Mi Ran Yun

Glycolysis inhibition sensitizes non-small cell lung cancer with T790M mutation to irreversible EGFR inhibitors via translational suppression of Mcl-1 by AMPK activation

The secondary EGF receptor (EGFR) T790M is the most common mechanism of resistance to reversible EGFR-tyrosine kinase inhibitors (TKI) in patients with non–small cell lung cancer (NSCLC) with activating EGFR mutations. Although afatinib (BIBW2992), a second-generation irreversible EGFR-TKI, was expected to overcome the acquired resistance, it showed limited efficacy in a recent phase III clinical study. In this study, we found that the inhibition of glycolysis using 2-deoxy-d-glucose (2DG) improves the efficacy of afatinib in H1975 and PC9-GR NSCLC cells with EGFR T790M. Treatment with the combination of 2DG and afatinib induced intracellular ATP depletion in both H1975 and PC9-GR cells, resulting in activation of AMP-activated protein kinase (AMPK). AMPK activation played a central role in the cytotoxicity of the combined treatment with 2DG and afatinib through the inhibition of mTOR. The alteration of the AMPK/mTOR signaling pathway by the inhibition of glucose metabolism induced specific downregulation of Mcl-1, a member of the antiapoptotic Bcl-2 family, through translational control. The enhancement of afatinib sensitivity by 2DG was confirmed in the in vivo PC9-GR xenograft model. In conclusion, this study examined whether the inhibition of glucose metabolism using 2DG enhances sensitivity to afatinib in NSCLC cells with EGFR T790M through the regulation of the AMPK/mTOR/Mcl-1 signaling pathway. These data suggest that the combined use of an inhibitor of glucose metabolism and afatinib is a potential therapeutic strategy for the treatment of patients with acquired resistance to reversible EGFR-TKIs due to secondary EGFR T790M. Mol Cancer Ther; 12(10); 2145–56. ©2013 AACR.

Phase II clinical and exploratory biomarker study of dacomitinib in patients with recurrent and/or metastatic squamous cell carcinoma of head and neck.

Purpose: The goals of this study were to investigate the clinical activity, safety, and biomarkers of dacomitinib, an irreversible tyrosine kinase inhibitor of EGFR, HER2, and HER4, in recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M-SCCHN). Experimental Design: Patients were eligible if the diseases were not amenable to curative treatment and had progressed on platinum-based chemotherapy, and were treated with dacomitinib 45 mg/day. The primary endpoint was objective response rate by RECISTv1.1. Exploratory analysis included the characterization of somatic mutation, gene copy number, gene expression, p16INK4A expression by IHC, and investigation of their relationship with clinical outcomes. Results: Forty-eight patients were evaluable for efficacy and toxicity. Ten patients (20.8%) had partial responses and 31 patients (65%) had stable diseases. The median progression-free survival (PFS) and overall survival (OS) were 3.9 months [95% confidence interval (CI), 2.9–5.0] and 6.6 months (95% CI, 5.4–10.3). Adverse events were mostly grade 1–2. Mutations in the PI3K pathway (PIK3CA, PTEN) and high expression of inflammatory cytokines (IL6, IL8, IL1A, IL1B, IL4, and TNF) were significantly associated with shorter PFS (2.9 vs. 4.9 months without mutations, P = 0.013; 2.8 vs. 9.9 months with low expression, P = 0.004). Those harboring PI3K pathway mutations or high inflammatory cytokine expression had shorter median OS (6.1 vs. 12.5 months lacking PI3K pathway mutations and with low inflammatory cytokine expression, P = 0.005). Conclusions: Dacomitinib demonstrated clinical efficacy with manageable toxicity in platinum-failed R/M-SCCHN patients. Screening of PI3K pathway mutation and inflammatory cytokine expression may help identify which R/M-SCCHN patients are likely to gain benefit from dacomitinib. Clin Cancer Res; 21(3); 544–52. ©2014 AACR.

Antitumor Activity and Acquired Resistance Mechanism of Dovitinib (TKI258) in RET-Rearranged Lung Adenocarcinoma

RET rearrangement is a newly identified oncogenic mutation in lung adenocarcinoma (LADC). Activity of dovitinib (TKI258), a potent inhibitor of FGFR, VEGFR, and PDGFR, in RET-rearranged LADC has not been reported. The aims of the study are to explore antitumor effects and mechanisms of acquired resistance of dovitinib in RET-rearranged LADC. Using structural modeling and in vitro analysis, we demonstrated that dovitinib induced cell-cycle arrest at G0–G1 phase and apoptosis by selective inhibition of RET kinase activity and ERK1/2 signaling in RET-rearranged LC-2/ad cells. Strong antitumor effect of dovitinib was observed in an LC-2/ad tumor xenograft model. To identify the acquired resistance mechanisms to dovitinib, LC-2/ad cells were exposed to increasing concentrations of dovitinib to generate LC-2/ad DR cells. Gene-set enrichment analysis of gene expression and phosphor-kinase revealed that Src, a central gene in focal adhesion, was activated in LC-2/ad DR cells. Saracatinib, an src kinase inhibitor, suppressed ERK1/2 phosphorylation and growth of LC-2/ad DR cells. Taken together, these findings suggest that dovitinib can be a potential therapeutic option for RET-rearranged LADC, in which acquired resistance to dovitinib can be overcome by targeting Src. Mol Cancer Ther; 14(10); 2238–48. ©2015 AACR.

EGFR-Mediated Reactivation of MAPK Signaling Induces Acquired Resistance to GSK2118436 in BRAF V600E–Mutant NSCLC Cell Lines

Although treatment of BRAF V600E–mutant non–small cell lung cancer (NSCLCV600E) with GSK2118436 has shown an encouraging efficacy, most patients develop resistance. To investigate the mechanisms of acquired resistance to GSK2118436 in NSCLCV600E, we established GSK2118436-resistant (GSR) cells by exposing MV522 NSCLCV600E to increasing GSK2118436 concentrations. GSR cells displayed activated EGFR–RAS–CRAF signaling with upregulated EGFR ligands and sustained activation of ERK1/2, but not MEK1/2, in the presence of GSK2118436. Treatment of GSR cells with GSK2118436 enhanced EGFR-mediated RAS activity, leading to the formation of BRAF–CRAF dimers and transactivation of CRAF. Interestingly, sustained activation of ERK1/2 was partly dependent on receptor-interacting protein kinase-2 (RIP2) activity, but not on MEK1/2 activity. Combined BRAF and EGFR inhibition blocked reactivation of ERK signaling and improved efficacy in vitro and in vivo. Our findings support the evaluation of combined BRAF and EGFR inhibition in NSCLCV600E with acquired resistance to BRAF inhibitors. Mol Cancer Ther; 15(7); 1627–36. ©2016 AACR.

Profiling of protein–protein interactions via single-molecule techniques predicts the dependence of cancers on growth-factor receptors

The accumulation of genetic and epigenetic alterations in cancer cells rewires cellular signalling pathways through changes in the patterns of protein–protein interactions (PPIs). Understanding these patterns may facilitate the design of tailored cancer therapies. Here, we show that single-molecule pull-down and co-immunoprecipitation techniques can be used to characterize signalling complexes of the human epidermal growth-factor receptor (HER) family in specific cancers. By analysing cancer-specific signalling phenotypes, including post-translational modifications and PPIs with downstream interactions, we found that activating mutations of the epidermal growth-factor receptor (EGFR) gene led to the formation of large protein complexes surrounding mutant EGFR proteins and to a reduction in the dependency of mutant EGFR signalling on phosphotyrosine residues, and that the strength of HER-family PPIs is correlated with the strength of the dependence of breast and lung adenocarcinoma cells on HER-family signalling pathways. Furthermore, using co-immunoprecipitation profiling to screen for EGFR-dependent cancers, we identified non-small-cell lung cancers that respond to an EGFR-targeted inhibitor. Our approach might help predict responses to targeted cancer therapies, particularly for cancers that lack actionable genomic mutations.The characterization of protein–protein interactions by combined single-molecule pull-down and single-molecule co-immunoprecipitation assays uncovers, for specific cancers, their dependence on signalling complexes of the human epidermal growth-factor receptor.

Establishment of a Conditional Transgenic Mouse Model Recapitulating EML4-ALK–Positive Human Non–Small Cell Lung Cancer

Background: Anaplastic lymphoma receptor tyrosine kinase gene (ALK) fusion is a distinct molecular subclassification of NSCLC that is targeted by anaplastic lymphoma kinase (ALK) inhibitors. We established a transgenic mouse model that expresses tumors highly resembling human NSCLC harboring echinoderm microtubule associated protein like 4 gene (EML)‐ALK fusion. We aimed to test an EML4‐ALK transgenic mouse model as a platform for assessing the efficacy of ALK inhibitors and examining mechanisms of acquired resistance to ALK inhibitors. Methods: Transgenic mouse lines harboring LoxP‐STOP‐LoxP‐FLAGS–tagged human EML4‐ALK (variant 1) transgene was established by using C57BL/6N mice. The transgenic mouse model with highly lung‐specific, inducible expression of echinoderm microtubule associated protein like 4–ALK fusion protein was established by crossing the EML4‐ALK transgenic mice with mice expressing Cre–estrogen receptor fusion protein under the control of surfactant protein C gene (SPC). Expression of EML4‐ALK transgene was induced by intraperitoneally injecting mice with tamoxifen. When the lung tumor of the mice treated with the ALK inhibitor crizotinib for 2 weeks was measured, tumor shrinkage was observed. Results: EML4‐ALK tumor developed after 1 week of tamoxifen treatment. Echinoderm microtubule associated protein like 4–ALK was strongly expressed in the lung but not in other organs. ALK and FLAGS expressions were observed by immunohistochemistry. Treatment of EML4‐ALK tumor–bearing mice with crizotinib for 2 weeks induced dramatic shrinkage of tumors with no signs of toxicity. Furthermore, prolonged treatment with crizotinib led to acquired resistance in tumors, resulting in regrowth and disease progression. The resistant tumor nodules revealed acquired ALK G1202R mutations. Conclusions: An EML4‐ALK transgenic mouse model for study of drug resistance was successfully established with short duration of tumorigenesis. This model should be a strong preclinical model for testing efficacy of ALK TKIs, providing a useful tool for investigating the mechanisms of acquired resistance and pursuing novel treatment strategies in ALK‐positive lung cancer.

ER2, a novel human anti-EGFR monoclonal antibody inhibit tumor activity in non-small cell lung cancer models.

OBJECTIVES The epidermal growth factor receptor (EGFR) abnormalities including amplification, mutation, and overexpression are frequent in non-small cell lung cancer (NSCLC). We investigated in vitro and in vivo antitumor activity of ER2, a novel human anti-EGFR monoclonal antibody, in NSCLC. METHODS A panel of NSCLC cell lines (A549, H460, H322, H358, H1299, HCC827, PC9, H1975, and PC9-GR) was used to evaluate in vitro antitumor activity of ER2 and cetuximab. The inhibitory effects of ER2 and cetuximab on downstream signaling were assessed by western blot. Secreted VEGF was measured by Human VEGF Quantikine ELISA kit. Antitumor effects of ER2 and cetuximab as single agents and in combination with cisplatin were evaluated in H322, HCC827 and A549 xenograft models. RESULTS ER2 efficiently inhibits EGFR and its downstream signaling molecules including Akt and Erk1/2 in NSCLC cell lines with wild-type or mutant EGFR. ER2 inhibited cell viability of H322, HCC827 and A549 cells in a dose-dependent manner by inducing cell cycle arrest and apoptosis. Also, ER2 suppressed EGF-stimulated VEGF production as efficiently as cetuximab in H322, HCC827 and A549 cells. Moreover, ER2 alone and in combination with cisplatin showed a significant anti-tumor efficacy in xenograft mouse models. CONCLUSION Taken together, ER2 has significant anti-tumor activity in in vitro and in vivo NSCLC models, suggesting a rationale for clinical development of ER2 in NSCLC.

Establishment of a platform of non-small-cell lung cancer patient-derived xenografts with clinical and genomic annotation

BACKGROUND Preclinical models that can better predict therapeutic activity in clinical trials are needed in this era of personalized cancer treatment. Herein, we established genomically and clinically annotated patient-derived xenografts (PDXs) from non-small-cell lung cancer (NSCLC) patients and investigated whether these PDXs would faithfully recapitulate patient responses to targeted therapy. METHODS Patient-derived tumors were implanted in immunodeficient mice and subsequently expanded via re-implantation. Established PDXs were examined by light microscopy, genomic profiling, and in vivo drug testing, and the successful engraft rate was analyzed with the mutation profile, histology, or acquisition method. Finally, the drug responses of PDXs were compared with the clinical responses of the respective patients. RESULTS Using samples from 122 patients, we established 41 NSCLC PDXs [30 adenocarcinoma (AD), 11 squamous cell carcinoma (SQ)], among which the following driver mutation were observed: 13 EGFR-mutant, 4 ALK-rearrangement, 1 ROS1-rearrangement, 1 PIK3CA-mutant, 1 FGFR1-amplification, and 2 KRAS-mutant. We rigorously characterized the relationship of clinical features to engraftment rate and latency rates. The engraft rates were comparable across histologic type. The AD engraft rate tended to be higher for surgically resected tissues relative to biopsies, whereas similar engraft rates was observed for SQ, irrespective of the acquisition method. Notably, EGFR-mutants demonstrated significantly longer latency time than EGFR-WT (86 vs. 37days, P = 0.007). The clinical responses were recapitulated by PDXs harboring driver gene alteration (EGFR, ALK, ROS1, or FGFR1) which regressed to their target inhibitors, suggesting that established PDXs comprise a clinically relevant platform. CONCLUSION The establishment of genetically and clinically annotated NSCLC PDXs can yield a robust preclinical tool for biomarker, therapeutic target, and drug discovery.

Enhancer Remodeling and MicroRNA Alterations Are Associated with Acquired Resistance to ALK Inhibitors

Anaplastic lymphoma kinase (ALK) inhibitors are highly effective in patients with ALK fusion-positive lung cancer, but acquired resistance invariably emerges. Identification of secondary mutations has received considerable attention, but most cases cannot be explained by genetic causes alone, raising the possibility of epigenetic mechanisms in acquired drug resistance. Here, we investigated the dynamic changes in the transcriptome and enhancer landscape during development of acquired resistance to ALK inhibitors. Histone H3 lysine 27 acetylation (H3K27ac) was profoundly altered during acquisition of resistance, and enhancer remodeling induced expression changes in both miRNAs and mRNAs. Decreased H3K27ac levels and reduced miR-34a expression associated with the activation of target genes such as AXL. Panobinostat, a pan-histone deacetylase inhibitor, altered the H3K27ac profile and activated tumor-suppressor miRNAs such as miR-449, another member of the miR-34 family, and synergistically induced antiproliferative effects with ALK inhibitors on resistant cells, xenografts, and EML4-ALK transgenic mice. Paired analysis of patient samples before and after treatment with ALK inhibitors revealed that repression of miR-34a or miR-449a and activation of AXL were mutually exclusive of secondary mutations in ALK. Our findings indicate that enhancer remodeling and altered expression of miRNAs play key roles in cancer drug resistance and suggest that strategies targeting epigenetic pathways represent a potentially effective method for overcoming acquired resistance to cancer therapy.Significance: Epigenetic deregulation drives acquired resistance to ALK inhibitors in ALK-positive lung cancer. Cancer Res; 78(12); 3350-62. ©2018 AACR.

Abstract 2104: ERK-dependent IL-6 autocrine signaling mediates adaptive resistance to pan-PI3K inhibitor in head and neck squamous cell carcinoma

Purpose: To investigate resistant mechanisms to NVP-BKM120, a pan-PI3K inhibitor, and potential combination therapies in head and neck squamous cell carcinoma (HNSCC). Experimental Design: A panel of 10 human HNSCC cell lines including NVP-BKM120-resistant HNSCC patient-derived cell lines were used to evaluate the antitumor activity of NVP-BKM120. Patient-derived tumor xenograft (PDTX) models were established from recurrent/metastatic HNSCC patients treated with and resistant to NVP-BKM120. Results: Treatment of NVP-BKM120 led to upregulation of interleukin 6 (IL-6) and subsequent activation of either extracellular signal-regulated kinase (ERK) or signal transducers and activators of transcription 3 (STAT3), resulting in a modest antitumor effects on HNSCC cells. In a subset of cell lines, blockade of IL-6 autocrine signaling with IL-6 receptor (IL-6R) siRNA or IL-6R neutralizing antibody completely abolished constitutive or NVP-BKM120-induced activation of ERK and STAT3 as well as expression of oncogenic factors including Bcl2 and cMYC, resulting in the enhanced sensitivity to NVP-BKM120 with a marked reduction of S phase and significant increment of G0/G1 arrest. Interestingly, trametinib, a MEK inhibitor, significantly reduced NVP-BKM120-induced IL-6 and cMYC upregulation, whereas it had no effect on STAT3 inhibition. Moreover, combination of NVP-BKM120 with trametinib yielded more potent anti-tumor effects than combination with a pharmacologic inhibitor of STAT3 or siSTAT3. Furthermore, combination of NVP-BKM120 with trametinib or tocilizumab, a humanised anti-IL-6 receptor antibody, synergistically suppressed the growth of NVP-BKM120-resistant PDTX models as compared with either agent alone, which was confirmed in two PDTX-derived cell lines. Conclusions: Collectively, these results suggested that IL-6/ERK signaling contributes to intrinsic and adaptive resistance to NVP-BKM120 in HNSCC, providing a rationale for a novel therapeutic strategy to overcome resistance to pan-PI3K inhibitors. Citation Format: Mi Ran Yun, Han Na Kang, Byoung Chul Cho. ERK-dependent IL-6 autocrine signaling mediates adaptive resistance to pan-PI3K inhibitor in head and neck squamous cell carcinoma. [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 2104.