Anti-HER3 Monoclonal Antibody Patritumab Sensitizes Refractory Non-Small Cell Lung Cancer to the Epidermal Growth Factor Receptor Inhibitor Erlotinib
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Oncogene (2016) 35, 878–886 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE Anti-HER3 monoclonal antibody patritumab sensitizes refractory non-small cell lung cancer to the epidermal growth factor receptor inhibitor erlotinib K Yonesaka1, K Hirotani2, H Kawakami1, M Takeda1, H Kaneda1, K Sakai3, I Okamoto4, K Nishio3, PA Jänne5,6,7 and K Nakagawa1 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. Oncogene (2016) 35, 878–886; doi:10.1038/onc.2015.142; published online 11 May 2015 INTRODUCTION HER3 is a promising oncotherapeutic target.12 Monoclonal Members of the human epidermal growth factor receptor (HER) antibodies targeting HER3 have been investigated preclinically 13–16 family of proteins—which includes HER1 (also known as epidermal and clinically. One of these, patritumab, is a novel, growth factor receptor (EGFR)), HER2, HER3 and HER4—are fully human monoclonal antibody directed against the receptor tyrosine kinases (RTKs)1–3 that homodimerize or hetero- extracellular domain of HER3. Early clinical trials designed to dimerize when activated by ligand binding. Aberrant HER evaluate patritumab monotherapy revealed that it was signalling, which is caused by overexpression or activating well tolerated and limited tumour growth in some cancers, mutations, has a critical role in the oncogenesis of some cancer including NSCLC.17 Despite these promising results, the drug’s types.4,5 HER3 is occasionally amplified and aberrantly upregulated mechanism of action is incompletely understood, and a in cancers such as non-small cell lung cancer (NSCLC),6,7 and the biomarker has not yet been identified for the selection of an latter is associated with a poor prognosis.7 Owing to structural optimal target patient subpopulation who can benefitfrom features that limit its intrinsic kinase activity, HER3 cannot be patritumab treatment. auto-phosphorylated but can be transphosphorylated through EGFR is also a drug target in NSCLC, and somatic mutations in heterodimerization or higher-order clustering with other family the tyrosine kinase domain cause constitutive activation of EGFR members, such as HER2.1–3,8 HER3 is a major mediator of the in a subset of NSCLC;18,19 this activity can be blocked by EGFR- phosphoinositide 3-kinase/AKT cell survival signalling pathway.9 TKIs.20 Although EGFR-TKIs show robust effects on NSCLC with The HER3 ligand heregulin is locally secreted and induces HER3 activating EGFR mutations, the cells eventually acquire resistance; activation through an autocrine mechanism.10 Alternatively, HER3 in approximately half of such cases, this is attributable to can also be activated independently of ligand binding by the a secondary mutation in the EGFR kinase domain, most frequently dysregulation of other tyrosine kinase receptors.9,11 For example, a T790M substitution in exon 20.21,22 In addition, HER3 has a key in NSCLC cases harbouring an activating EGFR mutation, EGFR can role in EGFR-TKI resistance.23,24 Additionally, amplification of the activate HER3 in trans through heterodimerization.9 EGFR tyrosine MET oncogene can activate HER3, thereby switching on down- kinase inhibitors (TKIs) such as gefitinib can prevent HER3 stream phosphoinositide 3-kinase/AKT survival mechanisms, even phosphorylation in this subset of NSCLC. in the presence of an EGFR-TKI.25 Thus targeting HER3 may help 1Department of Medical Oncology, Kinki University School of Medicine, Osaka, Japan; 2Daiichi-Sankyo Pharmaceutical Development, Tokyo, Japan; 3Department of Genome Biology, Kinki University School of Medicine, Osaka, Japan; 4Center for Clinical and Translational Research, Kyushu University, Fukuoka, Japan; 5Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; 6Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA and 7Department of Medicine, Brigham Women’s Hospital, Harvard Medical School, Boston, MA, USA. Correspondence: Dr K Yonesaka, Department of Medical Oncology, Kinki University School of Medicine, 377-2 Ohno-higashi Osaka-sayamashi, Osaka-sayama, Osaka 589-8511, Japan or Dr PA Jänne, Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, HIM223, Boston, MA 02215, USA. E-mail: [email protected] or [email protected] Received 18 October 2014; revised 17 March 2015; accepted 23 March 2015; published online 11 May 2015 Patritumab sensitizes lung cancer to erlotinib K Yonesaka et al 879 prevent the development of cancer cell resistance to EGFR-TKIs. Patritumab sensitivity is associated with heregulin expression level To investigate this hypothesis, we evaluated the antitumorigenic in NSCLC efficacy of patritumab in NSCLC as a single agent and in We hypothesized that the differential sensitivity to patritumab was combination with established clinical EGFR inhibitors. due to molecular differences in the HER3 signalling pathway. Indeed, heregulin and HER3 mRNA expression levels varied across the 48 NSCLC cell lines (Supplementary Figure S1). Cell lines were RESULTS stratified into three groups (n = 16 each) based on their heregulin NSCLC cell lines exhibit differential sensitivity to patritumab mRNA expression level. According to the HER3 mRNA expression To evaluate the efficacy of patritumab in vitro, cell proliferation level, cell lines were divided into high and low expression groups was assessed in 48 NSCLC cell lines with the soft agar colony- (n = 24 each); growth inhibition rates upon treatment with 100 μg/ml formation assay. Cells were treated with patritumab for 7 days at patritumab were compared between groups. Cell lines with high 100 μg/ml, which is comparable to the concentration in the heregulin expression were more sensitive to patritumab than those 13,26 fi with medium or low heregulin expression (16.5% vs 1.1% vs 3.8%; plasma of cancer patients in phase II clinical trials. The ef cacy o of patritumab varied across cell lines, with growth inhibition P 0.002, Figure 1c), and all six patritumab-sensitive cell lines had high heregulin expression. In contrast, there were no differences in rates ranging from 0% to 40% (Figure 1a). Although patritumab the growth inhibition rates between the low and high HER3 had a marginal antiproliferative effect on most cell lines, in six of — — expression groups (7.5% vs 5.2%; P = 0.500). Notably, A549 and these QG56, HARA, SW900, H1944, H358 and A549 a growth HARA cells had the lowest HER3 expression among cell lines but 4 inhibition rate of 25% was observed (Supplementary Table S1). were nonetheless sensitive to patritumab due to high heregulin fi These lines were de ned as patritumab-sensitive, while expression (Supplementary Figure S1). theotherlineswereclassified as patritumab-refractory. Cell Activating EGFR mutations were identified in 10 cell lines (PC9, viability was reduced in a dose-dependent manner in HARA, HCC827, H1650, HCC4006, Ma70, Ma70GR, 11-18, H1975, PC9ZD QG56, A549 and SW900 cells treated with patritumab, while in and HCC827GR5 cells; Supplementary Table S2). Five of these lines refractory cell lines such as H1437, PC3, H1573 and H520, (PC9, HCC827, HCC4006, Ma70 and 11-18) were sensitive to EGFR- patritumab did not inhibit cell growth even at the highest TKI (data not shown), and all cell lines were refractory to concentration (Figure 1b). patritumab (Figure 1c, Supplementary Table S1). To verify whether Figure 1. Patritumab sensitivity depends on heregulin expression in NSCLC in vitro.(a) Patritumab sensitivity was evaluated using a soft agar colony-formation assay. NSCLC cell lines (n = 48) were treated with 100 μg/ml patritumab, and the growth inhibition rate was calculated relative to untreated control cells. (b) Patritumab-sensitive or -refractory cell lines (n = 8) were treated with the indicated concentrations of patritumab for 3 days; cell viability was evaluated using the CellTiter-Glo viability assay and shown relative to untreated control cells (mean