Han Na Kang

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.

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.

Abstract 5194: A Mouse-Human co-clinical trial with patient-derived xenograft (PDX) models demonstrates a predictive signature for dovitinib (TKI258), an FGFR and VEGFR inhibitor,in lung squamous cell carcinomalung cancer (LSCC)

Background: We conducted a co-clinical trial with PDX models in conjunction with a phase II clinical trial with dovitinib in patients with fibroblast growth factor receptor 1 (FGFR1) amplified LSCC in order to identify a predictive biomarker for dovitinib, a FGFR inhibitor. Methods: PDX models were established using tumor samples obtained from LSCC patients enrolled in the trial. We conducted an in vivo efficacy test with dovitinib in PDX models and comprehensive molecular profiling by whole exome sequencing (WES), array comparative genomic hybridization (aCGH), and microarray-based gene expression. Results: The histological and genomic characteristics of xenograft tumors (F2) resembled case-matched original tumors (F0). Dovitinib treatment resulted in tumor shrinkage in PDXL01, but not in PDXL02-04, which corresponded to responses in LSCC patients enrolled in the clinical trial. The patient, from whom PDXL01 was derived, showed a partial response to dovitinib with progression-free survival of 6 months, whereas the patient, from whom PDXL04 was derived had rapid disease progression within 2 months of treatment. Mutation profiles of F0 and F2 were largely concordant with each other in at least 70% of somatic mutations. The genome-wide copy number alterations were also largely concordant between F0 and F2, suggesting that PDX tumors can successfully represent the genomic features of the tumors from LSCC patients. Notably, mutation in FGFR 1-3 genes was not observed. FGFR1 amplification largely due to arm-level 8p gain was consistently observed, regardless of sensitivity to dovitinib. This finding suggests that FGFR1 amplification may not be a predictor for dovitinib sensitivity. Finally, we compared gene expression between a responder and non-responders. In a Heatmap analysis of the top 50 significantly up- and down-regulated genes, FGFR gene signature including FGF3 and FGF19 was significantly up-regulated in the responder. Gene set enrichment analysis (GSEA) identified that gene sets, such as FGFR ligand binding and activation and SHC-mediated cascade pathway, were significantly enriched in the responder, highlighting FGFR pathway activation as a key molecular determinant for sensitivity to dovitinib. To identify predictive gene signatures from independent datasets, we further performed GSEA in dovitinib-sensitive lung cancer cell lines compared to resistant ones as available in the CCLE (Cancer Cell Line Encyclopedia) database. Among the four functional modules (FGFR signaling, Immune, Cell cycle, and RNA) that we identified, the FGFR signaling module performed well in the prediction of dovitinib sensitivity of PDXL01-04 Conclusions: FGFR gene expression signatures are predictors for response to dovitinib in LSCC. Citation Format: Hye Ryun Kim, Han Na Kang, Sung Moo Kim, Hwan Kim, Kyoung-Ho Pyo, Myung-Ju Ahn, Tae Min Kim, Byoung Chul Cho. A Mouse-Human co-clinical trial with patient-derived xenograft (PDX) models demonstrates a predictive signature for dovitinib (TKI258), an FGFR and VEGFR inhibitor,in lung squamous cell carcinomalung cancer (LSCC). [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 5194.