Anti-HER3 Monoclonal Antibody Patritumab Sensitizes Refractory Non-Small Cell Lung Cancer to the Epidermal Growth Factor Receptor Inhibitor Erlotinib

$Anti-HER3 Monoclonal Antibody Patritumab Sensitizes Refractory Non-Small Cell Lung Cancer to the Epidermal Growth Factor Receptor Inhibitor Erlotinib$ Oncogene (2015), 1–9 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 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 advance online publication, 11 May 2015; doi:10.1038/onc.2015.142 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, fully growth factor receptor (EGFR)), HER2, HER3 and HER4—are human monoclonal antibody directed against the extracellular receptor tyrosine kinases (RTKs)1–3 that homodimerize or hetero- domain of HER3. Early clinical trials designed to evaluate dimerize when activated by ligand binding. Aberrant HER patritumab monotherapy revealed that it was well tolerated and 17 signalling, which is caused by overexpression or activating limited tumour growth in some cancers, including NSCLC. mutations, has a critical role in the oncogenesis of some cancer Despite these promising results, the drug’s mechanism of action is types.4,5 HER3 is occasionally amplified and aberrantly upregulated incompletely understood, and a biomarker has not yet been in cancers such as non-small cell lung cancer (NSCLC),6,7 and the identified for the selection of an optimal target patient latter is associated with a poor prognosis.7 Owing to structural subpopulation who can benefit from patritumab treatment. features that limit its intrinsic kinase activity, HER3 cannot be EGFR is also a drug target in NSCLC, and somatic mutations in auto-phosphorylated but can be transphosphorylated through the tyrosine kinase domain cause constitutive activation of EGFR heterodimerization or higher-order clustering with other family in a subset of NSCLC;18,19 this activity can be blocked by EGFR- members, such as HER2.1–3,8 HER3 is a major mediator of the TKIs.20 Although EGFR-TKIs show robust effects on NSCLC with phosphoinositide 3-kinase/AKT cell survival signalling pathway.9 activating EGFR mutations, the cells eventually acquire resistance; The HER3 ligand heregulin is locally secreted and induces HER3 in approximately half of such cases, this is attributable to activation through an autocrine mechanism.10 Alternatively, HER3 a secondary mutation in the EGFR kinase domain, most frequently can also be activated independently of ligand binding by the a T790M substitution in exon 20.21,22 In addition, HER3 has a key dysregulation of other tyrosine kinase receptors.9,11 For example, role in EGFR-TKI resistance.23,24 Additionally, amplification of the in NSCLC cases harbouring an activating EGFR mutation, EGFR can MET oncogene can activate HER3, thereby switching on down- activate HER3 in trans through heterodimerization.9 EGFR tyrosine stream phosphoinositide 3-kinase/AKT survival mechanisms, even kinase inhibitors (TKIs) such as gefitinib can prevent HER3 in the presence of an EGFR-TKI.25 Thus targeting HER3 may help phosphorylation in this subset of NSCLC. prevent the development of cancer cell resistance to EGFR-TKIs. 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 Patritumab sensitizes lung cancer to erlotinib K Yonesaka et al 2 To investigate this hypothesis, we evaluated the antitumorigenic Patritumab sensitivity is associated with heregulin expression level efficacy of patritumab in NSCLC as a single agent and in in NSCLC combination with established clinical EGFR inhibitors. We hypothesized that the differential sensitivity to patritumab was 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 stratified into three groups (n = 16 each) based on their heregulin RESULTS mRNA expression level. According to the HER3 mRNA expression NSCLC cell lines exhibit differential sensitivity to patritumab level, cell lines were divided into high and low expression groups To evaluate the efficacy of patritumab in vitro, cell proliferation (n = 24 each); growth inhibition rates upon treatment with 100 μg/ml was assessed in 48 NSCLC cell lines with the soft agar colony- patritumab were compared between groups. Cell lines with high formation assay. Cells were treated with patritumab for 7 days at heregulin expression were more sensitive to patritumab than those μ with medium or low heregulin expression (16.5% vs 1.1% vs 3.8%; 100 g/ml, which is comparable to the concentration in the o plasma of cancer patients in phase II clinical trials.13,26 The efficacy P 0.002, Figure 1c), and all six patritumab-sensitive cell lines had high heregulin expression. In contrast, there were no differences in of patritumab varied across cell lines, with growth inhibition rates the growth inhibition rates between the low and high HER3 ranging from 0% to 40% (Figure 1a). Although patritumab had expression groups (7.5% vs 5.2%; P = 0.500). Notably, A549 and a marginal antiproliferative effect on most cell lines, in six of these HARA cells had the lowest HER3 expression among cell lines but — — QG56, HARA, SW900, H1944, H358 and A549 a growth were nonetheless sensitive to patritumab due to high heregulin 4 inhibition rate of 25% was observed (Supplementary Table expression (Supplementary Figure S1). S1). These lines were defined as patritumab-sensitive, while the Activating EGFR mutations were identified in 10 cell lines (PC9, other lines were classified as patritumab-refractory. Cell viability HCC827, H1650, HCC4006, Ma70, Ma70GR, 11-18, H1975, PC9ZD was reduced in a dose-dependent manner in HARA, QG56, A549 and HCC827GR5 cells; Supplementary Table S2). Five of these lines and SW900 cells treated with patritumab, while in refractory cell (PC9, HCC827, HCC4006, Ma70 and 11-18) were sensitive to EGFR- lines such as H1437, PC3, H1573 and H520, patritumab did not TKI (data not shown), and all cell lines were refractory to inhibit cell growth even at the highest 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 ± s.d. of six independent experiments). (c) Relationship between growth inhibition rate at 100 μg/ml patritumab and heregulin and HER3 expression levels among 48 NSCLC cell lines classified as high, medium or low expression (n = 16 each) for heregulin and high or low expression (n = 24 each) for HER3. Also shown are the growth inhibition rates for 10 NSCLC cell lines with activating EGFR mutations treated with 100 μg/ml patritumab. Patritumab-sensitive cell lines are highlighted in red. Data represent mean ± s.d. *Po0.002, unpaired t-test, high vs medium or low heregulin groups. (d) In a xenograft NSCLC model, cells expressing high (QG56, HARA and A549) or low (H1437, H520 and PC3) heregulin were treated with patritumab or vehicle.

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