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De-Repression of Pdgfrb Transcription Promotes Acquired

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De-Repression of Pdgfrb Transcription Promotes Acquired Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 RESEARCH ARTICLE De-Repression of PDGFR b Transcription Promotes Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Glioblastoma Patients David Akhavan1 , 2 , Alexandra L. Pourzia 3 , Alex A. Nourian 3 , Kevin J. Williams3 , 7 , David Nathanson 2 , Ivan Babic 9 , Genaro R. Villa 1 , 2 , 9 , Kazuhiro Tanaka 2 , Ali Nael 2 , Huijun Yang 9 , Julie Dang 2 , Harry V. Vinters 3 , William H. Yong 3 , Mitchell Flagg3 , Fuyuhiko Tamanoi 5 , Takashi Sasayama 12 , C. David James 8 , Harley I. Kornblum 4 , Tim F. Cloughesy 6 , Webster K. Cavenee 9 , 10 , Steven J. Bensinger 2 , 3 , 7 , and Paul S. Mischel 9 , 10 , 11 Downloaded from cancerdiscovery.aacrjournals.org on September 23, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 ABSTRACT Acquired resistance to tyrosine kinase inhibitors (TKI) represents a major chal- lenge for personalized cancer therapy. Multiple genetic mechanisms of acquired TKI resistance have been identifi ed in several types of human cancer. However, the possibility that cancer cells may also evade treatment by co-opting physiologically regulated receptors has not been addressed. Here, we show the fi rst example of this alternate mechanism in brain tumors by show- ing that EGF receptor (EGFR)-mutant glioblastomas (GBMs) evade EGFR TKIs by transcriptionally de-repressing platelet-derived growth factor receptor β (PDGFRβ). Mechanistic studies show that EGFRvIII signaling actively suppresses PDGFR b transcription in an mTORC1- and extracellular signal– regulated kinase-dependent manner. Genetic or pharmacologic inhibition of oncogenic EGFR renders GBMs dependent on the consequently de-repressed PDGFRβ signaling for growth and survival. Impor- tantly, combined inhibition of EGFR and PDGFRβ signaling potently suppresses tumor growth in vivo . These data identify a novel, nongenetic TKI resistance mechanism in brain tumors and provide compel- ling rationale for combination therapy. SIGNIFICANCE: These results provide the fi rst clinical and biologic evidence for receptor tyrosine kinase (RTK) “switching” as a mechanism of resistance to EGFR inhibitors in GBM and provide a molecu- lar explanation of how tumors can become “addicted” to a nonamplifi ed, nonmutated, physiologically regulated RTK to evade targeted treatment. Cancer Discov; 3(5); 1–14. ©2013 AACR. INTRODUCTION PTEN ( 2 ), PIK3CA ( 5 ), or KRAS ( 6 ); and (iii) co-occurrence of other amplifi ed or mutated receptor tyrosine kinases The EGF receptor, EGFR, is commonly amplifi ed and/ (RTK), including C-MET and platelet-derived growth factor or mutated in many types of solid cancer, including a receptor α (PDGFRα; refs. 7, 8 ). In addition to these genetic variety of epithelial cancers and glioblastoma (GBM) ( 1–3 ). resistance-promoting mutations, it is suspected that EGFR- Despite compelling evidence for EGFR addiction in experi- dependent cancers may escape targeted therapy by devel- mental models, the clinical benefi t of most EGFR tyrosine oping dependence on other nonamplifi ed, nonmutated kinase inhibitors (TKI) has been quite limited. Multiple RTKs ( 9 ). However, the mechanisms by which cancers, genetic resistance mechanisms enable cancer cells to main- including GBM, evade EGFR TKI treatment by coopt- tain signal fl ux to critical downstream effector pathways, ing other physiologically regulated receptors has not been and thus evade EGFR-targeted therapy, including: (i) addressed. acquisition and/or selection for secondary EGFR muta- Herein, we integrate studies in cell lines, patient-derived tions conferring EGFR TKI resistance ( 4 ); (ii) additional tumor cultures, xenotransplants, and tumor tissue from activating mutations in downstream effectors, such as patients with GBM in a phase II clinical trial to provide the fi rst demonstration of EGFR TKI resistance mediated by transcrip- Authors’ Affi liations: 1 Medical Scientist Training Program, David Geffen tional de-repression of PDGFRb. We show that the persist- School of Medicine; Departments of 2 Molecular and Medical Pharmacol- ently active EGFR mutation, EGFRvIII, suppresses PDGFRβ ogy, 3 Pathology and Laboratory Medicine, 4 Psychiatry and Biobehavioral expression via mTORC1- and extracellular signal–regulated Sciences, and 5 Microbiology, Immunology & Molecular Genetics; 6 Neuro- 7 kinase (ERK)-dependent mechanisms. We show in cells, mice, Oncology Program; Institute for Molecular Medicine, University of Cali- β, fornia Los Angeles, Los Angeles; 8 Brain Tumor Research Center, University and patients that EGFR TKI treatment de-represses PDGFR of California San Francisco, San Francisco; 9 Ludwig Institute for Cancer rendering GBMs dependent on PDGFRβ signaling for growth. Research; 10 Moores Cancer Center; 11 Department of Pathology, Univer- We further show that combined abrogation of EGFRvIII and sity of California San Diego, La Jolla, California; and 12 Department of PDGFRβ potently prevents GBM growth in vivo . These results Neurosurgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, identify a novel physiologic EGFR TKI resistance mechanism Japan in GBM and suggest a clinically actionable approach to sup- Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). press it. Corresponding Authors: Paul S. Mischel, Ludwig Institute for Can- cer Research, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0753. Phone: 858-534-6080; Fax: 858-534-7750; RESULTS E-mail: [email protected] ; and Steven J. Bensinger, Institute for Molecular EGFR Inhibition Promotes PDGFRb Medicine Department of Pathology and Laboratory Medicine, 36-128 Upregulation in Glioma Center for Health Sciences, University of California Los Angeles, Los Angeles, CA 90095. Phone: 310-825-9885; Fax: 310-267-6267; To better understand how malignant glioma acquires resist- E-mail: [email protected] ance to EGFR inhibitors in vivo , U87 glioma cells expressing doi: 10.1158/2159-8290.CD-12-0502 the EGFRvIII gain-of-function mutation (designated U87- © 2013 American Association for Cancer Research. EGFRvIII herein) were placed in the fl anks of severe combined MAY 2013CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on September 23, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 RESEARCH ARTICLE Akhavan et al. ABDays post-injection ) 3 600 Drug Drug Drug 500 20181614121086420 400 EGFRvIII 300 Tumor injection MeasureMeasure Measure 200 EGFRvIII 100 Erlotinib 150 mg/kg/d Tumor volume (mm 0 Day 10 Day 13 Day 17 C D U87vIII pEGFR U87 U87vIIIU87vIIIU87vIII Erlot. K.D. PDGFRβ pPDGFRβ Y751 U87vIII erlotinib pEGFR Y1086 150 mg/kg, 18 d β EGFR pPDGFR PI3K-p85 pEGFR pAXL E F GBM39 GBM6 HK296 pEGFR Y1086 PDGFRβ Erlotinib (5 μmol/L) –+ –+ –+ PDGFRα EGFRvIII Vehicle PDGFRβ pEGFR Y1068 PI3K-p85 pEGFR Y1086 PDGFRβ GBM12 HK242 Erlotinib (5 μmol/L) – + –+ Wild-type EGFR PDGFRα Erlotinib PDGFRβ pEGFR Y1068 PI3K-p85 G GBM39 Vehicle Erlotinib Tumor 1 Tumor 2 Tumor 1 Tumor 2 pPDGFRb Y751 PDGFRβ pEGFR Y1086 PI3K-p85 Figure 1. A reciprocal relationship between EGFR and PDGFRβ in glioma. A, experimental design of a mouse model of EGFR inhibitor resistance. U87- EGFRvIII cells were subcutaneously implanted in the mouse fl ank on day 0. Mice were treated with erlotinib (150 mg/kg) on day 2 and as indicated there- after. B, tumor growth curve of U87-EGFRvIII xenografts in mice treated with erlotinib (150 mg/kg as indicated in A) or vehicle. C, immunoblot of indicated tumor lysates determining total and phospho-PDGFRβ or EGFR from U87, U87-EGFRvIII ± eroltinib treatment and U87-EGFRvIII kinase dead xenografts harvested on day 21. PI3K-p85 is used as a loading control in this and subsequent immunoblots. D, RTK array of 42 RTKs conducted on U87-EGFRvIII xenograft lysates on day 21 from mice treated with erlotinib or vehicle as described in A. E, immunohistochemistry (IHC) of PDGFRβ and phospho-EGFR in vehicle- and erlotinib-treated U87-EGFRvIII orthotopic xenografts. F, immunoblot of PDGFRα, PDGFRβ, and phospho-EGFR (pEGFR) from patient-derived GBM neurospheres expressing EGFRvIII or wild-type EGFR as indicated. Whole-cell lysates were collected after 24 hours of erlotinib or vehicle treatment. G, immunoblot of tumor lysates from EGFRvIII expressing GBM39 xenografts following oral gavage with vehicle or erlotinib for 10 days. immunodefi cient (SCID) mice. EGFRvIII, the most common vehicle control, and tumor growth was assessed over 18 days EGFR mutation in GBM, arises from an in-frame genomic ( Fig. 1A ). As expected, erlotinib treatment slowed U87- deletion of exons 2 to 7, resulting in a persistently active, EGFRvIII tumor growth relative to control; however, tumors highly oncogenic protein ( 10 ). Tumor-bearing mice were retained a substantial growth rate despite continued erlotinib gavaged with the EGFR inhibitor erlotinib (150 mg/kg) or treatment ( Fig. 1B ). Immunoblots of tumor lysates confi rmed OF3 | CANCER DISCOVERYMAY 2013 www.aacrjournals.org Downloaded from cancerdiscovery.aacrjournals.org on September 23, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst March 26, 2013; DOI: 10.1158/2159-8290.CD-12-0502 An RTK Switch in Malignant Glioma RESEARCH ARTICLE that erlotinib treatment of mice signifi cantly reduced EGFR a strong inverse correlation between EGFR (total and phos-
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