. Further pre-clinical evaluations and development are ongoing. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C111. Citation Format: Kimihiro Ito, Kazutaka Miyadera, Yoshimi Aoyagi, Masanori Kato, Kazuhiko Yonekura, Yoshikazu Iwasawa, Teruhiro Utsugi. In vitro characterization of TAS-121, a novel, highly potent, and mutant-specific EGFR-TKI. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C111.

Abstract A148: Modeling patient responses to targeted therapy with rAAV mediated gene editing.

Abstract Successful drug development in oncology requires a deeper understanding of the functional consequences of the diverse genetic changes observed in human cancers. For example, responses to epidermal growth factor receptor (EGFR) inhibitors are observed in patients whose tumors express EGFR alleles with activating mutations, rather than in tumors overexpressing EGFR. Furthermore, antibodies against EGFR are ineffective in tumors bearing certain activating alleles of KRAS. Horizon Discovery has used its proprietary rAAV gene engineering technology to generate isogenic cell lines covering a range of mutations commonly found in cancer patients. Use of a non-tumorigenic ‘clean’ cell line background such as MCF10A allows specific evaluation of the mutations without any confounding factors due to the presence of other genetic alterations. Mutations introduced into cancer cell line backgrounds allow the contextual evaluation of a cancer related gene. Here we describe the use of isogenic cell line panels as powerful tools for investigating sensitivity and resistance markers to cancer therapeutics. Some 50% of human tumors exhibit p53 loss or inactivation. To investigate how p53 loss in combination with other common cancer-driving mutations may influence therapeutic responses, we have generated a suite of MCF10A isogenic cell lines covering some of the major cancer genotypes, either in isolation or on a TP53 (-/-) background. These genotypes include EGFR (delE746-A750/+), EGFR (L858R/+), KRAS (G12V/+), BRAF (V600E/+), BRAF (V600K/+) and PIK3CA (H1047R/+). Thus, we have been able to investigate the interaction effects of discrete mutations in molecularly defined, but more tumor-like cell models. One data highlight arose from the profiling of the EGFR mutant panel using small molecule EGFR inhibitors; in isolation, the introduction of common activating EGFR mutations L858R or deletion of E746-A750 led to increased sensitivity, recapitulating clinical findings. However, combining EGFR mutation with loss of p53 further enhanced the cell response. Through systematic profiling of this panel to targeted therapeutic agents such as gefinitib, selumetinib, vemurafenib, and pictilisib, we have identified interesting differential sensitivities, which can be directly attributable to introduction of a given mutation. Results such as these can enable better patient stratification for anticancer agents, and allow incorporation of molecular markers into clinical trial design for personalised therapeutic regimens. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A148. Citation Format: Annette S. Little, Jessica Hunt, David Hughes, Ruth Feltell, Daniel Gitterman, Rachel Leah, Holly Astley, Ramu Mangena, Kyla Grimshaw, Christopher Torrance. Modeling patient responses to targeted therapy with rAAV mediated gene editing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A148.

Abstract C202: Probing EGFR, HER2 and c-Met protein-protein interactions using an antibody array.

Abstract The receptor tyrosine kinase (RTK) family comprises important disease drivers and validated drug targets. Cooperation between certain RTKs is often achieved through physical interactions and is thought be a contributing factor to some cases of tumor resistance to targeted therapies. Epidermal Growth Factor Receptor (EGFR), HER2/ErbB2, and c-Met are prominent examples of disease driver RTKs that cooperate and form heterodimers. Current methods to detect RTK protein-protein interactions lack proper throughput, are not quantitative, or rely on highly engineered systems and cumbersome workflows. We have developed a planar surface antibody array-based sandwich assay that enables the detection and relative quantification of interactions between EGFR, HER2, and c-Met. The antibody-based assay revealed co-existing combinatorial receptor interactions that were unique to each cell type tested. The assay also pointed to an existence of distinct topological configurations of EGFR-c-Met and EGFR-HER2 heterodimers, unveiling previously not described nuances of these interactions. Lastly, the array-based assay allowed monitoring the disruption of receptor complexes using small molecule inhibitors and revealed differential sensitivity of EGFR-HER2 and EGFR-c-Met complexes to a small molecule EGFR kinase inhibitor, gefitinib. The antibody array sandwich assay described here allows utilizing larger panel of affinity reagents and can be applied to broader range of cases with various topological requirements, thus offering an advantage over technologies that have strict proximity requirement. This novel protein interaction array enables parallel detection of RTK protein-protein interactions, allowing a more systematic monitoring of response to activators or inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C202. Citation Format: Jacob Knowles, Stephen Capodilupo, Richard Noring, Roberto Campos-González, Julio Herrera, Zev Gechtman. Probing EGFR, HER2 and c-Met protein-protein interactions using an antibody array. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C202.

Adjuvant molecularly targeted therapy-epidermal growth factor tyrosine kinase inhibition and beyond.

In stage IV non-small cell lung cancer (NSCLC), DNA molecular testing for mutations in epidermal growth factor receptor ( EGFR ) and gene rearrangements of anaplastic lymphoma kinase ( ALK ) has become the new standard of care. This is based on the unprecedented efficacy of small molecule EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib against EGFR mutant NSCLC (1,2), and the ALK TKI crizotinib against ALK positive NSCLC (3). While these highly active drugs should conceptually be effective as a component of the curative treatment of earlier stage NSCLC, the presently available evidence is minimal as pivotal studies are either underway or still in development. This article reviews current evidence about the use of adjuvant therapy for molecular targets in NSCLC, in particular regarding the use of EGFR TKIs in the treatment of early stage NSCLC harboring EGFR mutations.

SOX2 plays a critical role in EGFR-mediated self-renewal of human prostate cancer stem-like cells

SOX2 is an essential transcription factor for stem cells and plays a role in tumorigenesis, however its role in prostate cancer stem cells (PCSCs) remains unclear. We report here a significant upregulation of SOX2 at both mRNA and protein levels in DU145 PCSCs propagated as suspension spheres in vitro. The expression of SOX2 in DU145 PCSCs is positively regulated by epidermal growth factor receptor (EGFR) signaling. Activation of EGFR signaling, following the addition of epidermal growth factor (EGF) or ectopic expression of a constitutively-active EGFR mutant (EGFRvIII), increased SOX2 expression and the self-renewal of DU145 PCSCs. Conversely, a small molecule EGFR inhibitor (AG1478) blocked EGFR activation, reduced SOX2 expression and inhibited PCSC self-renewal activity, implicating SOX2 in mediating EGFR-dependent self-renewal of PCSCs. In line with this notion, ectopic SOX2 expression enhanced EGF-induced self-renewal of DU145 PCSCs, while SOX2 knockdown reduced PCSC self-renewal with EGF treatment no longer capable of enhancing their propagation. Furthermore, SOX2 knockdown reduced the capacity of DU145 PCSCs to grow under anchorage-independent conditions. Finally, DU145 PCSCs generated xenograft tumors more aggressively with elevated levels of SOX2 expression compared to xenograft tumors derived from non-PCSCs. Collectively, we provide evidence that SOX2 plays a critical role in EGFR-mediated self-renewal of DU145 PCSCs.

Neoadjuvant erlotinib, erlotinib-sulindac, or placebo: A randomized, double-blind biomarker modulation study in operable head and neck squamous cell carcinoma (HNSCC).

6051 Background: The epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) pathways are upregulated in HNSCC. Crosstalk potentiates growth, proliferation and invasion. Preclinical models show synergistic anti-tumor effects from dual blockade. We evaluated biomarker modulation in paired tumor specimens from a 3-arm phase 0 trial of erlotinib, a small molecule EGFR inhibitor; erlotinib + sulindac, a non-selective COX inhibitor; vs. placebo. Methods: Patients (pts) with untreated stage II-IVb HNSCC were randomized 5:5:3 to neoadjuvant erlotinib 150 mg daily (Arm 1, n = 19), erlotinib + sulindac 150 mg twice daily (Arm 2, n = 16), or placebo (n = 12). Pts were treated for 7-14 days. Tumor specimens were collected pre- and post-treatment. The primary endpoint was change in Ki-67 proliferative index. We hypothesized an ordering in Ki-67 down-modulation, with Arm 2 &gt; Arm 1 &gt; placebo. A Jonckheere-Terpstra test evaluated trend; a Wilcoxon test was applied to pairwise contrasts. We prepared tissue microarrays for IHC, to explore pharmacodynamic modulation of 21 EGFR and COX-2 pathway constituents including EGFR, Src, STAT3, Akt, EP2 and EP4. Results: From Dec 2005 – Dec 2008, 48 pts enrolled; 28 tumor pairs were evaluable for biomarker modulation. Class toxicities of EGFR inhibition, including acneiform rash and diarrhea, were observed on Arms 1 and 2. Compared to placebo, Ki-67 was reduced by erlotinib (p = 0.0005) or by erlotinib-sulindac (p = 0.0001). There was a trend in ordering of Ki-67 down-modulation, with greater effect from the addition of sulindac (exact Jonckeheere-Terpstra, Arm 1 vs. 2, p = 0.003). Among 21 protein candidates tested for association with Ki-67, only low baseline p-Src correlated with greater Ki-67 reduction in both erlotinib groups (R2 = 0.312, p = 0.024). Conclusions: Relative to placebo, brief neoadjuvant treatment with erlotinib or erlotinib-sulindac significantly decreased the proliferative index in operable HNSCC. Sulindac enhanced Ki-67 down-modulation. Efficacy studies of dual EGFR-COX inhibition are justified. Baseline p-Src warrants further study as a resistance biomarker for anti-EGFR therapy. Clinical trial information: NCT01515137.

Abstract 4128: A novel approach to the measurement of tyrosine phosphorylation dynamics in intact single cells using capillary electrophoresis.

Abstract Receptor tyrosine kinases (RTKs) are key components of physiological and pathological cell signaling, and are normally subject to tight regulation by protein tyrosine phosphatases (PTPs). Irregularities on either side of this equilibrium can lead to inflammation, metabolic disease, and cancer. While many established and emerging techniques have been used to characterize these signaling pathways, few approaches are well adapted to the analysis of single primary cells, and fewer still measure enzyme activity directly. We report a new method for measuring the balance between RTK and PTP activity using fluorescent peptide substrates in conjunction with capillary electrophoresis. Briefly, live cells are visualized on an inverted microscope, microinjected with a fluorescent peptide substrate which is acted upon by RTKs and/or PTPs. After the desired time interval, a single cell is lysed using a microsecond laser pulse and cell contents are aspirated into a fused-silica capillary where electrophoresis is performed and reporter species are separated and quantified based on phosphorylation status. We have applied this technique to the characterization of PTP inhibition in single A431 cells by several components of diesel exhaust particles, an important component of air pollution linked to cancer. We have also adapted this system to measure the effects of small-molecule epidermal growth factor receptor (EGFR) inhibitors on RTK activity in tumor cell lines as well as tumor xenograft models, also at the single cell level. This approach provides a unique set of advantages over alternative techniques and is a valuable tool for the study of cell signaling in cancer. The integrated microscopy permits selection of cells for analysis based on morphology, viability, or vital staining, an important consideration for analysis of heterogeneous samples such as tumor specimens, especially when total sample size is small. High-sensitivity detection allows chemical cytometry data from individual cells to be gathered and the presence of phenotypic subpopulations is not lost by observing only the average behavior of a large population. Additionally, peptide substrate reporters can be tuned for enzyme specificity, and the analytical separation allows multiple reporter pairs to be separated and detected in a single experiment, permitting simultaneous analysis of multiple enzymes. The introduction of reporter peptides by microinjection preserves cellular architecture, while being equally applicable to immortalized and primary cells since no genetic manipulation is necessary. This array of advantages makes our approach uniquely well suited to the study of RTK and PTP signaling in both cultured tumor cell lines and patient specimens and a valuable new tool for cancer research. Citation Format: Ryan M. Phillips, David S. Lawrence, Christopher E. Sims, Nancy L. Allbritton. A novel approach to the measurement of tyrosine phosphorylation dynamics in intact single cells using capillary electrophoresis. [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 4128. doi:10.1158/1538-7445.AM2013-4128