Tumor Biol. DOI 10.1007/s13277-014-2252-y

RESEARCH ARTICLE

Blocking the PI3K pathway enhances the efficacy of ALK-targeted therapy in EML4-ALK-positive nonsmall-cell lung cancer Lin Yang & Guangchao Li & Likun Zhao & Fei Pan & Jiankun Qiang & Siqi Han

Received: 20 April 2014 / Accepted: 18 June 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Targeted therapy based on ALK tyrosine kinase inhibitors (ALK-TKIs) has made significant achievements in individuals with EML4-ALK (echinoderm microtubuleassociated protein-like 4 gene and the anaplastic lymphoma kinase gene) fusion positive nonsmall-cell lung cancer (NSCLC). However, a high fraction of patients receive inferior clinical response to such treatment in the initial therapy, and the exact mechanisms underlying this process need to be further investigated. In this study, we revealed a persistently activated PI3K/AKT signaling that mediates the drug ineffectiveness. We found that genetic or pharmacological inhibition of ALK markedly abrogated phosphorylated STAT3 and ERK, but it failed to suppress AKT activity or induce apoptosis, in EML4-ALK-positive H2228 cells. Furthermore, targeted RNA interference of PI3K pathway components restored sensitivity to TAE684 treatment at least partially due to

Lin Yang and Guangchao Li contributed equally to this work and are joint first authors. L. Yang Department of Clinical Laboratory, Hubei Maternal and Child Health Hospital, Wuhan 430070, China G. Li : L. Zhao School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China F. Pan PLA General Hospital Cancer Center, PLA Postgraduate School of Medicine, Beijing 100853, China J. Qiang School of Biology and Basic Medical Sciences, Soochow University, Suzhou 215006, China S. Han (*) Department of Medical Oncology, Jinling Hospital, 305 Zhongshan North Road, Nanjing 210002, China e-mail: [email protected]

increased apoptosis. Combined TAE684 with PI3K inhibitor synergistically inhibited the proliferation of EML4-ALKpositive cells in vitro and significantly suppressed the growth of H2228 xenografts in vivo, suggesting the potential clinical application of such combinatorial therapy regimens in patients with EML4-ALK positive lung cancer. Keywords Nonsmall-cell lung cancer . EML4-ALK . PI3K pathway . Combination therapy

Introduction Anaplastic lymphoma kinase (ALK) is a member of the insulin receptor superfamily (IRK) of receptor tyrosine kinases (RTKs) [1]. The chromosomal structural rearrangements which are the most common form of constitutively activated ALK have played a key role in the tumorigenic process. Two significant ALK-fusion proteins NPM-ALK and EMLA-ALK have been elaborately described and implicated in the development in about 75 % of anaplastic large-cell lymphoma (ALCL) [2] and 4–7 % of nonsmall-cell lung cancers (NSCLC) [3, 4], respectively. Aberrant ALK activation as a result of gene amplification also participates in the pathogenesis of neuroblastomas (NB) [5]. Additionally, activating ALK point mutations have the potential to cause certain types of cancer, such as neuroblastomas and anaplastic thyroid cancer (ATC) [6, 7]. Cancer cells harboring the genomic ALK translocations exhibit high sensitivity to ALK tyrosine kinase inhibitors (ALK-TKIs). Two of these inhibitors, crizotinib [8] (now FDA-approved as Xalkori) and TAE684 [9], have already been employed in numbers of scientific studies. The real patient benefit of crizotinib in treatment of ALK-positive NSCLC provides a potential avenue for therapeutic intervention towards against tumors with ALK alterations.

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Unfortunately, a number of patients carrying EML4-ALKpositive NSCLC derive inferior responses to these inhibitors, even in the initial therapy (intrinsic resistance) [10]. Although recent publications have proposed the possibility of rare events for the initial resistance, such as (1) concurrency of ALK rearrangement and EGFR/KRAS mutation [11, 12] or (2) preexisting genetic mutations in EML4-ALK that block crizotinib binding [13]. However, such events are real but minimal, and there must be other more important sources that get overlooked. To improve initial response to ALK-TKIs that expands the beneficiary population from ALK-targeted therapy, urgent studies are desperately needed to investigate the potential mechanisms and explore clinically applicable regimens. Here, our work proposes a novel mechanism driving insensitivity to ALK inhibitors in EML4-ALK-positive NSCLC. We identify PI3K pathway as the limit of drug responsiveness, which is constitutively activated in cells under treatment. Notably, targeting PI3K pathway effectively sensitizes cancer cells to ALK-TKIs treatment in vitro and in vivo. For the first time, our findings indicate that combinatorial regimen of ALK-targeted therapy plus PI3K inhibitors could be a potential therapeutic strategy for EML4-ALK-positive NSCLC.

lentiviral viruses were harvested and then transduced into the BEAS-2B cells. The stably transfected cells were selected in the presence of 3.0 μg/ml puromycin (Sigma) and validated by flow cytometry using an rabbit anti-ALK D5F3 antibody (Cell Signaling Technology).

Cell proliferation assay Cells were seeded into 96-well plates at 3×103 cells per well. The medium in the wells was replaced the next day with fresh medium containing drugs as indicated. After 4 days of continuous culture, cell proliferation in response to drugs was determined using CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. IC50 values were calculated by nonlinear regression using GraphPad Prism (Castro Valley, CA, USA). The nature of the drug interactions were determined by the method of Chou and Talalay [14, 15]. The combination index (CI) was calculated to determine the presence of synergism (CI 1), or additive effect (CI=1).

Immunoblot analysis Materials and methods Compounds and cell lines Crizotinib (ALK inhibitor), AZD6244 (ERK inhibitor), and S3I-201 (STAT3 inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA). TAE684 (ALK inhibitor) and BKM120 (PI3K inhibitor) were synthesized by Shanghai Biochempartner (Shanghai, China). KARPAS-299, H2228, A549 cell lines and normal human bronchial epithelial cells BEAS-2B were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) supplemented with 10 % fetal bovine serum (HyClone) at 37 °C in 5 % CO2. Generation of the BEAS-2BEML4-ALK cell line EML4-ALK variant 1 clone and a mutant version of the fusion (ALK-KD), where the kinase domain of ALK is inactivated by a point mutation (K589M), were synthesized by Sangon Biotech (Shanghai, China). The lentiviral plasmid PLVXIRES-puro (Clontech) expressing the variant 1 EML4-ALK or the kinase dead (K589M) variant 1 EML4-ALK, PCMVDR8.91 (Clontech) and PCMV-VSV-G (Clontech) were cotransfected into 293 T cells using Lipofectamine 2000 (Invitrogen). After transfection for 2 days, the recombinant

1×106 cells were seeded in 6-well plates in the inhibitor-free medium. Cells were incubated the next day with the indicated concentrations of inhibitors for 24 h. Cell lysates were prepared and subjected to immunoblot analysis according to the protocols provided by the antibody suppliers. Primary antibodies to phosphorylated ALK (p-ALK, Y1608), phosphorylated AKT (p-AKT, S473), phosphorylated ERK (p-ERK, T202/Y204), phosphorylated STAT3 (p-STAT3, Y705), ALK, AKT, ERK, STAT3, and caspase3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against GAPDH and secondary HRP-conjugated antibodies were purchased from CoWin Biotech (Beijing, China).

Gene silencing 3×105 cells were seeded in 6-well plates in 2 ml medium. After cells have attached overnight, cultures were changed into serum-free medium. Small interfering RNA (siRNA) specifically targeting p110α (Cell Signaling Technology, Beverly, MA, USA) and AKT (Cell Signaling Technology) were prepared in opti-MEM (Invitrogen, Carlsbad, CA, USA) mixed with Lipofectamine 2000 (Invitrogen) for 25 min before transferring to culture mixture. After 6 h incubation, the medium was removed, then replaced with fresh medium and continue to culture. After another 24 h, cells were prepared for indicated detection.

Tumor Biol.

Annexin V binding assay Cells (5×105/well in 6-well plates) were cultured overnight and treated with inhibitors at indicated doses for 60 h. For some experiments, cells were preincubated in the presence or absence of 50 μM Z-VAD-fmk (a pan-caspase inhibitor) for 1 h. Then cells were collected by centrifugation and stained with annexin V following the manufacturer’s procedures (Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit, Invitrogen, CA, USA). Binding of annexin V to cells was measured by flow cytometry. In vivo studies 5×106 cells were injected subcutaneously into the flank of 6week-old BALB/c nu/nu female mice (Weitonglihua Biotechnology, Beijing, China). When tumors reached a size of ~100 mm3, the mice were randomized into four groups (six mice per group) for the following treatments: control (0.5 % (w/v) aqueous solution of hydroxypropylmethylcellulose], TAE684 (10 mg/kg, in DMSO), BKM120 (15 mg/kg, in 0.5 % (w/v) aqueous solution of hydroxypropylmethylcellulose), or combination of TAE684 (10 mg/kg) and BKM120 (15 mg/kg). Each agent was administered by oral gavage (og) daily. Tumor volumes were measured every 3 days using caliper measurements and calculated by the formula π (length×width2)/6. Statistical analyses Quantitative data were expressed as mean±s.e.m. The significance of differences between groups was assessed by twotailed t-test or one-way ANOVA using the GraphPad Prism program version 5 (GraphPad Software, USA). The asterisk indicates a statistically significant difference: *p < 0.05, **p

Blocking the PI3K pathway enhances the efficacy of ALK-targeted therapy in EML4-ALK-positive nonsmall-cell lung cancer.

Targeted therapy based on ALK tyrosine kinase inhibitors (ALK-TKIs) has made significant achievements in individuals with EML4-ALK (echinoderm microtu...
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