Receptor Tyrosine Kinase Inhibitor Resistance Research Articles

An observational study of the epidermal growth factor receptor-tyrosine kinase inhibitor resistance mechanism in epidermal growth factor receptor gene mutation-positive non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation show a high response to EGFR-tyrosine kinase inhibitor (EGFR-TKI). Clinically, EGFR-positive NSCLC acquires several resistance mechanisms during EGFR-TKI treatment, such as the emergence of a secondary mutation (T790M), MET gene amplification, and transformation to small cell lung cancer. However, the mechanism of resistance to afatinib, a second-generation EGFR-TKI, remains unclear. In this study, we prospectively investigate the mechanism of resistance to afatinib using proteomic analyses.In total, 35 EGFR-positive NSCLC patients of both sexes and ≥20 years old will be included. NSCLC patients with major obstacles in major organs, such as bone marrow, heart, lung, liver, and kidney, will be excluded. Eligible patients will be administered afatinib or gefitinib until disease progression and proteomic analysis will be performed with biopsy samples before treatment and at disease progression.The primary outcome is to detect the potential predictive anomalies in proteins that can be candidates for the resistance factor of afatinib. The secondary outcome is to detect gene and protein abnormalities affecting progression-free survival, response rate, and rate of disease control in afatinib therapy.The protocol was approved by the institutional review boards of Kyoto Prefectural University of Medicine and all the participating hospitals. Written informed consent was obtained from all patients before registration, in accordance with the Declaration of Helsinki. The results of the study will be disseminated via publications in peer-reviewed journals.Trial registration number is UMIN000031013.

MEF2C promotes gefitinib resistance in hepatic cancer cells through regulating MIG6 transcription.

Mitogen-inducible gene 6 ( MIG6) holds a special position in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance. As MIG6 regulates the activity of EGFR signal pathway negatively, high level of MIG6 can increase the EGFR TKI resistance of cancer cells, and limit the therapeutic action of EGFR TKI, such as gefitinib or erlotinib. Therefore, better understanding of the molecular mechanisms underlying the regulation of EGFR TKI resistance holds great value in cancer therapy. In our study, we mainly explored the function of transcription activator, myocyte enhancer factor 2C (MEF2C), on MIG6 expression as well as gefitinib-resistant ability of hepatic cancer cells. Our results indicated that both MEF2C and MIG6 could be upregulated in gefitinib-resistant cancer tissues and cancer cell lines compared with gefitinib-sensitive ones. Chromatin immunoprecipitation assay and dual luciferase assay showed that MEF2C could bind to the MEF2C element in the promoter sequence of MIG6 and promote the transcription of MIG6. This effect increased the gefitinib-resistant ability of cancer cells. Therefore, MEF2C knockdown inhibited the gefitinib resistance and limited the proliferation of hepatic cancer cells in vitro and in vivo, while overexpression of MEF2C showed opposite effect on cancer cell proliferation. Our study provides novel insight into the regulation mechanism of MIG6 and suggests potential implications for the therapeutic strategies of gefitinib resistance through inhibiting MEF2C in hepatic cancer cells.

Novel approaches against epidermal growth factor receptor tyrosine kinase inhibitor resistance.

BackgroundThe identification and characterization of molecular biomarkers has helped to revolutionize non-small-cell lung cancer (NSCLC) management, as it transitions from target-focused to patient-based treatment, centered on the evolving genomic profile of the individual. Determination of epidermal growth factor receptor (EGFR) mutation status represents a critical step in the diagnostic process. The recent emergence of acquired resistance to “third-generation” EGFR tyrosine kinase inhibitors (TKIs) via multiple mechanisms serves to illustrate the important influence of tumor heterogeneity on prognostic outcomes in patients with NSCLC.DesignThis literature review examines the emergence of TKI resistance and the course of disease progression and, consequently, the clinical decision-making process in NSCLC.ResultsMolecular markers of acquired resistance, of which T790M and HER2 or MET amplifications are the most common, help to guide ongoing treatment past the point of progression. Although tissue biopsy techniques remain the gold standard, the emergence of liquid biopsies and advances in analytical techniques may eventually allow “real-time” monitoring of tumor evolution and, in this way, help to optimize targeted treatment approaches.ConclusionsThe influence of inter- and intra-tumor heterogeneity on resistance mechanisms should be considered when treating patients using resistance-specific therapies. New tools are necessary to analyze changes in heterogeneity and clonal composition during drug treatment. The refinement and standardization of diagnostic procedures and increased accessibility to technology will ultimately help in personalizing the management of NSCLC.

Selective gene amplification to detect the T790M mutation in plasma from patients with advanced non-small cell lung cancer (NSCLC) who have developed epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance.

The epidermal growth factor receptor (EGFR) T790M mutation is associated with resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). However, tissues for the genotyping of the EGFR T790M mutation can be difficult to obtain in a clinical setting. The aims of this study were to evaluate a blood-based, non-invasive approach to detecting the EGFR T790M mutation in advanced NSCLC patients using the PointMan™ EGFR DNA enrichment kit, which is a novel method for the selective amplification of specific genotype sequences. Blood samples were collected from NSCLC patients who had activating EGFR mutations and who were resistant to EGFR-TKI treatment. Using cell-free DNA (cfDNA) from plasma, EGFR T790M mutations were amplified using the PointMan™ enrichment kit, and all the reaction products were confirmed using direct sequencing. The concentrations of plasma DNA were then determined using quantitative real-time PCR. Nineteen patients were enrolled, and 12 patients (63.2%) were found to contain EGFR T790M mutations in their cfDNA, as detected by the kit. T790M mutations were detected in tumor tissues in 12 cases, and 11 of these cases (91.7%) also exhibited the T790M mutation in cfDNA samples. The concentrations of cfDNA were similar between patients with the T790M mutation and those without the mutation. The PointMan™ kit provides a useful method for determining the EGFR T790M mutation status in cfDNA.

Correlation between expression of C-met and epidermal growth factor receptor-tyrosine kinase inhibitor resistance in lung adenocarcinoma

Objective To detect C-met protein expression and gene amplification in lung adenocarcinoma, and to analyze their relationship with epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance and prognosis. Methods A total of 120 cases of lung adenocarcinoma diagnosed in Shanxi Provincial Cancer Hospital from January 2011 to May 2013 were selected. The expressions of C-met protein and C-met gene amplification were conducted by immunohistochemistry (IHC) method and fluorescence in situ hybridization (FISH), and all patients were followed up. The relationship between the expression of C-met protein and gene amplification with clinicopathological features and EGFR-TKI resistance and prognosis were analyzed. Results The high expression of C-met protein and gene amplification in 120 tissues were 17.5 % (21/120), 10.83 % (13/120). Of the 80 patients treated with EGFR-TKI, the incidence of C-met protein high expression was 30.43 % (14/46) in patients with drug resistance, which was significantly higher than that in patients without drug resistance (11.76 %, 4/34), the difference was statistically significant (χ2 = 3.908, P= 0.048). The rate of C-met gene amplification was 19.57 % (9/46) in patients with drug resistance, which was significantly higher than that in patients without drug resistance (2.94%, 1/34) the difference was statistically significant (P= 0.038). The expression of C-met protein in 46 patients with drug resistance was positively correlated with gene amplification (r= 0.388, P= 0.008), but in 40 patients without TKI, the expression of C-met protein was not correlated with gene amplification (r= 0.279, P= 0.081). The high expression of C-met protein was correlated with age, pathological grade and clinical stage (all P < 0.05), while C-met gene amplification was related to clinical stage (P= 0.036). Cox regression analysis suggested that C-met gene amplification was an independent prognostic factor (P= 0.034). Conclusions C-met protein expression and gene amplification are risk factors for EGFR-TKI resistance. C-met gene amplification suggests poor prognosis, and can be used as an independent factor for prognostic evaluation. Key words: Lung neoplasms; Proto-oncogene proteins c-met; Receptor, epidermal growth factor; Tyrosine kinase inhibitor; Prognosis; Drug resistance, neoplasm

Targeting EHMT2 reverses EGFR-TKI resistance in NSCLC by epigenetically regulating the PTEN/AKT signaling pathway

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Epigenetic alterations have been shown to be involved in NSCLC oncogenesis; however, their function in EGFR-TKI resistance remains uncharacterized. Here, we found that an EHMT2 inhibitor, UNC0638, can significantly inhibit cell growth and induce apoptosis in EGFR-TKI-resistant NSCLC cells. Additionally, we also found that EHMT2 expression and enzymatic activity levels were elevated in EGFR-TKI-resistant NSCLC cells. Moreover, we determined that genetic or pharmacological inhibition of EHMT2 expression enhanced TKI sensitivity and suppressed migration and tumor sphere formation in EGFR-TKI-resistant NSCLC cells. Further investigation revealed that EHMT2 contributed to PTEN transcriptional repression and thus facilitated AKT pathway activation. The negative relationship between EHMT2 and PTEN was confirmed by our clinical study. Furthermore, we determined that combination treatment with the EHMT2 inhibitor and Erlotinib resulted in enhanced antitumor effects in a preclinical EGFR-TKI-resistance model. We also found that high EHMT2 expression along with low PTEN expression can predict poor overall survival in patients with NSCLC. In summary, our findings showed that EHMT2 facilitated EGFR-TKI resistance by regulating the PTEN/AKT pathway in NSCLC cells, suggesting that EHMT2 may be a target in the clinical treatment of EGFR-TKI-resistant NSCLC.

Clinical strategies for acquired epidermal growth factor receptor tyrosine kinase inhibitor resistance in non-small-cell lung cancer patients.

Epidermal growth factor receptor (EGFR) mutations (EGFRm+) occur in 10–35% of non-small-cell lung cancer (NSCLC) cases and confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs). EGFR TKIs are standard treatments for NSCLC patients harboring EGFR exon 19 deletions or exon 21 L858R point mutations. Despite initial benefit, most patients develop drug resistance, posing a challenge to oncologists. The secondary T790M point mutation in EGFR exon 20 contributes to approximately 60% of resistance cases. Optimum strategies for overcoming acquired EGFR TKI resistance are not clearly defined, although current common practice is to switch to platinum-based chemotherapy following resistance onset. While the second-generation EGFR TKIs, including afatinib, dacomitinib, and neratinib, exhibit promising preclinical activity against T790M mutants, dose-limiting toxicities in patients have limited clinical success. However, third generation EGFR TKIs appear able to overcome this mutation. Other treatment options aimed at EGFR TKI resistance include use of an EGFR TKI beyond progression, and chemotherapy plus an EGFR TKI. This review focuses on improved anticancer agents and therapy options for NSCLC patients with acquired EGFR TKI resistance.

Abstract 94: Association of xCT overexpression with RTKI resistance and metastases in clear cell renal cell carcinoma

Abstract Background: Cystine/glutamate exchanger xCT is a catalytic component of system xc- involved in transport of ‘conditionally indispensable’ amino acid cystine. Cystine transport is a rate limiting step for the synthesis of glutathione, a major intracellular redox regulator. Recently, we and other groups reported the overexpression of xCT and its association with drug resistance in several human cancers including bladder, glioma, breast, and colon. There are no studies to show the xCT expression in clear cell renal cell carcinoma (ccRCC) and its association with tyrosine kinase inhibitors resistance and metastasis. In the current study we have evaluated xCT expression in human ccRCC tumors arranged in tissue microarray (TMA) and determined its role in receptor tyrosine kinase inhibitor (RTKI) resistance and metastases using the patient derived tumor xenografts (PDX) models and TKI resistance ccRCC cells. Methods: Human Renal cell carcinoma tumor nephrectomy specimens arranged in tissue microarray (TMA) were used to determine xCT expression by immunohistochemistry. Patient derived tumor xenografts (PDX) of primary, metastasis, and sunitinib resistance were used to determine the role of xCT in RCC. To understand the molecular alterations associated with RTKI resistance, we have generated sunitinib resistance 786 OR ccRCC cells and performed RNAseq analysis. To determine the xCT inhibition effect on ccRCC tumor metastases, sulfasalazine, an inhibitor of xCT was used to treat metastatic ccRCC tumor xenografts transplanted in SCID mice. Results: Immunohistochemical evaluation of xCT in RCC TMA revealed that 70 % (19 out of 27) of the tumors express different levels of xCT. Association with tumor response to RTKI will be presented. RTKI less responsive PDX RP-R-02 was developed using the dose escalation treatment strategy and was found to have an upregulation of xCT when the tumors become less responsive to sunitinib. RNAseq analysis revealed differential gene expression of various pathway genes including lysosome biogenesis and function such as SLC7A11, HPS4, HPS5, CTSB, IFI30, PPT1, SCPEP1, TPP1, ATP6AP1L, MCOLN1, PRKAG2, and VPS18 in sunitinib resistant 786 OR cells compared to parental cells supports the role of lysosomes function in RTKI drug resistance. Furthermore, xCT inhibitor sulfasalazine treatment significantly decreased the metastasis lung nodules of RP-R-02LM in SCID mice demonstrated the xCT role in RCC tumor metastasis. Conclusions: We found preliminary evidence that overexpression of xCT may be associated with RTKI resistance in ccRCC. These results suggest that targeting the xCT in ccRCC may reverse the resistance and enhance the efficacy of RTKI. Additional studies using larger numbers of ccRCC tumors are required to identify xCT as a potential predictive biomarker for response/resistance to RTKI in ccRCC patients. Citation Format: Sreenivasulu Chintala, Remi Adelaiye-Ogala, Ashley Orillion, Sreevani Arisa, May Elbanna, Nur P. Damayanti, Roberto Pili. Association of xCT overexpression with RTKI resistance and metastases in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 94. doi:10.1158/1538-7445.AM2017-94

Phosphatase and tensin homolog deleted on chromosome 10 degradation induced by NEDD4 promotes acquired erlotinib resistance in non-small-cell lung cancer.

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors, such as gefitinib and erlotinib, is a critical issue in the treatment of patients with epidermal growth factor receptor mutant-positive non-small-cell lung cancer. Recent evidence suggests that downregulation of gene of phosphatase and tensin homolog deleted on chromosome 10 plays an important role in acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors in various types of cancers, including lung cancer. It was reported that the E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated gene (NEDD4) (also known as NEDD4-1) negatively regulated phosphatase and tensin homolog deleted on chromosome 10 protein levels through poly-ubiquitination and proteolysis in carcinomas of the prostate, lung, and bladder. Whether this process plays a role in epidermal growth factor receptor-tyrosine kinase inhibitors resistance in non-small-cell lung cancer has not been studied extensively. In view of this, we investigated the involvement of NEDD4 and phosphatase and tensin homolog deleted on chromosome 10 in acquired erlotinib resistance with tyrosine kinase inhibitor-sensitive (HCC827) or tyrosine kinase inhibitor-resistant (Erlotinib-resistant HCC827/ER cells which harbored exon 19 deletion. Overexpression of NEDD4 in HCC827/ER cells was detected, and the reverse correlation between NEDD4 and phosphatase and tensin homolog deleted on chromosome 10 expression in these cells was also revealed. In HCC827/ER cells with knockdown of NEDD4, phosphatase and tensin homolog deleted on chromosome 10 and p-Akt expressions were decreased; the sensitivity of HCC827/ER cells to erlotinib was partially restored. Similar results were also observed in vivo. In H1650/ER cells harboring both exon 19 and phosphatase and tensin homolog deleted on chromosome 10 deletion, expression of p-Akt and sensitivity to erlotinib were not affected by simple knockdown of NEDD4 but affected after transfection of phosphatase and tensin homolog deleted on chromosome 10 into H1650/ER cells. Our results demonstrate that NEDD4 may promote the acquired resistance of non-small-cell lung cancer cells to erlotinib by decreasing phosphatase and tensin homolog deleted on chromosome 10 protein expression. Targeted decrease in NEDD4 expression may be a potential therapeutic strategy for tyrosine kinase inhibitor-resistant non-small-cell lung cancer.

Treatment After First-Generation Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small-Cell Lung Cancer.

Systemic treatment is the basic treatment approach to advanced-stage non-small-cell lung cancer (NSCLC), and chemotherapy and targeted treatments are commonly employed in these patients. Recently, positive results achieved with immunotherapy have led to a growing number of treatment options and prolonged survival time. Today, specific tyrosine kinase inhibitors (TKIs), such as erlotinib, gefitinib, and afatinib, which target the epidermal growth factor receptor (EGFR), and the TKI crizotinib, which targets anaplastic lymphoma kinase gene rearrangement, have become the standard treatment among targeted therapies for patients with sensitive molecular anomalies. However, resistance develops against all these agents after a while. Numerous genetic mutations, T790M+ in particular, have been identified as resistance mechanisms against EGFR-TKIs, and researchers are developing specific inhibitors against them. Among those inhibitors, third-generation EGFR-TKIs such as osimertinib and rociletinib have gained prominence due to their high level of effectiveness and low toxicity profile. Besides, systemic chemotherapy and immunotherapy are proper alternatives. A second biopsy during the progression stage and better clarification of the mechanisms causing secondary resistance will enable more successful treatments in the future.

Establishment of a Novel Method for Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance Mutations in Lung Cancer.

Background:Drug resistance to targeted therapies occurs in lung cancer, and resistance mechanisms related to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are continuously being discovered. We aimed to establish a novel method for highly parallel multiplexed detection of genetic mutations related to EGFR TKI-resistant lung cancer using Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on the MassARRAY mass spectrometry platform.Methods:A review of the literature revealed 60 mutation hotspots in seven target genes (EGFR, KRAS, PIK3CA, BRAF, ERBB2, NRAS, and BIM) that are closely related to EGFR TKI resistance to lung cancer. A total of 183 primers comprised 61 paired forward and reverse amplification primers, and 61 matched extension primers were designed using Assay Design Software. The detection method was established by analyzing nine cell lines, and by comparison with LungCarta™ kit in ten lung cancer specimens. EGFR, KRAS, and BIM genes in all cell lines and clinical samples were subjected to Sanger sequencing for confirming reproducibility.Results:Our data showed that designed panel was a high-throughput and robust tool, allowing genotyping for sixty hotspots in the same run. Moreover, it made efficient use of patient diagnostic samples for a more accurate EGFR TKIs resistance analysis. The proposed method could accurately detect mutations in lung cancer cell lines and clinical specimens, consistent with those obtained by the LungCarta™ kit and Sanger sequencing. We also established a method for detection of large-fragment deletions based on single-base extension technology of MassARRAY platform.Conclusions:We established an effective method for high-throughput detection of genetic mutations related to EGFR TKI resistance based on the MassARRAY platform, which could provide more accurate information for overcoming cancers with de novo or acquired resistance to EGFR-targeted therapies.

Anti-cancer Effects of Polyphenolic Compounds in Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor-resistant Non-small Cell Lung Cancer.

Background:Polyphenolic phytochemicals are natural compounds, easily found in fruits and vegetables. Importantly, polyphenols have been intensively studied as excellent antioxidant activity which contributes to anticancer function of the natural compounds. Lung cancer has been reported to mainly account for cancer-related deaths in the world. Moreover, epidermal growth factor receptor tyrosine kinase inhibitor (TKI) resistance is one of the biggest issues in cancer treatment, especially in nonsmall cell lung cancer (NSCLC). Even though several studies both in preclinical and clinical trials have showed promising therapeutic effects of polyphenolic compounds in anticancer therapy, the function of the natural compounds in TKI-resistant (TKIR) lung cancer remains poorly studied.Objective:The aim of this study is to screen polyphenolic compounds as potential anticancer adjuvants which suppress TKIR lung cancer.Materials and Methods:Colony formation and thiazolyl blue tetrazolium blue assay were performed in the pair-matched TKI-sensitive (TKIS) versus TKIR tumor cell lines to investigate the therapeutic effect of polyphenolic compounds in TKIR NSCLC.Results:Our data show that equol, kaempferol, resveratrol, and ellagic acid exhibit strong anticancer effect in HCC827 panel. Moreover, the inhibitory effect of most of tested polyphenolic compounds was highly selective for TKIR lung cancer cell line H1993 while sparing the TKIS one H2073.Conclusion:This study provides an important screening of potential polyphenolic compounds for drug development to overcome TKI resistance in advanced lung cancer.SUMMARY The study provides an important screening of potential polyphenolic compounds for drug development to overcome tyrosine kinase inhibitor (TKI) resistance in advance lung cancerEquol, kaempferol, resveratrol, and ellagic acid show strong anticancer effect in HCC827 panel, including TKI-sensitive (TKIS) and TKI-resistant clonesThe inhibitory effect of polyphenolic compounds such as equol, kaempferol, resveratrol, ellagic acid, gallic acid, p-Coumaric, and hesperidin is highly selective for TKI-resistant lung cancer cell line H1993 while sparing the TKIS one H2073. Abbreviations used: EGFR: Epidermal growth factor receptor, EMT: Epithelial-to-mesenchymal transition, GTP: Green tea polyphenols, IGF1R: Insulin-like growth factor 1 receptor, MET: Met proto-oncogene, MTT: Thiazolyl blue tetrazolium blue, NSCLC: Non-small cell lung cancer, ROS: Reactive oxygen species, RTK: Receptor tyrosine kinase, STAT3: Signal transducer and activator of transcription 3, TKIR: TKI-resistant, TKIs: Tyrosine kinase inhibitors, TKIS: TKI-sensitive.

Overcoming drug resistance to receptor tyrosine kinase inhibitors: Learning from lung cancer.

There are various receptor tyrosine kinase (TK)-targeted drugs that are currently used in the treatment of patients with non-small cell lung cancer (NSCLC). Among them, the epidermal growth factor receptor (EGFR) TK inhibitors (TKIs) are the most extensively studied. Receptor TKIs including EGFR TKIs have shown dramatic therapeutic efficacies in malignant tumors, which harbor activating mutations in the EGFR gene. However, within 1 or 2years after treatment, patients harboring these mutations often develop resistance to TKI therapy. This review article is aimed at drawing attention to the fact that we must first understand how receptor TKI resistance is acquired to develop strategies for overcoming resistance to TKIs. Furthermore, an insight into the specific molecules or signaling pathways that mediate resistance is a key factor for understanding and overcoming acquired drug resistance. Finally, we present our views on the continuing battle against "drug resistance," and provide further guidelines and strategies on how to minimize the development of drug-resistant tumors.

Mechanism of TRIM24 to Regulate Resistance of Gefitinib in NSCLC cells

背景与目的表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitor, EGFR-TKI)的原发和继发性耐药已成为其在临床肺癌治疗中的拦路虎，在肺癌组织中研究发现TRIM24呈高表达状态，在肺癌细胞中能调控细胞增殖、周期和细胞凋亡，本研究旨在进一步探讨TRIM24调控肺癌细胞吉非替尼耐药的分子机制。方法应用MTT方法和流式细胞仪检测干扰TRIM24及干扰TRIM24后加入吉非替尼对肺癌细胞系增殖能力及凋亡率的抑制率的变化，同时应用Western blot方法检测凋亡相关基因的变化及AKT信号通路中蛋白的表达。结果在A549细胞中使用干扰TRIM24可以提高吉非替尼对肿瘤增殖能力的抑制率，增加吉非替尼诱导的凋亡水平，同时干扰TRIM24及干扰TRIM24加入吉非替尼后促凋亡相关基因p-BAD、Bcl-2和AKT及AKT信号通路相关蛋白PIK3CA均出现降低。结论TRIM24能够调控肺癌细胞吉非替尼耐药，并且通过AKT信号通路引起肺癌细胞EGFRTKI耐药。

HSP90 inhibition blocks ERBB3 and RET phosphorylation in myxoid/round cell liposarcoma and causes massive cell death in vitro and in vivo.

Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS.

Posttranslational modifications of FOXO1 regulate epidermal growth factor receptor tyrosine kinase inhibitor resistance for non-small cell lung cancer cells.

Epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are effective clinical therapies for advanced non-small cell lung cancer (NSCLC) patients, while resistance to TKIs remains a serious problem in clinical practice. Recently, it has been proposed that targeting mTOR could overcome TKI resistance in NSCLC cells. Forkhead box class O1 (FOXO1) has emerged as an important rheostat that modulates the activity of Akt and mTOR signaling pathway. However, the role of FOXO1 and related regulatory mechanism in TKI resistance in NSCLC remain largely unknown. Here, we find that mTOR-AKT-FOXO1 signaling cascade is deregulated in TKI-resistant NSCLC cells and that FOXO1 was highly phosphorylated and lowly acetylated upon erlotinib treatment. Combination of mTOR or PI3K inhibitor and erlotinib overcomes TKI resistance to inhibit cell growth and induce apoptosis in TKI-resistant NSCLC cells. Furthermore, the phosphorylation and acetylation of FOXO1 are reversely modulated by mTORC2-AKT signaling pathway. FOXO1 mutation analyses reveal that FOXO1 acetylation inhibits cell proliferation and promotes NSCLC cell apoptosis, while the phosphorylation of FOXO1 plays opposite roles in NSCLC cells. Importantly, increasing FOXO1 acetylation by a HDAC inhibitor, depsipeptide, overcomes TKI resistance to effectively induce TKI-resistant NSCLC cell apoptosis. Together, FOXO1 plays dual roles in TKI resistance through posttranslational modifications in NSCLC and this study provides a possible strategy for treatment of TKI-resistant NSCLC patients.

Biomarker analysis of a phase II trial of cabozantinib and erlotinib in patients (pts) with EGFR-mutant NSCLC with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance: A California Cancer Consortium Phase II Trial (NCI 9303).

8087 Background: Modulation of the MET and VEGFR signaling pathways are associated with resistance to EGFR TKI therapy in addition to acquired T790M mutation (mt). Cabozantinib is a TKI that targets MET and VEGFR2, and RET, AXL, IT and TIE-2. Third generation EGFR inhibitors are effective in T790M mt-pos tumors, although treatment of T790M-neg disease remains an area of unmet need. Cabozantinib and erlotinib in EGFRmt-positive NSCLC following PD on EGFR TKI showed benefit, and we evaluated potential mechanisms of resistance to therapy. Methods: Pts with EGFR mt and PD on an EGFR TKI immediately prior to enrollment were eligible. Erlotinib 150 mg daily + cabozantinib 40 mg daily were given on a 28 day cycle and ORR was the primary objective. Pts were required to have archival tissue available, and a subset had biopsy samples post-progression on EGFR TKI. MET amplification was determined by FISH and EGFRmt, including T790M, was analyzed by next-generation sequencing. Results: 37 pts were enrolled and baseli...

Anti-HER3 monoclonal antibody patritumab sensitizes refractory non-small cell lung cancer to the epidermal growth factor receptor inhibitor erlotinib

Human epidermal growth factor receptor (HER) 3 is aberrantly overexpressed and correlates with poor prognosis in non-small cell lung cancer (NSCLC). Patritumab is a monoclonal antibody against HER3 that has shown promising results in early-phase clinical trials, but an optimal target population for the drug has yet to be identified. In the present study, we examined whether heregulin, a HER3 ligand that is also overexpressed in a subset of NSCLC, can be used as a biomarker to predict the antitumorigenic efficacy of patritumab and whether the drug can overcome the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) resistance induced by heregulin. Patritumab sensitivity was associated with heregulin expression, which, when abolished, resulted in the loss of HER3 and AKT activation and growth arrest. Furthermore, heregulin overexpression induced EGFR TKI resistance in NSCLC cells harbouring an activating EGFR mutation, while HER3 and AKT activation was maintained in the presence of erlotinib in heregulin-overexpressing, EGFR-mutant NSCLC cells. Sustained HER3-AKT activation was blocked by combining erlotinib with either anti-HER2 or anti-HER3 antibody. Notably, heregulin was upregulated in tissue samples from an NSCLC patient who had an activating EGFR mutation but was resistant to the TKI gefitinib. These results indicate that patritumab can overcome heregulin-dependent EGFR inhibitor resistance in NSCLC in vitro and in vivo and suggest that it can be used in combination with EGFR TKIs to treat a subset of heregulin-overexpressing NSCLC patients.

Tumor heterogeneity and resistance to EGFR-targeted therapy in advanced nonsmall cell lung cancer: challenges and perspectives.

Lung cancer, mostly nonsmall cell lung cancer, continues to be the leading cause of cancer-related death worldwide. With the development of tyrosine kinase inhibitors that selectively target lung cancer-related epidermal growth factor receptor mutations, management of advanced nonsmall cell lung cancer has been greatly transformed. Improvements in progression-free survival and life quality of the patients were observed in numerous clinical studies. However, overall survival is not prolonged because of later-acquired drug resistance. Recent studies reveal a heterogeneous subclonal architecture of lung cancer, so it is speculated that the tumor may rapidly adapt to environmental changes via a Darwinian selection mechanism. In this review, we aim to provide an overview of both spatial and temporal tumor heterogeneity as potential mechanisms underlying epidermal growth factor receptor tyrosine kinase inhibitor resistance in nonsmall cell lung cancer and summarize the possible origins of tumor heterogeneity covering theories of cancer stem cells and clonal evolution, as well as genomic instability and epigenetic aberrations in lung cancer. Moreover, investigational measures that overcome heterogeneity-associated drug resistance and new assays to improve tumor assessment are also discussed.

205P - Efficacy Analysis of Gefitinib +/- Ficlatuzumab in Serum Proteomic Based Subgroups of Patients with Previously Untreated Lung Adenocarcinoma

ABSTRACT Aim: Ficlatuzumab (F), a humanized anti-hepatocyte growth factor (HGF) IgG1 mAb, inhibits c-MET receptor activation by neutralizing HGF. HGF/c-MET pathway activation has been implicated in epithelial growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer. A randomized Phase 2 study of gefitinib (G) +/- F in Asian patients with previously untreated lung adenocarcinoma (±EGFR mutations) failed to demonstrate improved overall survival (OS) or progression-free survival (PFS) over G alone (NCT01039948) in the intent-to-treat population. VeriStrat® (VS) is a multivariate serum proteomic test that utilizes mass spectrometry to assign a “GOOD” (VSG) or “POOR” (VSP) classification and has demonstrated broad prognostic and predictive utility for EGFR TKI treatments. In part, VS appears to measure a host inflammatory state that may stimulate tumors via alternative pathways, including HGF secretion. Methods: 183 blinded unique patient pretreatment serum samples from the study were sent for VS analysis. Clinical outcomes of F + G vs G alone were analyzed in VS subgroups. Results: 183 serum samples were assigned a VS label (VSG = 145, VSP = 35, and 3 “Indeterminate”). Addition of F to G provided significant clinical benefit to the VSP subgroup: OS hazard ratio (HR) 0.41, P = .032; medians = 23.9 mo for F + G (N = 18) vs 5.8 mo for G alone (N = 17) and PFS HR 0.41, P = .014; medians = 7.4 mo for F + G vs 2.3 mo for G alone. No benefit was observed in the VSG subgroup in either OS (HR 1.18, P = .492; median 24.7 mo for F + G [N = 69] vs not reached for G alone [N = 76]) or PFS (HR 1.06, P = .753; median 5.6 mo for F + G vs 5.6 mo for G alone). Unadjusted interaction test for treatment and VS was statistically significant for OS (HR = 3.31, P = .013) and PFS (HR = 2.51, P = .019). Despite the small sample sizes, similar patterns in OS and PFS based on VS stratification were also observed in patients with known EGFR mutations (n = 71). Conclusions: VS may be predictive for clinical benefit of F + G over G (OS and PFS) in patients classified as VSP. A prospective confirmatory study using VS as predictive biomarker on the combination of F + EGFR-TKI in EGFR mutation–positive patients is planned. Disclosure: J.C. Yang: Advisory boards: Boehringer Ingelheim, Eli Lilly, Pfizer, Novartis, Roche/Genetech, Astrazeneca, Merck, Bayer, Clovis Oncology Corporate sponsored research: Boehringer Ingelheim; J. Gyuris, M.Han, F. Payumo and Z. Weng have declared: AVEO Oncology employee • Stock • Salary; P. Komarnitsky: (former) AVEO Oncology employee; G. Pestano, J. Roder and D. Spinella have declared: Biodesix employee • Stock • Salary; K. Park: Advisory boards: AVEO, Astellas, AZ, BI, Clovis, Daiichi Sankyo, Eli Lilly, Kyowa Hakko Kirin, Novartis, Roche. All other authors have declared no conflicts of interest.