Crizotinib-resistant Cell Line Research Articles

Establishment of an acquired lorlatinib-resistant cell line of non-small cell lung cancer and its mediated resistance mechanism.

Although lorlatinib, the third generation of echinoderm microtubule protein 4-anaplastic lymphoma kinase (EML4-ALK) tyrosine kinase inhibitor (TKI), overcame the previous generation ALK-TKIs' drug resistance problems, but the mechanism of lorlatinib resistance remained unclear. Furthermore, optimal chemotherapy for lorlatinib-resistant non-small cell lung cancer (NSCLC) patients was still unknown. A lorlatinib-resistant NSCLC cell line SNU-2535LR was generated by gradually increasing dose of lorlatinib to crizotinib-resistant cell line SNU-2535 in vitro. To study the resistance mechanism of SNU-2535LR cells, we applied CCK-8 assay to detect the sensitivity of crizotinib and the reverse effect of APR-246, a p53 activator, on lorlatinib-induced resistance and different chemotherapy drugs to SNU-2535LR cells. We also detected the expressions of EML4-ALK-related proteins of SNU-2535LR cells via western blot.Please confirm that author names have been identified correctly and are presented in the right order.Dear Editor: I have carefully confirmed that the author names have been identified correctly and are presented in right order.Thank you very much! Your sincerely BoXie RESULTS: The sensitivity of SNU-2535LR cells to lorlatinib was decreased significantly than that of SNU-2535 cells. EML4-ALK fusion was decreased both at protein level and DNA level in SNU-2535LR cells. More interesting, the crizotinib-resistant mutation ALK p.G1269A disappeared, while new TP53 mutation emerged in SNU-2535LR cells. APR-246 can reverse the lorlatinib resistance in SNU-2535LR cells, with a reversal index of 4.768. Compared with SNU-2535 cells, the sensitivity of SNU-2535LR cells to gemcitabine, docetaxel and paclitaxel was significantly increased (P < 0.05), but decreased to cisplatin (P < 0.05). This study demonstrated that the combination of p53 protein agonist and lorlatinib may provide a new therapeutic strategy for NSCLC patients with lorlatinib resistance and TP53 mutation. Furthermore, the results also provide guidance for selecting optimal chemo-regimens for NSCLC patients after ALK-TKIs failure.

Impact of STAT1 polymorphisms on crizotinib-induced hepatotoxicity in ALK-positive non-small cell lung cancer patients.

Crizotinib is the first-line small molecule tyrosine kinase inhibitor for ALK-positive non-small cell lung cancer. In this study, a retrospective pharmacogenomics investigation was conducted to explore the relationship between genes related to RTK downstream signaling pathways and crizotinib-induced hepatic toxicity in ALK-positive NSCLC patients. The variable importance analysis of random forest algorithm was applied to identify the significant features which contribute to the crizotinib sensitivity in Cancer Cell Line Encyclopedia (CCLE) database. The KEGG and reactome pathway enrichment analysis were conducted with EnrichR. The differential expression genes were identified with R package DESeq2 in CCLE liver derived cell lines between crizotinib sensitive and resistant groups. From 2012 to 2015, 42 NSCLC patients were enrolled in this study. 90 polymorphisms were genotyped using the Sequenom Massarray system. Sequencing of HGFR (c-Met) genes was carried out on the Ion Torrent Proton. In total, 66.7% NSCLC patients suffered from crizotinib-induced liver toxicity and 11.9% progressed to severe hepatic toxicity. The features with the top importance from classification and regression random forest model were enriched in RTK downstream signaling pathways (JAK/STAT, RAS/RAF/MAPK, PI3K/AKT pathways) and immune system-related pathways. Collagen family genes (COL1A1, COL1A2, COL6A1, COL5A1) and other extracellular matrix protein (TNC, TAGLN, TENM2, EDIL3, VCAN, CNN1, SH3BP4, TAGLN), which were closely related to MAPK-ERK signaling pathways, were significantly enriched in crizotinib resistant cell lines. In multiple logistic regression, STAT1 rs10208033 (T > C) was significantly associated with crizotinib-induced liver toxicity (OR = 6.733, 95% CI 1.406-32.24, P = 0.017). Compared with non-CC, OR is 5.5 (95% CI 1.219-24.81, P = 0.027) for STAT1 rs10208033 CC genotypeto develop crizotinib-induced liver toxicity. Further cell viability test in human fetal hepatocyte line, L-02, reveals that the STAT1 inhibitor might protect hepatocyte cells from the toxicity caused by crizotinib. Polymorphism of rs10208033 is a potential biomarker for predicting crizotinib-induced hepatotoxicity. These results suggest that STAT1 plays an important role in crizotinib-induced hepatotoxicity. Further studies are needed to confirm our finding and understand the underlying mechanisms.

ROS1-fusion protein induces PD-L1 expression via MEK-ERK activation in non-small cell lung cancer

ABSTRACT Introduction Despite some of the oncogenic driver mutations that have been associated with increased expression of programmed death-ligand 1 (PD-L1), the correlation between PD-L1 expression and ROS1 fusion in NSCLC cells, especially for those with Crizotinib resistance has not been fully addressed. Materials and Methods The expression of PD-L1 in 30 primary NSCLC tumors with/without ROS1-fusion protein was evaluated by immunohistochemical (IHC) analysis. To assess the correlation between ROS1 fusion and PD-L1 expression, we down-regulated ROS1 with RNA interference or specific inhibitor (Crizotinib) in ROS1-fusion positive NSCLC cell line HCC78; or up-regulate ROS1-fusion gene in an immortalized human bronchial epithelial cell line (HBE). Mouse xenograft models were also used to determine the effect of ROS1 expression on PD-L1 expression in vivo. Crizotinib-resistant cell line was generated for measuring the association between Crizotinib resistance and PD-L1 expression. Results ROS1-rearrangement in primary NSCLC tumor was significantly associated with up-regulated PD-L1 expression. PD-L1 expression was significantly up-regulated in bronchial epithelial cells after forced expression of ROS1 fusion and was eliminated when HCC78 xenograft mouse models were treated with Crizotinib. We found PD-L1 expression was modulated by MEK-ERK pathway signaling in both parental and Crizotinib-resistant NSCLC cells with ROS1 fusion. Conclusions The correlation between ROS1-fusion and PD-L1 overexpression suggested that PD-L1/PD-1 blockade could be the second-line treatment option for the Crizotinib-resistant NSCLC with ROS1 rearrangement.

GSK1838705a, an IGF-1R/ALK Inhibitor, Overcomes Resistance to Crizotinib in ALK-Positive ALCL

Anaplastic large cell lymphoma (ALCL) is a type of CD30-expressing non-Hodgkin's lymphoma (NHL), which accounts for 2% to 3% of adult non-Hodgkin's lymphoma,accounting for 15% to 30% of children with large cell lymphoma. Anaplastic lymphoma kinase (ALK) positive ALCL is highly invasive, and currently it is generally based on CHOP combined with chemotherapy. The proportion of patients with complete relief of symptoms is as high as 90%, but the proportion of recurrence is also as high as 40%. Crizotinib is the first generation of ALK inhibitors that have been approved for the treatment of ALK+ ALCL. Unfortunately, most patients treated with crizotinib relapse after a significant initial response. The median progression-free survival of clinical trials was 10.5 months. Various mutations in the ALK kinase domain and amplification of the ALK gene copy number, activation of the alternative pathway, and tumor heterogeneity are major causes of crizotinib resistance. Studies have shown that IGF-1R interacts with NPM-ALK to promote ALK+ALCL transformation, proliferation and migration. GSK is a small molecule kinase inhibitor that inhibits both IGF-IR and ALK. Therefore, GSK with simultaneous inhibition of the bidirectional potential of IGF-IR and ALK has a promising prospect in the targeted therapy of NPM-ALK+ALCL. This study explored the inhibitory effects of GSK on NPM-ALK+ALCL and crizotinib-resistant NPM-ALK+ALCL by in vivo and in vitro experiments. In vitro experiments: The sensitivity of ALCL cell line to GSK1838705a was detected by CCK8 and flow cytometry. The expression of phosphorylation of IGF-1R and NPM-ALK signaling pathway in Karpas299 and SR786 cell lines stimulated by GSK was detected by WB method. In order to study the crizotinib resistance mutation, we established ALK+ALCL crizotinib-resistant cell lines Karpas299-R and SR786-R, and identified the resistance of Karpas299-R and SR786-R cell lines by CCK8 and flow cytometry. The drug-resistant and non-resistant strains were stimulated with gradient concentrations of crizotinib and gradient GSK, and the IC50 of the two were compared by CCK8. The WB method was used to compare the phosphorylation levels of downstream signaling pathways in drug-resistant and non-resistant strains. In vivo experiment: The ALK+ALCL and resistant-ALK+ALCL mouse model was established, and three groups of mice treated with control, GSK single drug 30 mg/kg, GSK single drug 60 mg/kg, were established. The tumor volume and body weight of the four groups were compared. Immunohistochemistry was used to compare the expression levels of key signaling molecules and apoptotic proteins in each group. SPSS statistical software draws survival curves. As the concentration of GSK gradually increases, the survival rate of ALCL cells gradually decreases. The expression of pIGF-1R, pNPM-ALK, pSTAT3, pAKT, casepase3 and other molecules decreased in the downstream signaling pathway, and the expression level of cleaved-casepase3 increased.In the crizotinib-resistant cell line, with the increase of the concentration of GSK, the apoptosis rate of the cells increased and the phosphorylation level of the downstream molecules gradually decreased. Tumor volume of three groups of mouse models: control&gt;GSK single drug 30 mg/kg&gt;GSK single drug 60 mg/kg. Immunohistochemistry results showed that the expression level of key signaling molecules in GSK-treated CHOP-treated mice decreased, and the expression level of apoptotic proteins increased. In this research, we explored the effects of GSK1838705A on proliferation, apoptosis, and clonogenesis of ALCL cell lines. Subsequently, we established a crizotinib-resistant cell line and noticed that GSK1838705A can effectively reduce the viability of resistant ALCL cells and significantly restrain the transmission of downstream survival signaling pathways induced by IGF1R/IR phosphorylation. Besides, we discovered that GSK1838705A inhibited the development of both crizotinib-sensitive and crizotinib-resistant ALCL tumors in the ALCL mouse model established by subcutaneous tumorigenesis. Based on the results of previous clinical trials, we put forward to use GSK1838705A as an alternative treatment strategy to overcome crizotinib-resistant ALCL. Disclosures No relevant conflicts of interest to declare.

Abstract 2640: DKK1 overexpression might induce crizotinib resistance

Abstract NSCLC patients with pleural effusions tend to show progressive status and malignant features. Newly established lung cancer cell lines from pleural effusions, SNU-2550 and SNU-2563 were harboring ALK rearrangement and they were originally sensitive to crizotinib. After treated with regular crizotinib treatment and recovery at least 1 year, crizotinib-resistant cell lines (SNU-2550CR and SNU-2563CR) that became resistant to crizotinib more than 5 times were established. And then, whole exome sequencing (WES) and RNA-seq were operated in order to understand way to acquire resistance. According to WES result, there were differences in mutations between parental cell lines and resistant cell lines. For example, mutations in MKNK2, IGFN1, PRSS1 and FOXA2 genes were only present in SNU-2550CR not SNU-2550. Variations of ESX1, PRSS1, PCDHA4 and AGAP3 genes were observed in only SNU-2563CR. When the differences were studied and visualized via SPIA two-way evidence plot and heatmap, genes related with cell cycle, p53 and Wnt pathway were commonly changed in RNA expression level. In fact, there were expressional differences in genes related those pathways such as MAPK2, DKK1, APC and GSK3b genes. Among them, Dkk1 expression level was more than doubled in two set. Through those analysis, we examined differences between parental cell lines that have sensitive reactivity and resistant cell lines that acquired resistance. When crizotinib resistance was induced, Dkk1 expression was elevated. Augmentation of Dkk1 expression level induces the level of overall β-catenin expression, and cytoplasmic β-catenin tends to be concentrated adjacent to membrane and into nucleus region. Therefore, Dkk1 can be considered as a factor that is related to crizotinib resistance by up-regulating Wnt/β-catenin pathway in lung cancer. Acquired resistance to crizotinib is major predicament especially when there is ALK rearrangement. Studying the mechanism of crizotinib resistance therefore improves the prognosis of ALK rearrangement lung cancer. We found that Dkk1 overexpression might be correlated to crizotinib resistance. Note: This abstract was not presented at the meeting. Citation Format: Ha Young Seo. DKK1 overexpression might induce crizotinib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2640.

Abstract 4761: Transcriptomic overview of EML4-ALK lung cancer cells resistant to ALK inhibitors highlights a vulnerability to CDK inhibitors

Abstract Introduction: EML4-ALK-driven lung cancer represents about 5% of lung adenocarcinomas. Despite the constant expansion of the armamentarium to inhibit ALK, patients typically develop acquired resistance to ALK inhibitors. In the case of resistance mediated by ALK mutations, sequencing of ALK inhibitors is recommended, but in ALK wild-type patients, a better understanding of acquired resistance, as well as new therapeutic strategies are needed. Here, we aimed to characterize the transcriptional network of EML4-ALK lung cancer and used these data to address acquired resistance to ALK inhibitors. Methods: We utilized EML4-ALK cell lines treated long-term with ALK inhibitors as a model of acquired resistance. Transcriptomic changes were quantified through means of RNA-seq or RT-qPCR. Results: We have found a dysregulation of several microRNAs in crizotinib-resistant cells. Specifically, the oncogenic miR-25 and miR-30c were upregulated and their inhibition resulted in cell cycle arrest. We analyzed plasma samples from patients with EML4-ALK lung cancer and found increased circulating levels of these miRNAs in 2 patients upon progression with crizotinib. In addition, the known tumour-suppressing miRNAs miR-103a and miR-149 were downregulated in crizotinib-resistant cells and restoration of miR-149 resulted in apoptotic induction. We show that CDK6 is a direct target of miR-103 and an indirect target of miR-149. Moreover, by comparing the transcriptome of parental and crizotinib-resistant cells we uncovered an upregulation of genes implicated in cell cycle. Treatment with Cyclin-Dependent Kinase (CDK) inhibitors led to suppression of cell proliferation and potent induction of apoptosis. Moreover, these inhibitors spared normal epithelial cells while they induced higher levels of apoptosis in EML4-ALK cells compared with other NSCLC cell lines, suggesting a preferential targeting of EML4-ALK cells .Lastly, a pilot xenograft experiment with a crizotinib-resistant cell line showed in vivo anti-tumor activity of CDK inhibition and we are currently expanding this in a larger mouse cohort. Conclusions: Crizotinib-resistant cells may exhibit several concurrent oncogenic alterations including miRNA- or coding gene-dysregulation, in the absence of ALK mutations. Moreover, the contribution of these alterations to acquired resistance cannot be easily and quickly assessed. A therapeutic intervention which does not rely on identifying the driver of resistance would be of high practical value. We suggest that CDK inhibition may be a new avenue to target relapsed EML4-ALK lung cancer refractory to ALK inhibitors. Citation Format: Athanasios R. Paliouras, Peter Magee, Sudhakar Sahoo, Hui Sun Leong, Matthew Krebs, Fiona Blackhall, Christine M. Lovly, Michela Garofalo. Transcriptomic overview of EML4-ALK lung cancer cells resistant to ALK inhibitors highlights a vulnerability to CDK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4761.

MiR-200c regulates crizotinib-resistant ALK-positive lung cancer cells by reversing epithelial-mesenchymal transition via targeting ZEB1.

Crizotinib is an orally administered drug for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive locally advanced or metastatic non-small cell lung cancer (NSCLC). Despite the impressive efficacy of crizotinib in the treatment of ALK-positive lung cancer, acquired resistance eventually develops in the majority of patients. The microRNA (miR)-200c reverses the resistance of lung cancer cells to various chemotherapeutic drugs and molecular targeted drugs, however, whether it can reverse the resistance of crizotinib remains unknown. The present study established a crizotinib resistant cell line (NCI-2228/CRI), which was derived from the parental NCI-2228 cell line by long-term exposure to increasing concentrations of crizotinib. Through overexpression and suppression of miR-200c expression, the characteristics associated with epithelial-mesenchymal transition (EMT), including morphology, EMT marker proteins and cellular mobility, were investigated. Cell viability and invasion assays demonstrated that high expression of miR-200c significantly inhibited the proliferation, migration and invasion of NCI-2228 cells compared with the negative control. A luciferase reporter assay indicated that miR-200c directly targeted the 3′-untranslated region of zinc finger E-box binding homeobox 1. Additionally, reverse transcription-quantitative polymerase chain reaction analysis demonstrated that the mRNA levels of N-cadherin and Vimentin were decreased in NCI-2228 cells transfected with miR-200c mimic compared with negative control cells, whereas the mRNA level of E-cadherin was increased. In addition, EMT was reversed by miR-200c, which suggests that miR-200c may serve a role in mediating the sensitivity of NCI-2228/CRI cells to crizotinib. The present study may therefore contribute to improving the sensitivity of ALK positive lung cancer cells to crizotinib.

Abstract 1889: AXL and EGFR signaling mediate resistance to Crizotinib in non-small cell lung cancer cells harboring the ROS1 fusion gene

Abstract Background: The multi-tyrosine kinase inhibitor (TKI) crizotinib elicits a dramatic response in patients with non-small cell lung cancer (NSCLC) harboring the c-ros oncogene 1 (ROS1) fusion gene (Shaw et al. NEJM 2014); however, these patients inevitably develop resistance to crizotinib within a year, which limits the efficacy of crizotinib. Although reported molecular changes include ROS1 tyrosine kinase mutations, epidermal growth factor receptor (EGFR) activation, and epithelial-to mesenchymal transition, the detailed mechanism of crizotinib resistance has not been elucidated. Methods: To explore the molecular mechanisms of acquired crizotinib resistance in detail, we used a cell line model. HCC78 cells harboring the SLC34A2-ROS1 fusion gene, which are sensitive to crizotinib, were exposed continuously to increasing concentrations of crizotinib in a step-wise manner. A crizotinib-resistant cell line designated HCC78R was established and assessed by MTT assay, Western blotting, a receptor tyrosine kinase (RTK) array, quantitative PCR (RT-PCR), ELISA, and an RNA kinome targeted kinome panel (612 genes) with next-generation sequencing. Results: Crizotinib-resistant HCC78R (50% inhibitory concentration [IC50] 4085.9 nmol/L) was 47-fold more resistant to crizotinib than was parental HCC78 (IC50 85.8 nmol/L) (p = 0.0145) according to MTT assay. The RTK array revealed that EGFR phosphorylation was upregulated in HCC78R cells. Western blotting confirmed the activation of EGFR and its downstream signaling pathways. RT-PCR screening of EGFR ligand family member expression showed increased heparin-binding EGF-like growth factor (HB-EGF) in HCC78R compared with HCC78 cells. Consistently, addition of the HB-EGF or conditioned medium from HCC78R cells rendered the HCC78 parental cells moderately resistant to crizotinib. EGFR overexpression or activating mutations were not detected. Furthermore, RNA kinome sequencing revealed 16-fold higher AXL mRNA expression and 1.68-fold lower ROS1 fusion gene expression in HCC78R compared with HCC78. Overexpression of the AXL protein and downregulation of the ROS1 protein were confirmed by Western blotting. Monotherapy with gefitinib (EGFR-TKI) or R428 (AXL inhibitor) or cabozantinib (AXL inhibitor) moderately inhibited the growth of HCC78R cells, while combined therapy suppressed proliferation more significantly compared with each monotherapy. Conclusions: AXL and EGFR signaling mediate resistance to crizotinib, and combination treatment of an EGFR-TKI and AXL inhibitor may be an alternative strategy in ROS1 fusion gene-driven lung cancer cells. Citation Format: Yuka Kato, Kadoaki Ohashi, Eiki Ichihara, Shuuta Tomida, Hiroe Kayatani, Kenichiro Kudo, Daisuke Minami, Takashi Ninomiya, Toshio Kubo, Toshiyuki Kozuki, Katsuyuki Hotta, Nagio Takigawa, Mitsune Tanimoto, Katsuyuki Kiura. AXL and EGFR signaling mediate resistance to Crizotinib in non-small cell lung cancer cells harboring the ROS1 fusion gene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1889.

Methodology of Establishing and Identifying NCI-H2228/Crizotinib-resistant Cell Lines In Vitro

背景与目的小分子靶向药物发生耐药的机制及寻找克服耐药的手段是目前提高临床疗效需要迫切解决的问题。本研究探讨采用不同方法建立对Crizotinib耐药的非小细胞肺癌NCI-H2228/Crizotinib细胞株的可行性及鉴定分析，为深入研究Crizotinib耐药发生的机制并寻找克服耐药的手段提供实验基础和理论依据。方法采用逐步增加药物浓度和化学诱变剂处理NCI-H2228细胞，诱导细胞对Crizotinib耐药。MTT法检测亲本细胞和耐药细胞的50%抑制浓度（50% inhibitory concentration, IC50）和群体倍增时间。RT-PCR和Western blot实验检测棘皮动物微管相关蛋白样4-间变性淋巴瘤激酶（echinoderm microtubule-associated protein like 4-anaplastic lymph kinase, EML4-ALK）基因表达。对耐药细胞和亲本细胞的EML4-ALK基因全长测序并对比分析发生耐药的机制。结果逐步增加药物浓度的方法耗时过长，细胞恢复生长缓慢，不能有效诱导NCI-H2228细胞对Crizotinib耐药；化学诱变剂ENU可以在短时间内诱导NCI-H2228细胞对Crizotinib耐药[IC50=（3.810±1.100）μmol/L，P=0.002, 9，vs亲本细胞]。耐药细胞EML4-ALK基因发生点突变的频率高于亲本细胞。结论化学诱变剂诱导细胞耐药操作简便，可有效缩短实验流程，为深入研究耐药发生机制，寻找克服靶向药物耐药的手段提供了前期技术方法和实验依据。

Highlights of This Issue

TCR-gene T-cell therapy has been studied in refractory melanoma and sarcoma patients, and the regimens usually include lymphodepleting preconditioning. Kageyama and colleagues treated 10 MAGE-A4+ recurrent esophageal cancer patients with wild-type TCR-gene transduced T-cell transfer. They show that the transferred T cells persisted long, trafficked to tumor sites, and maintained tumor-specific reactivity and that 3 of 10 patients who had minimal disease long-remained free from disease progression. All the patients did not encounter any serious toxicity. Wild-type TCRs with physiologically high avidity may represent a safe approach in clinical use.The development of novel anticancer agents is central to improving cancer treatment. Lolkema and colleagues present a study exploring a novel selective C-Met inhibitor and report unexpected renal toxicity in humans. Extensive preclinical analysis showed that species-specific metabolism of quinoline ring structures causes the renal toxicity. This integrated clinical and preclinical analysis elucidated the mechanism of toxicity allowing future quinoline ring structure containing drugs to be tested for species specific metabolism.Cell-penetrating peptides represent a promising class of anticancer drugs; however, these agents have had limited applicability because of poor pharmacologic and intracellular delivery properties. Hasegawa and colleagues report that these challenges can be circumvented when targeting the widely overexpressed MUC1-C oncoprotein by encapsulating the GO-203 inhibitor peptide in novel biodegradable PLA-PEG-PPG-PEG tetrablock nanoparticles (NPs). They show that these NPs are highly effective for inhibiting MUC1-C by the sustained intracellular delivery of GO-203 in diverse carcinoma cells growing in vitro and in vivo. This NP-based approach may be functional for the targeting of other intracellular oncoproteins with peptide drugs.Crizotinib showed marked antitumor activity against ROS1-rearranged NSCLC. Despite the efficacy of crizotinib, development of resistance is inevitable. Song and colleagues discerned the mechanisms of crizotinib resistance in ROS1-rearranged NSCLC using samples from two patients and crizotinib-resistant cell lines. ROS1 kinase mutations (G2032R and L2155S), EGFR activation, and epithelial-to-mesenchymal transition were associated with acquired crizotinib resistance. Secondary ROS1-mutant NSCLC cells were sensitive to foretinib. In addition, EGFR/ROS1-activated NSCLC cells were inhibited by irreversible EGFR inhibitors. These might define subsequent treatment strategies in patients with ROS1-rearraged NSCLC who showed acquired resistance to crizotinib.

Abstract 5530: OTX015, a novel pan BET-BRD inhibitor is active in non-small-cell lung cancer (NSCLC) cell lines bearing the fusion protein EML4-ALK

Abstract Background: Various inhibitors targeting the activity of BET proteins have been recently developed and have shown potent anti-proliferative effects in several tumors, including NSCLC. The fusion of the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) genes results in the chimeric oncogene EML4-ALK, identified as a distinct entity of NSCLC patients that enables effective ALK-targeted therapy. However, most of these patients invariably acquire resistance within few months. Herein, we report preclinical findings obtained with a novel oral pan-BET-BRD inhibitor, OTX015, in a panel of NSCLC cell lines, some of which bear the fusion protein EML4-ALK. Materials and Methods: Five established NSCLC cell lines (i.e. H2228, H3122, A549, HOP62 and HOP92) were exposed to increasing concentrations of OTX015 (OncoEthix SA, Switzerland). In each case, the effect on cell viability was determined by the MTT assay after 72 h of exposure. GI50 values, representing 50% growth inhibition concentration, were calculated using GraphPad Prism 5.0 software. Protein levels were determined by Western blot using commercial antibodies. RNA was extracted with the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit following manufacturer's instructions. RT-PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. Results: OTX015 displayed anti-proliferative activity in EML4-ALK-positive H2228 and H3122 cells after a 72-h treatment, with GI50 values of 629 and 627nM, respectively. The corresponding GI50 values for the benchmark compound, JQ-1, were 2161 and 1265 nM. Interestingly, OTX015 was also active in the EML4-ALK-negative A549 cell line, with a GI50 of 432 nM. BRD4/3/2, c-MYC, BCL-2, p21 and CyclinD1 were detected at protein and mRNA levels in all cell lines. Both OTX015-sensitive and -resistant cells exhibited similar basal expression levels for the aforementioned proteins. EML4-ALK variants 1 and 3 were identified in H3122 and H2228 cells, respectively. Assessment of the signaling pathways involved in the anti-proliferative activity of OTX015 in these cells showed a transient up-regulation of STAT3, followed by a down-regulation after 24 h and up to 72 h of exposure. These results indicate that the key downstream effector of OTX015 is STAT3, frequently found to be overexpressed in crizotinib-resistant cell lines. Interestingly, c-MYC protein and mRNA levels were not altered by OTX015. EML4-ALK-positive H3122 cells showed down-regulation of n-MYC mRNA levels after OTX015 treatment. Conclusion: Our results indicate that NSCLC cell lines with genomic ALK alterations are sensitive to BET-BRD inhibition by OTX015, suggesting its potential clinical use as anticancer agent in EML4-ALK positive NSCLC patients. Citation Format: Ramiro Vázquez, Lucile Astorgues-Xerri, Mohamed Bekradda, Esteban Cvitkovic, Patrice Herait, Michela Boi, Giorgio Inghirami, Maurizio D'Incalci, María E. Riveiro, Eric Raymond. OTX015, a novel pan BET-BRD inhibitor is active in non-small-cell lung cancer (NSCLC) cell lines bearing the fusion protein EML4-ALK. [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 5530. doi:10.1158/1538-7445.AM2014-5530

Design of Potent and Selective Inhibitors to Overcome Clinical Anaplastic Lymphoma Kinase Mutations Resistant to Crizotinib

Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients. Under pressure of crizotinib treatment, point mutations arise in the kinase domain of ALK, resulting in resistance and progressive disease. The successful application of both structure-based and lipophilic-efficiency-focused drug design resulted in aminopyridine 8e, which was potent across a broad panel of engineered ALK mutant cell lines and showed suitable preclinical pharmacokinetics and robust tumor growth inhibition in a crizotinib-resistant cell line (H3122-L1196M).

Abstract 919: AZD3463, a novel ALK/IGF1R inhibitor, overcomes multiple mechanisms of acquired resistance to crizotinib.

Abstract Genomic rearrangement of Anaplastic Lymphoma Kinase (ALK) has been observed in several tumor types including 60-80% anaplastic large cell lymphoma (ALCL) and 3-6% of non small cell lung cancer (NSCLC). Although the ALK inhibitor crizotinib has clinical efficacy in selected ALK positive NSCLC patients, the majority of patients who show initial responses eventually relapse. Various mechanisms leading to resistance have been proposed and include ALK amplification and resistance mutations, as well as alternative pathway drivers including EGFR, cKIT and, more recently, IGF1R. We have discovered a novel and potent inhibitor of ALK, AZD3463 with a Ki value of 0.75nM which also inhibits additional receptor tyrosine kinases including insulin growth factor receptor (IGF1R) with equivalent potency. AZD3463 inhibits ALK in cells as demonstrated by its ability to decrease ALK autophosphorylation in tumor cell lines containing ALK fusions including DEL (ALCL NPM-ALK), H3122 (NSCLC EML4-ALK) and H2228 (NSCLC EML4-ALK). Inhibition of ALK is associated with perturbations in downstream signaling including ERK, AKT and STAT3 pathways leading to preferential inhibition of proliferation in the ALK fusion containing cell lines in vitro. AZD3463 also demonstrates the ability to dose dependently inhibit pALK in xenograft tumors in vivo resulting in stasis (H3122) or regression (DEL, H2228). AZD3463 retains good activity against a number of clinically relevant crizotinib resistant mutations including the gatekeeper mutant L1196M where equivalent potency to wild type ALK is observed in vitro and in vivo in EML4-ALK containing BAF3 cell lines. To further assess the potential ability of AZD3463 to overcome additional resistance mechanisms, antiproliferative activity was assessed in multiple crizotinib resistant cell lines independently derived in vitro from H3122 cells as well as a patient derived crizotinib relapsed model. These resistant cell lines contain multiple resistance mechanisms including the L1196M gatekeeper and T115Ins mutations, ALK amplification and/or secondary drivers including EGFR and IGF1R. AZD3463 retains antiproliferative potency within 4 fold of parental H3122 cells for 10 out of 12 of these acquired resistance models in vitro. In summary, AZD3463 is a potent ALK inhibitor which inhibits additional kinases including IGF1R and has activity in a number of crizotinib resistant models driven by multiple resistance mechanisms. Citation Format: Lisa Drew, Jane Cheng, Jeffrey Engelman, Douglas Ferguson, Ryohei Katayama, Brenda McDermott, Jamal Saeh, Alice Shaw, Minhui Shen, Dan Widzowski, Allan Wu, Graeme Smith. AZD3463, a novel ALK/IGF1R inhibitor, overcomes multiple mechanisms of acquired resistance to crizotinib. [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 919. doi:10.1158/1538-7445.AM2013-919