Dual Targeting of Insulin Receptor and KIT in Imatinib-Resistant

Published OnlineFirst July 31, 2017; DOI: 10.1158/0008-5472.CAN-17-0917

Cancer Therapeutics, Targets, and Chemical Biology Research

Dual Targeting of Insulin Receptor and KIT in Imatinib-Resistant Gastrointestinal Stromal Tumors Weicai Chen1, Ye Kuang1, Hai-Bo Qiu2, Zhifa Cao1, Yuqing Tu1, Qing Sheng1, Grant Eilers3, Quan He1, Hai-Long Li4, Meijun Zhu3, Yuexiang Wang3, Rongqing Zhang4, Yeqing Wu1, Fanguo Meng4, Jonathan A. Fletcher3, and Wen-Bin Ou1,3,4

Abstract

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) S6 in GIST430 and GIST48, but not in GIST882, exerting mutations are compelling therapeutic targets in gastrointestinal minimal effect on KIT phosphorylation in these cell lines. stromal tumors (GIST), and treatment with the KIT/PDGFRA Additive effects showing increased apoptosis, antiproliferative inhibitor imatinib is the standard of care for patients with effects, cell-cycle arrest, and decreased pAKT and pS6 expres- metastatic GIST. Most GISTs eventually acquire imatinib resis- sion, tumor growth, migration, and invasiveness were observed tance due to secondary mutations in the KIT kinase domain, in imatinib-resistant GIST cells with IR activation after coordi- but it is unclear whether these genomic resistance mechanisms nated inhibition of IR and KIT by linsitinib (or IR shRNA) and require other cellular adaptations to create a clinically mean- imatinib, respectively, compared with either intervention ingful imatinib-resistant state. Using phospho-RTK and immu- alone. IGF2 overexpression was responsible for IR activation noblot assays, we demonstrate activation of KIT and insulin in imatinib-resistant GIST cells, whereas IR activation did not receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and result from IR amplification, IR mutation, or KIT phosphory- GIST48) and biopsies with acquisition of KIT secondary muta- lation. Our findings suggest that combinatorial inhibition of tions, but not in imatinib-sensitive GIST cells (GIST882 and IR and KIT warrants clinical evaluation as a novel therapeutic GIST-T1). Treatment with linsitinib, a specificIRinhibitor, strategy in imatinib-resistant GISTs. Cancer Res; 77(18); 5107–17. inhibited IR and downstream intermediates AKT, MAPK, and 2017 AACR.

Introduction third-line therapies, respectively, in patients with inoperable GIST (9–11), and adjuvant imatinib is used in patients with localized Gastrointestinal stromal tumors (GIST) are the most common GIST with a high risk of recurrence (12). mesenchymal and solid tumors of the gastrointestinal tract. The Insulin receptor (IR) is a member of the RTK family that majority of GISTs contain gain-of-function oncogenic mutations includes the homologous type 1 and type 2 insulin-like growth of KIT (approximately 85%) or PDGFRA (approximately 5%– factor receptors (IGF1R and IGF2R; refs. 13, 14). IR and IGF1/2R 10%; refs. 1–5). The KIT and PDGFRA mutant oncoproteins are have essential roles in energy metabolism, cell growth, division, crucial for GIST tumorigenesis, proliferation, and survival, as and differentiation (15–17), and various studies suggest that demonstrated by the clinical successes of small-molecule thera- aberrant IR/IGF1/2R signaling drives tumorigenesis in breast, peutics targeting KIT and PDGFRA (6–8). Therefore, imatinib, lung, hepatocellular, and pancreatic cancers (18–22). IGF1R has sunitinib, and regorafenib are the standard ﬁrst-, second-, and been shown to be overexpressed in wild-type and pediatric GIST (23, 24) and is a potential therapeutic target in GIST lacking KIT and PDGFRA mutations (25). In addition, IGF1 and IGF2 expres- 1Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, sion has prognostic relevance for GIST patients; strong IGF1 College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China. expression is correlated with higher mitotic index, larger, higher 2 Department of Gastric Surgery, Sun Yat-sen University Cancer Center, Guangz- risk, metastatic, and relapsed GIST, and strong IGF2 expression is 3 hou, China. Department of Pathology, Brigham and Women's Hospital and correlated with higher mitotic index and higher risk GIST (26). Harvard Medical School, Boston, Massachusetts. 4Zhejiang Provincial Key Lab- oratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua Studies have shown that IGF2 signaling is an autocrine survival University, Jiaxing, Zhejiang, China. pathway in GISTs (27). Targeting KIT/PDGFRA with imatinib results in response rates Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). of 80% among unresectable or metastatic GIST (28, 29). Despite this high response rate, most patients with inoperable GIST W. Chen, Y. Kuang, and H.-B. Qiu contributed equally to this article. eventually experience clinical progression due to the development Corresponding Author: Wen-Bin Ou, College of Life Sciences, Zhejiang Sci-Tech of imatinib resistance. Imatinib resistance mechanisms include University, Hangzhou, Zhejiang 310018, China. Phone: 86-573-82586633; Fax: secondary mutations in the KIT or PDGFRA kinase domains, 86-571-86843303; E-mail: [email protected] genomic ampliﬁcation of KIT, loss of former KIT expression, or doi: 10.1158/0008-5472.CAN-17-0917 activation of an alternate kinase oncoprotein (4, 30–32). There- 2017 American Association for Cancer Research. fore, we hypothesize that multiple mechanisms can contribute to

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imatinib resistance in individual GIST cells, such as the coordi- Phospho-RTK array analysis nated activity of multiple kinases (KIT and other RTKs). In this The Human Phospho-RTK Array Kit (R&D Systems) was used study, coactivation of IR and KIT was identified in imatinib- to determine the relative levels of tyrosine phosphorylation resistant GIST cell lines and biopsies, but not in imatinib-sensitive of 42 distinct RTKs, according to the manufacturer's protocol. GIST cell lines. Additive antiproliferative and proapoptotic effects Briefly, after a 6-hour incubation in serum-free medium, GIST were obtained after combination inhibition of IR and KIT in cells were lysed with lysis buffer (1% NP-40, 50 mmol/L Tris-HCl imatinib-resistant GIST cell lines. IR inactivation increased ima- pH 8.0, 100 mmol/L sodium fluoride, 30 mmol/L sodium pyro- tinib sensitivity in drug-resistant GIST cells, indicating that inhi- phosphate, 2 mmol/L sodium molybdate, 5 mmol/L EDTA, bition of the IGF2/IR signaling pathway is a rational therapeutic 2 mmol/L sodium orthovanadate) containing protease inhibitors strategy in imatinib-resistant GIST. (10 mg/mL aprotinin, 10 mg/mL leupeptin, and 1 mmol/L phe- nylmethylsulfonyl fluoride). The arrays were blocked for 1 hour Materials and Methods with Array Buffer 1, and then incubated with 500 mg of protein lysate overnight at 4C. The arrays were washed and incubated Antibodies, plasmids, and reagents with a horseradish peroxidase–conjugated phospho-tyrosine Polyclonal antibodies to KIT and IR were from Dako, Santa detection antibody (1:5,000). Detection was by chemilumines- Cruz Biotechnology, and Millipore, respectively. All phospho- cence (Immobilon Western, Millipore Corporation), and signals antibodies, polyclonal antibodies to IGF-1R and AKT, and the were captured using a GE Healthcare ImageQuant LAS4000 Mini mAb to S6 were from Cell Signaling Technology. Monoclonal Chemiluminescence Imaging System. mouse antibodies were to IR (Santa Cruz Biotechnology) and b IGF2 IR -actin (Sigma-Aldrich). Lentiviral and shRNA constructs Protein lysate preparations and immunoblotting were from The RNAi Consortium (TRC), and included IGF2:50- 0 0 Immunoblotting was performed after 6 hours of treatment with GCATCGTTGAGGAGTGCTGTT-3 (shRNA1), 5 -CAGAAGCC- linsitinib, imatinib, or sunitinib in serum-free medium. Whole- CAAAGAGCCAAAT-30 (shRNA2); and IR:50-CGTGTTGAACAAA- 0 cell lysates from cell lines were prepared using lysis buffer. Frozen GATGACAA-3 . Imatinib mesylate, sunitinib, and linsitinib were tumor samples were diced into small pieces in cold lysis buffer on purchased from LC Laboratories. Puromycin, Crystal violet, pro- ice and homogenized with a Tissue Tearor (Model 398, Biospec pidium iodide solution, and polybrene were from Sigma-Aldrich. Products Inc.) for three seconds, 3–5 times, on ice, and then rocked overnight at 4C. Lysates were cleared by centrifugation at Cell lines GIST882 is a human cell line established from an untreated 14,000 rpm for 30 minutes at 4 C, and supernatant protein GIST with a primary homozygous missense mutation in KIT exon concentrations were determined using a Bio-Rad protein assay 13, encoding a K642E-mutant KIT oncoprotein (33). GIST-T1 was (Bio-Rad Laboratories). Electrophoresis and Western blotting a gift from Dr. Takahiro Taguchi (Kochi University, Kochi, Japan). were performed as described previously (36). The hybridization GIST430 and GIST48 are human cell lines established from GIST signals were detected by chemiluminescence and captured using that had progressed on imatinib therapy, after initial clinical ImageQuant LAS4000. response. GIST430 has a heterozygous primary mutation in KIT exon 11, accompanied by a secondary exon 13 missense mutation Immunoprecipitation (V654A). GIST48 has a homozygous KIT exon 11 mutation Sepharose-protein G beads with mouse mAb and sepharose- (V560D) and a heterozygous KIT exon 17 mutation (D820A; protein A with rabbit polyclonal antibody were used. 1 mg of m ref. 34). GIST430, GIST48, GIST882, and GIST-T1 cell lines were protein lysate was preadsorbed for 30 minutes using 20 Lof m obtained from J.A. Fletcher's Laboratory in 2014, and maintained protein G or protein A beads at 4 C. Then, 2 gofprimary antibody against IR were added to each supernatant and rocked and used in W.-B. Ou's laboratory for three years. Cells were regularly screened for mycoplasma contamination using Myco- for 2 hours at 4 C (anti-mouse IR antibody was used as the plasma Stain Assay Kit (Beyotime Biotechnology), and authenti- control for IR immunoprecipitation). Twenty microliters of KIT protein G or protein A beads were added and rocked overnight cated by SNP array analysis prior to these studies and by sequencing, before and after the studies, to validate presence of at 4 C. The lysates were then spun at 10,000 rpm for 2 minutes m the unique exon 11, 13, and 17 mutations. at 4 C and beads were washed 3 times with 750 LofIPbuffer for 25 minutes followed once by 750-mL10mmol/LTris-Cl Frozen tumor specimens buffer (pH 7.6). Twenty microliters of loading buffer was added All frozen tumor specimens were analyzed histologically, and to the beads and boiled for 5 minutes at 95 C. IR activation was shown to be composed of >90% neoplastic cells. Tumor samples subsequently evaluated by phosphotyrosine and specificanti- GIST 1–6 are from six GIST progressing after treatment with body immunoblotting. imatinib/sunitinib, which have a heterozygous primary mutation in KIT exon 9 (A502_Y503dup; transmembrane domain) Lentiviral IGF2 and IR shRNA constructs and a secondary point mutation in the KIT tyrosine kinase Lentivirus preparations were produced by cotransfecting domain 1/2 (TYD1/2) or in the juxtamembrane domain. All pLKO.1puro (empty vector or containing IGF2 or IR shRNAs), tumor samples progressed clinically on imatinib or sunitinib, and helper virus packaging plasmids pCMVDR8.91 and VSVG (at according to the Southwest Oncology Group Response Evalu- a 10:10:1 ratio) into 293T cells. Transfections were carried out ation Criteria in solid tumors (35). The studies were conducted using PolyJet In Vitro DNA Transfection Reagent (SignaGen Lab- in accordance with recognized ethical guidelines (U.S. Common oratories). Lentiviruses were harvested at 24, 36, 48, and 60 hours Rule), were approved by the Brigham and Women's Hospital posttransfection, and frozen at 80C in aliquots of appropriate Institutional Review Board under a discarded tissues protocol, amounts for single-use infection. Well-validated shRNAs were and informed consent was obtained from all subjects. used for IGF2 and IR knockdowns.

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Cell culture and virus infection GIST430-expressing IR shRNA were treated twice daily for another The GIST882 cell line was maintained in RPMI1640 medium 8 weeks with imatinib 50 mg/kg by intragastric administration. with 15% FBS containing penicillin/streptomycin and L-gluta- The remaining 4 mice injected with GIST430 expressing IR shRNA mine. GIST48 and GIST430 were maintained in DMEM/F-12 (n ¼ 4) and the 4 mice injected with GIST430 expressing the containing 15% FBS, penicillin/streptomycin, and L-glutamine. scramble shRNA control were maintained for 8 weeks with no GIST cells were seeded in 6-well plates. Infections were carried out treatment. Mice were sacriﬁced by CO2 inhalation and necropsied in the presence of 8 mg/mL polybrene. Following transduction, 16 weeks after injection to evaluate tumor volume, IR, and KIT GIST430 and GIST882 were treated with 2 mg/mL puromycin to signaling. All mouse experiments were conducted in accordance select for stable expression of the IR shRNA or IGF2 shRNAs. with an Institutional Animal Care and Use Committee.

Cell viability and apoptosis analysis In vitro wound-healing assays GIST cells were plated at 15,000 cells/well in a 96-well flat Wound-healing studies were carried out as described previous- bottom plate (BD Falcon) and cultured in RPMI1640 or DMEM/ ly (38). Briefly, slashes were created in near-confluent cell cultures F-12 for 24 hours before treatment with linsitinib, imatinib, or using the tip of a P-100 pipetman after addition of inhibitors sunitinib. Viability studies were performed 3 or 6 days after (linsitinib or imatinib). Plates were photographed at day 0, 5, and inhibitor treatment in GIST cell lines (GIST430, GIST48, and 9 using a Leica DMI 3000B inverted microscope (Leica Micro- GIST882) or in IR/IGF2-silenced GIST cell lines (GIST430 and systems). Experiments were performed in triplicate. GIST882) with stable shRNA expression using the CellTiter-Glo luminescence assay from Promega. Viability was quantified using Cell migration and invasion assays a Veritas Microplate Luminometer from Turner Biosystems. Data Migration and invasiveness of GIST cells were evaluated by the were normalized to the control group (DMSO/pLKO). All assays Matrigel assay (Collaborative Research Inc.), as described previ- were performed in quadruplicate wells, and were averaged from ously (39). Briefly, Matrigel was diluted with RPMI1640/F12 in a two independent transductions in each cell line. 1:2 ratio and then coated onto 24-well inserts (Boyden chamber) Apoptosis was evaluated using the PE Annexin V Apoptosis with a 12-mm pore size, and then incubated at 37 C overnight. Detection Kit I (BD Pharmingen). Briefly, GIST430 and GIST882 Cells (4 104) were treated with linsitinib or imatinib, followed cells in 6-well plates were treated with linsitinib (2.5 mmol/L) or by suspension in 0.5 mL of 0.5% serum-containing RPMI/F12 and imatinib (1 mmol/L) for 96 hours, trypsinized, and washed twice seeded on the top chamber of each well with 1.5 mL of 15% with cold PBS buffer, and then treated with 5 mL of PE Annexin V serum-containing medium added to the bottom chamber, the and 5 mL 7-AAD in 1 binding buffer for 15 minutes at room higher serum content in the bottom chamber providing a che- temperature (25 C) in the dark. The stained cells were analyzed motactic gradient. After 48 hours, noninvasive cells that remained in a flow cytometer (BD FACSAria, Special Order System) within on the top surface of the filter were removed using a cotton swab 1 hour. Flowjo software version 7.6 (Flowjo LLC) was used to and cells that remained adherent to the underside of the mem- analyze the data. brane were fixed in 4% formaldehyde and stained with 0.1% Crystal violet. Invasive cells were quantified in five contiguous Cell-cycle analysis fields of a fluorescence microscope, using a 20 objective to GIST430 and GIST882 cells in 6-well plates were trypsinized obtain a representative number of cells. Experiments were per- and washed with PBS buffer at 72 hours after treatment with formed in triplicate. linsitinib (2.5 mmol/L) or imatinib (1 mmol/L). Nuclear staining was with a propidium iodide solution and the cell suspension was RNA preparation and qRT-PCR immediately analyzed in a flow cytometer. Data analysis was Total RNA was prepared using the RNAprep Pure Cell/Bacteria performed using Flowjo software version 7.6. kit (TIANGEN). RT-PCR was performed using 1 mg RNA with the SuperScript First-Strand Synthesis System (Invitrogen). qPCR Colony formation assay was performed using TaqMan chemistry on the ABI Step One Colony formation assays were performed as published previ- Plus (Applied Biosystems). Reactions (20 mL) contained 1 mL ously with minor modifications (37). In brief, GIST430 cells were cDNA, 200 nmol/L of each primer, and 10 mL2 KAPA SYBR FAST plated at 10,000 cells/well in 6-well plates and cultured in DMEM/ qPCR Kit Master Mix (Kapa Biosystems). After 3 minutes at 95C, F-12 for 14 days before treatment with linsitinib or imatinib. After each of the 40 PCR cycles consisted of denaturation for 5 seconds treatment with inhibitors for 7 days, the medium was removed, at 95C and hybridization of primers and SYBR Green as well the cells were washed with PBS, and the cells were then stained as DNA synthesis for 1 minute at 60C. The qRT-PCR assays with 0.5% Crystal violet in methanol for 5 minutes. Excess stain for IGF1 and IGF2 were performed using the following primers: was removed by washing with distilled water. Colonies were IGF1 (NM_053056) sense: 50-GCAATGGGAAAAATCAGCAG-30 photographed and counted. The experiments were performed in and anti-sense: 50-GAGGAGGACATGGTGTGCA-30 (40); IGF2 duplicate wells and repeated three times. (NM_002228) sense: 50-CCCCTCCGACCGTGCT-30 and anti- sense: 50-TCATATTGGAAGAACTTGCCCA-30 (41). As controls, Xenotransplant murine models GAPDH was amplified using the following primers: GAPDH Female adult athymic nude mice (6–8 weeks old) were housed (NM_002046) sense: 50-GAAGGTGAAGGTCGGAGTCAAC-30 in a specific pathogen-free facility. Mice were injected subcutane- and anti-sense: 50-TGGAAGATGGTGATGGGATTTC-30. All pri- 6 ously with 1 10 GIST430 cells suspended in BD Matrigel mers were obtained from Invitrogen. The comparative Ct (thresh- expressing IR-targeting shRNA (n ¼ 8), scramble shRNA control old cycle) method was used to determine RNA expression differ- (n ¼ 4), or untreated GIST430 (n ¼ 4). After 8 weeks, 4 mice ences in GIST cell lines. Data from triplicate assays were normal- injected with untreated GIST430 and 4 mice injected with ized to GAPDH.

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Whole transcriptome sequencing positive result. Subsequently, we found that the phospho-array After RNA extraction, rRNA was depleted from 5 mg of total RNA EGFR activation signal was overly sensitive in other human using biotinylated oligonucleotides (Ribominus, Invitrogen), cancers (42). Immunoblot studies confirmed strong activation and libraries were constructed from the rRNA-depleted RNA of IR in GIST430 and GIST48 cell lines, but not in GIST882 and according to the SOLiD Total RNA-seq Kit Protocol. Briefly, RNA GIST-T1 cells (Fig. 1B). IR activation in GIST430 and GIST882 was fragmented by RNAse III to an average size of 150 bases, cells was further evaluated by IR immunoprecipitation followed ligated to adaptors in a directed orientation, and the resulting by phosphotyrosine immunoblotting (Fig. 1C). IR was strongly library served as a template for cDNA synthesis and PCR ampli- tyrosine-phosphorylated in GIST430. In contrast, IR phosphory- fication. Approximately 50 bases were sequenced from one end of lation was undetectable in GIST882 (Fig. 1C). In addition, phos- each fragment using either the SOLiD 3þ or SOLiD 4 instrument photyrosine staining demonstrated that IR was activated in GIST and reagents (Applied Biosystems). The resulting sequence data biopsy samples (GIST 1–6; Fig. 1D). were mapped to the human reference genome, hg18, using Bio- scope v1.2. Sequences that mapped to unique locations were Inhibition of IR signaling in imatinib-resistant GIST quantified per transcriptional unit, as defined in RefSeq (release IR signaling inhibition was evaluated by phospho-immuno- 35), as "reads per kilobase of transcript per million mapped reads blot analysis of total cell lysates in imatinib-resistant cell lines (RPKM)" of total sequence. (GIST430 and GIST48) and an imatinib-sensitive cell line (GIST882) after 6 hours of linsitinib treatment (a specific inhib- Statistical analysis itor of IR/IGF1R) in serum-free conditions (Fig. 2). As expected, Student t tests were performed on data from cells treated linsitinib treatment inactivated IR in GIST430 and GIST48, and with inhibitors/shRNAs or DMSO/pLKO (control). Statistically inactivated downstream signaling intermediates AKT and S6 in a significant differences between control and treatment were dose-dependent manner. We observed unexpected activation of defined as , P < 0.05; , P < 0.01; and , P < 0.001. MAPK in GIST430, whereas MAPK was inactivated in GIST48. In contrast, IR inhibition had less effect on levels of phospho-AKT, Results -MAPK, and -S6 in GIST882 (Fig. 2). Linsitinib treatment showed Activation of IR in imatinib-resistant GIST little effect on KIT phosphorylation in the three GIST cell lines Phospho-RTK arrays in imatinib-resistant GIST cell lines (GIST430, GIST48, and GIST882; Fig. 2). (GIST430 and GIST48) and in a GIST patient sample demonstrated coordinated activation of IR, EGFR, and KIT (Fig. 1A). In Additive antiproliferative effects via dual targeting of IR and KIT contrast, imatinib-sensitive GIST cell lines (GIST882 and GIST- in imatinib-resistant GIST T1) demonstrated apparent activation of EGFR, PDGFRA, and KIT Additive effects of combined IR and KIT inhibition were dem- (Fig. 1A), but EGFR activation was not corroborated by immu- onstrated by immunoblot, viability, cell cycle, apoptosis, and noblot analysis (Supplementary Fig. S1), indicating that the colony formation assays. IR and KIT signaling was evaluated by phospho-EGFR expression observed in the array was a false- immunoblots in GIST430, GIST48, and GIST882 after 6 hours of

Figure 1. Coactivation of multiple RTKs in GIST cell lines and primary tumor tissue. A, Total cell lysates (500 mg) from imatinib-resistant GIST cell lines (GIST430 and GIST48), imatinib- sensitive GIST cell lines (GIST882 and GIST-T1), and a GIST biopsy were analyzed by phospho-RTK array. Each RTK is spotted in duplicate, with the spots at each corner serving as positive controls. Eight spots at the lower right serve as negative controls. B, Immunoblot evaluations of IR, IGF1R, phospho-IR/IGF1R, KIT, and phospho- KIT in GIST cell lines. Actin stain was used as a loading control. C, IR activation in GIST430 and GIST882 was validated by IR immunoprecipitation, followed by phosphotyrosine immunoblotting. D, Immunoblot evaluations of IR, IGF1R, and phospho- IR/IGF1R in GIST patient tumor tissues (GIST 1–6). Actin stain was used as a loading control.

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treatment with linsitinib and/or imatinib in serum-free condi- GIST430 and GIST882 cells were further evaluated by colony tions (Fig. 3A). Imatinib treatment inactivated KIT and down- formation and apoptosis assays. Colonies of GIST430 cells treated stream signaling intermediates AKT, MAPK, and S6 in GIST882, with imatinib were fewer and smaller in size when compared with whereas imatinib treatment partially decreased levels of phospho- DMSO-treated cells, but not linsitinib-treated cells (Fig. 3D). AKT, -MAPK, and –S6 in GIST48, and phospho-S6 expression in Combination inhibition further decreased the size and number GIST430. Combination inhibition of IR and KIT by linsitinib and of colonies in GIST430, as compared with either intervention imatinib resulted in greater inactivation of PI3K/AKT/mTOR alone (Fig. 3D). Relative to DMSO, colony formation decreased signaling in GIST430 and GIST48, and MAPK in GIST48, com- by 20% in cells treated with linsitinib, by 50% in cells treated with pared with linsitinib/imatinib treatment alone. An additive effect imatinib, and by 80% in cells treated with linsitinib and imatinib was not observed in GIST882 (Fig. 3A). (Fig. 3D). Treatment of GIST430 with both linsitinib and imatinib Additive effects on cell viability and cell-cycle inhibition were for 96 hours showed a greater increase in apoptotic cells (31.1%), observed after coordinated inhibition of IR and KIT (Fig. 3B and C; as compared with each intervention alone (linsitinib 3.49%; Supplementary Fig. S2). In all GIST cell lines (GIST430, GIST48, imatinib 6.15%; Fig. 3E). Levels of apoptosis in GIST882 cells and GIST882), KIT inhibition by imatinib or sunitinib resulted in after combination linsitinib and imatinib treatment (27.54%) a 40%–70% reduction in cell viability, as compared with the was comparable with imatinib treatment alone (24.89%; Fig. 3E). DMSO control (Fig. 3B; Supplementary Fig. S2). Treatment with Additive effects on cell viability were further demonstrated after linsitinib showed minimal impact on viability (Fig. 3B). How- combination of IR shRNA knockdown and KIT inhibition (Fig. ever, combination treatment with linsitinib and imatinib/suniti- 3F). Cotargeting of IR and KIT by shRNA and imatinib resulted in nib resulted in 60% and 80% reduction in viability for GIST430 greater inactivation of PI3K/AKT/mTOR signaling and viability and GIST48 cell lines, respectively, whereas combination treat- reduction in GIST430, as compared with IR shRNA or imatinib ment in GIST882 cells was comparable with imatinib treatment treatment alone. Additive effects were not observed in GIST882 alone (Fig. 3B; Supplementary Fig. S2). (Fig. 3F). IR and KIT inhibition in imatinib-resistant GIST430 resulted in In a mouse xenograft model, combination of shRNA-mediated a minimal increase of G1 peaks, from 72% in the DMSO-treated IR knockdown and imatinib treatment in GIST430 cells resulted cells to 74% and 78% in linsitinib and imatinib-treated cells, in a greater decrease in tumor growth than either intervention respectively. Combination treatment with linsitinib and imatinib alone (Fig. 4A). IR knockdown was evaluated by immunoblot in induced greater apoptosis in GIST430 than each intervention GIST430 with stable expression of a shRNA targeting IR before alone: nuclear fragmentation was demonstrated in 0% of cells injection into mice (Fig. 4B) and after harvesting xenograft tissue treated with DMSO/linsitinib, in 4.36% of those treated with (Fig. 4C). The shRNA-mediated knockdown resulted in a 70%– imatinib, and in 32.29% of those treated with both linsitinib and 80% reduction of IR expression in GIST430 and in mouse xeno- imatinib (Fig. 3C). Cell-cycle analysis also demonstrated apopto- graft tumor tissues (Fig. 4B and C), and KIT expression was sis in imatinib-sensitive GIST882 cells after imatinib treatment, partially inhibited in xenograft tumors after treatment with ima- but not after linsitinib treatment. Nuclear fragmentation was tinib (Fig. 4C). observed in 0% of cells treated with DMSO control, but in 24.98% of cells treated with imatinib. Induction of apoptosis in Additive antiinvasive effects via dual targeting of IR and KIT in GIST882 cells after combination imatinib and linsitinib treatment imatinib-resistant GIST was comparable with KIT suppression alone, with nuclear frag- Assays were performed in GIST430 and GIST882 to evaluate the mentation observed in 24.98% of cells treated with imatinib, and effects of IR and KIT inhibition on GIST cell migration and in 28% of cells treated with linsitinib and imatinib (Fig. 3C). invasion. Wound-healing assays in IR-phosphorylated GIST430

Figure 2. Inactivation of IR and signaling intermediates in GIST cell lines after treatment with linsitinib (LST). Immunoblot evaluations of p-IR, IR, p-KIT, KIT, p-AKT, AKT, p-MAPK, MAPK, p-S6, and S6 in GIST430, GIST48, and GIST882 after 6 hours of linsitinib treatment in serum-free medium. Actin stain was used as a loading control.

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Figure 4. Combination of shRNA-mediated IR knockdown and KIT inhibition by imatinib (IM) slows growth of GIST xenografts in mice. A, Tumors were resected and photographed, demonstrating decreased tumor growth when treated with a combination of IR shRNA and IM. B, Western blot conﬁrming IR knockdown in GIST430 cells infected with IR-targeting shRNA, as compared with an empty pLKO lentiviral vector. C, Western blot conﬁrming IR knockdown in resected GIST430 xenografts expressing IR-targeting shRNA, as compared with an empty pLKO lentiviral vector.

cells demonstrated that combination treatment with imatinib and ever, IR phosphorylation was unaffected after sunitinib treat- linsitinib impaired wound closure at 5 days to a greater extent ment (Fig. 6A). Furthermore, IR immunoprecipitation did not than either intervention alone, whereas complete wound closure show interaction between IR and KIT in GIST882 or GIST430 was seen in DMSO-treated control cells (Fig. 5A). In contrast, (Fig. 1C). inhibition of wound closure at 9 days in GIST882 was similar in IGF1, IGF2, INS, IGF1R, IGF2R, and IR transcript levels, cells treated with imatinib or a combination of imatinib and expressed as RPKM values, are shown in Table 1, and the whole linsitinib, while DMSO-treated cells showed complete healing of transcriptome data for these three GIST cell lines (GIST882, the wound (Fig. 5A). Matrigel assays demonstrated similar results, GIST430, and GIST48) are archived at http://thorn15.net/mod with 35% inhibition of invasiveness after linsitinib treatment, ules.php?op¼modload&name¼Publications&file¼index. and 75% inhibition after combination treatment with linsitinib Whole transcriptome sequencing at >25 million mappable reads (2.5 mmol/L) and imatinib (0.5 mmol/L) in imatinib-resistant demonstrated low levels of IGF2 mRNA (RPKM ¼ 393) in GIST430, as compared with the DMSO control (Fig. 5B and C). In imatinib-sensitive GIST882, whereas IGF2 was abundantly imatinib-sensitive GIST882, treatment with linsitinib alone did expressed in imatinib-resistant GIST48 and GIST430, with RPKM not inhibit invasiveness, while treatment with a combination of ¼ 2743 and 1758, respectively. IGF1, INS, IGF1R, and IR transcript imatinib and linisitinib was comparable with inhibition with expression was comparable between imatinib-sensitive GIST882 imatinib alone (Fig. 5B and C). and imatinib-resistant GIST48 and GIST430. The transcriptome data was confirmed by qRT-PCR, which Induction of IR activation by IGF2 overexpression in GIST demonstrated elevated expression of IGF2 mRNA in imatinib- To determine the mechanism of activation of IR signaling in resistant GIST48 and GIST430, as compared with imatinib-sen- GIST cells, we first sequenced GIST48, GIST430, and GIST882, sitive GIST882 (Fig. 6B). In contrast, IGF1 mRNA expression was but did not find mutations in IR.SNPprofiles did not show comparable between imatinib-resistant and imatinib-sensitive IGF1, IGF2, INS, IGF1R, IGF2R, or IR genomic amplification lines (Fig. 6B). (SNP data not shown). Next, to rule out the possibility that IR To determine whether increased expression of IGF2 is respon- activation was due to hyperactive KIT in GIST430, we evaluated sible for IR activation in imatinib-resistant GIST430, IGF2 was the phosphorylation of KIT and IR after treatment with suni- silenced by shRNA knockdown. The level of IGF2 silencing was tinib, a second-line drug for GIST clinical management. How- evaluated by qRT-PCR in GIST430 at 15 days after IGF2 shRNA1 or

Figure 3. Additive effects of coordinated inhibition of IR and KIT as demonstrated by immunoblotting (A and F), cell viability (B and F), cell cycle (C), colony growth (D), and apoptosis assays (E), showing that combined inhibition of IR and KIT further decreased p-AKT and p-S6 expression, induced greater antiproliferative and apoptotic effects, and cell-cycle arrest in imatinib-resistant GIST cell lines (GIST430 and GIST48), as compared with either intervention alone, but not in imatinib-sensitive GIST882. A, The IR/KIT signaling and downstream intermediates (AKT, MAPK, and S6) were evaluated by immunoblotting at 6 hours after treatment with linsitinib (LST) and imatinib (IM). Actin stain was used as a loading control. B, Cell viability was evaluated by a CellTiter Glo ATP-based luminescence assay in GIST cell lines (GIST430, GIST882, and GIST48), 3 days after treatment with linsitinib and imatinib. Data were normalized to DMSO and represent the mean values (SD) from quadruplicate cultures. Statistically signiﬁcant differences between untreated control and inhibitor treatments are presented as , P < 0.05; , P < 0.01; , P < 0.001. C, Cell-cycle analysis was performed 72 hours after treatment with linsitinib (2.5 mmol/L) and imatinib (1 mmol/L). GIST430 and GIST882 showed substantial nuclear fragmentation after combination inhibition of IR and KIT, and KIT inhibition alone, respectively. D, Colony growth assays were performed 7 days after treatment with linsitinib (2.5 mmol/L) and imatinib (1 mmol/L). Combined IR and KIT inhibition led to a greater reduction in colony formation and size in GIST430 than either intervention alone. E, Apoptosis assays following linsitinib (2.5 mmol/L) and/or imatinib (1 mmol/L) treatment for 96 hours were performed with the PE Annexin V Apoptosis Detection Kit I. F, Left, p-IR, IR, p-KIT, KIT, and downstream intermediates were evaluated by immunoblotting at 6 hours after treatment with imatinib in IR stably silenced GIST430 and GIST882. Right, cell viability was evaluated by a CellTiter Glo ATP-based luminescence assay in IR stably silenced GIST cell lines (GIST430 and GIST882), 3 days after treatment with imatinib. Data were normalized to DMSO and pLKO and represent the mean values (SD) from quadruplicate cultures. Statistically signiﬁcant differences between control and inhibitor treatments are presented as , P < 0.01; , P < 0.001.

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Figure 5. Combination treatment with linsitinib (LST) and imatinib (IM) inhibits migration and invasion in imatinib-resistant GIST430, but not in imatinib- sensitive GIST882. In vitro wounding assays (A) and Transwell migration assays (B) show that combination treatment with linsitinib and imatinib more effectively inhibit migration and invasion of GIST430 than either intervention alone. An additive effect was not observed in GIST882. Transwell experiments were performed in triplicate. C, Quantitation of GIST cell invasiveness after treatment with linsitinib and imatinib for 48 hours. Statistically signiﬁcant differences between untreated control and inhibitor treatments are presented as , P < 0.05; , P < 0.01; , P < 0.001.

shRNA2 transduction (Fig. 6C). Immunoblotting demonstrates of unresectable and advanced GIST, achieving at least a partial that IGF2 knockdown decreases IR phosphorylation in GIST430 response in approximately 80% of patients with metastatic dis- cells (Fig. 6D). ease (7). However, within 2 to 3 years from the start of treatment, Further studies showed that KIT inhibition by imatinib the majority of patients develop imatinib resistance (43), which (1 mmol/L) resulted in a 30% reduction of cell viability in IR- remains the biggest challenge in the clinical management of GIST. activated GIST430, as compared with the DMSO control (Fig. 6E). Thus, new treatment targets need to be identified in GIST to IGF2 shRNA knockdown decreased viability by 10%–25%; how- expand the treatment options for patients with resistance to ever, combination treatment with IGF2 shRNA1 or shRNA2 and small-molecule tyrosine kinase inhibitors such as imatinib. Four imatinib (1 mmol/L) resulted in 60% and 50% reduction in main imatinib-resistant mechanisms have been characterized in a viability for GIST430, respectively (Fig. 6E). variety of GIST: (i) acquisition of a secondary point mutation in KIT or PDGFRA; (ii) genomic amplification of KIT; (iii) activation of an alternate kinase; and (iv) loss of KIT oncoprotein expression, Discussion but it is likely that other mechanisms contribute to imatinib Most GISTs harbor activating oncogenic "driver" mutations in resistance. KIT, or less frequently PDGFRA. Imatinib mesylate potently In this study, we evaluated phosphorylation and expression inhibits KIT kinase activity as the first-line drug for the treatment of RTKs in imatinib-resistant (GIST430 and GIST48) and

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IR and KIT Signaling Inhibition in Imatinib-Resistant GISTs

Figure 6. Regulation of IR activation by IGF2 overexpression in imatinib-resistant GIST cells. A, Immunoblot evaluations of IR and KIT signaling in imatinib- resistant GIST430 after treatment with sunitinib for 6 hours in serum-free media. Actin stain was used as a loading control. B, Quantiﬁcation of IGF1 and IGF2 mRNA transcripts by transcriptome sequencing in imatinib- resistant GIST cells (GIST430 and GIST48) and in the imatinib-sensitive GIST cell (GIST882). Data were normalized to a mesothelioma cell line (MESO257) that expresses IGF1 and IGF2 at normal levels. C, IGF2 silencing was evaluated by qRT-PCR in GIST430 15 days after IGF2 shRNA1 or shRNA2 transduction in the presence of puromycin selection. D, Immunoblotting demonstrating that IGF2 knockdown decreases IR phosphorylation in GIST430 cells. Actin stain was used as a loading control. E, Cell viability was evaluated by a CellTiter Glo ATP-based luminescence assay in IGF2 stably silenced GIST430, 6 days after treatment with imatinib (IM). Data were normalized to DMSO and pLKO, and represent the mean values (SD) from quadruplicate cultures. Statistically signiﬁcant differences between control and inhibitor treatments are presented as , P < 0.01; , P < 0.001.

imatinib-sensitive GIST cell lines (GIST882 and GIST-T1) and but not in imatinib-sensitive GIST882 and GIST-T1 (Fig. 1). frozen tumor samples by phospho-RTK assays, immunoprecipi- Coactivation of KIT and IR suggests that activated IR may con- tation, and immunoblotting. Our results show simultaneous tribute to drug resistance in these cells. activation of KIT and IR in imatinib-resistant GIST430, GIST48, Approximately 15% of GIST in adults and 85% in children are and GIST patient biopsies taken after imatinib treatment failure, so-called wild-type GIST, lacking mutations in KIT and PDGFRA. Wild-type GIST from adults and children express high levels of Table 1. Receptor and growth factor expression in imatinib-sensitive (GIST882) IGF1R (23, 24, 44). In this study, we did not detect high levels of and imatinib-resistant (GIST48 and GIST430, in bold) GIST from whole IGF1R in GIST cell lines or tumor biopsies, despite strong expres- transcriptome sequencing sion of IR (Fig. 1B and D). We evaluated activation of IR by Transcript levels (RPKM) IGF1 IGF2 INS IGF1R IGF2R IR phospho-IGF1R (Tyr1135/1136)/phospho-IR (Tyr1150/1151) GIST882 0 393 0 0.2 33 4.5 immunoblotting. IR was found to be strongly phosphorylated GIST48 0.04 2,743 0.5 0.8 27 8.4 in imatinib-resistant cell lines (GIST48 and GIST430), but not in GIST430 0 1,758 0 0.1 102 10 imatinib-sensitive cell lines (GIST-T1 and GIST882). Inhibition of MESO257 0.1 4.0 0 25 155 6.2 IR by linsitinib, a speciﬁc IR/IGF1R inhibitor, inhibited PI3K/ Abbreviation: RPKM, reads per kilobase of transcript per million mapped reads. AKT/mTOR signaling in imatinib-resistant cells (GIST430 and

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GIST48) in a KIT-independent manner, indicating that IR activa- In conclusion, these studies demonstrate that IR is activated in tion may be responsible for activating pathways downstream of imatinib-resistant GIST cells, and IR activation is due to over- KIT, even in the presence of a KIT secondary mutation (Fig. 2). expression of IGF2. On the basis of the evidence presented in this Additive effects were observed in imatinib-resistant GIST430 report, we believe IR to be an essential proliferation mediator in and GIST48 cell lines with simultaneous KIT (imatinib) and IR imatinib-resistant GIST, and that dual targeting of IR and KIT (linsitinib/shRNA) or IGF2 (shRNAs) inhibition as shown by an warrants evaluation as a novel therapeutic strategy in this chal- increase in apoptosis, cell-cycle arrest, AKT and S6 inactivation, lenging subset of GIST. and inhibition of proliferation, migration, invasiveness, and xenograft tumor growth (Figs. 3–5 and 6E). Additive effects were Disclosure of Potential Conflicts of Interest not observed in imatinib-sensitive GIST882. These findings high- No potential conflicts of interest were disclosed. light that combination inhibition of KIT and IR warrants clinical evaluation as a therapeutic strategy in imatinib-resistant GIST. Authors' Contributions A recent study showed that knockdown of either "big"-IGF-II or Conception and design: Y. Wang, J.A. Fletcher, W.-B. Ou IR decreased levels of p-AKT and p-MAPK and reduced survival in Development of methodology: H.-L. Li, Y. Wang, J.A. Fletcher, W.-B. Ou IR-unphosphorylated cell line GIST882, and that combination Acquisition of data (provided animals, acquired and managed patients, inhibition of KIT and big-IGF-II signaling had additive cytotoxic provided facilities, etc.): W. Chen, Y. Kuang, H.-B. Qiu, Z. Cao, Y. Tu, M. Zhu, effects in these cells (27). In this study, IR phosphorylation was Y. Wang, R. Zhang, Y. Wu, J.A. Fletcher, W.-B. Ou not observed in GIST882 (Figs. 1–3), and mRNA levels of IR Analysis and interpretation of data (e.g., statistical analysis, biostatistics, IGF2 computational analysis): W. Chen, Y. Kuang, H.-B. Qiu, Y. Tu, Q. He, M. Zhu, ligand were lower in GIST882 than in GIST430 or GIST48 Y. Wang, R. Zhang, Y. Wu, F. Meng, W.-B. Ou (Fig. 6B; Table 1), suggesting that IGF2-IR signaling has particular Writing, review, and/or revision of the manuscript: H.-B. Qiu, Q. Sheng, importance in imatinib-resistant GIST. G. Eilers, J.A. Fletcher, W.-B. Ou Finally, we investigated the following mechanisms of activa- Administrative, technical, or material support (i.e., reporting or organizing tion of IR signaling in imatinib-resistant GIST: (i) IR point data, constructing databases): J.A. Fletcher, W.-B. Ou mutation; (ii) IR gene amplification; (iii) cross-phosphorylation Study supervision: Q. He, J.A. Fletcher, W.-B. Ou of IR by KIT oncoprotein; or (iv) IR ligand (insulin, IGF1, or IGF2) overexpression. Sequencing and SNP data ruled out IR gene Grant Support mutation or amplification, and IR immunoprecipitation followed This work was supported by Zhejiang Provincial Top Key Discipline of by KIT immunostaining did not reveal interaction between IR and Biology and Open Foundation, the Major Science and Technology Special Project of Zhejiang Province (2014C03004 to W.B. Ou), Science Foundation KIT (Fig. 1C). Furthermore, sunitinib, a second-line KIT inhibitor of Zhejiang Sci-Tech University (14042107-Y to W.B. Ou), Graduate research in GIST, decreased KIT phosphorylation in imatinib-resistant and innovation projects of Zhejiang Sci-Tech University (YCX16036 to W.B. GIST430, with little effect on IR activation (Fig. 6A), indicating Ou), National Natural Science Foundation of China (81602061 to H. Qiu), the that IR activation is KIT-independent in imatinib-resistant GIST Natural Science Foundation of Zhejiang Province (LY13H160029 and cells. IGF2 transcripts were found to be overexpressed by tran- LQ16C050002 to Y. Wu), China and by the US NIH (1P50CA127003 and scriptome sequencing (Table 1) and qRT-PCR (Fig. 6B) in ima- 1P50CA168512 to J.A. Fletcher). The costs of publication of this article were defrayed in part by the payment of tinib-resistant GIST430 and GIST48, but not in imatinib-sensitive advertisement IGF1, insulin, IR, IGF1R IGF2R page charges. This article must therefore be hereby marked in GIST882, whereas , and were accordance with 18 U.S.C. Section 1734 solely to indicate this fact. expressed at normal levels. Furthermore, IGF2 knockdown inhibited IR phosphorylation in GIST430, demonstrating that IGF2 Received April 6, 2017; revised June 22, 2017; accepted July 21, 2017; overexpression is responsible for IR activation (Fig. 6C and D). published OnlineFirst July 31, 2017.

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Dual Targeting of Insulin Receptor and KIT in Imatinib-Resistant Gastrointestinal Stromal Tumors

Weicai Chen, Ye Kuang, Hai-Bo Qiu, et al.

Cancer Res 2017;77:5107-5117. Published OnlineFirst July 31, 2017.

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