Bruton's Tyrosine Kinase Inhibitors Prevent Therapeutic Escape in Breast Cancer Cells Xianhui Wang1, Jason Wong1, Christopher J

Published OnlineFirst June 2, 2016; DOI: 10.1158/1535-7163.MCT-15-0813

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Bruton's Tyrosine Kinase Inhibitors Prevent Therapeutic Escape in Breast Cancer Cells Xianhui Wang1, Jason Wong1, Christopher J. Sevinsky1, Leila Kokabee1,2, Faiza Khan1, Yan Sun1, and Douglas S. Conklin1

Abstract

þ We have reported that a novel isoform of BTK (BTK-C) factor–driven lapatinib resistance in HER2 breast cancer cells. þ expressed in breast cancer protects these cells from apoptosis. In HER2 breast cancer cell proliferation is blocked by ibrutinib even this study, we show that recently developed inhibitors of BTK, in the presence of these factors. AVL-292, which has no effect on such as ibrutinib (PCI-32765), AVL-292, and CGI-1746, reduce EGFR family activation, prevents NRG- and EGF-dependent þ breast cancer cell survival and prevent drug-resistant clones from growth factor–driven resistance to lapatinib in HER2 breast þ þ arising. Ibrutinib treatment impacts HER2 breast cancer cell cancer cells. In vivo, ibrutinib inhibits HER2 xenograft tumor viability at lower concentrations than the established breast growth. Consistent with this, immunoﬂuorescence analysis of cancer therapeutic lapatinib. In addition to inhibiting BTK, ibru- xenograft tumors shows that ibrutinib reduces the phosphoryla- tinib, but not AVL-292 and CGI-1746, efﬁciently blocks the tion of HER2, BTK, Akt, and Erk and histone H3 and increases activation of EGFR, HER2, ErbB3, and ErbB4. Consequently, the cleaved caspase-3 signals. As BTK-C and HER2 are often coex- activation of AKT and ERK signaling pathways are also blocked pressed in human breast cancers, these observations indicate leading to a G1–S cell-cycle delay and increased apoptosis. Impor- that BTK-C is a potential therapeutic target and that ibrutinib þ tantly, inhibition of BTK prevents activation of the AKT signaling could be an effective drug especially for HER2 breast cancer. pathway by NRG or EGF that has been shown to promote growth Mol Cancer Ther; 15(9); 2198–208. 2016 AACR.

Introduction lymphoma, chronic lymphocytic leukemia, and Waldenstrom€ macroglobulinemia (14). An alternate isoform of BTK, BTK-C, Bruton's tyrosine kinase (BTK) belongs to the TEC family of was identified as a novel survival factor for breast cancer cells in a cytoplasmic tyrosine kinases (1). BTK was identified in 1993 as a large-scale loss-of-function analysis of human tyrosine kinases novel nonreceptor protein tyrosine kinase that is mutated in X- using an RNA interference library (15). This study showed that linked agammaglobulinaemia (XLA; refs. 2, 3). It is predominant- although BTK-C is expressed at relatively low levels in several ly expressed in hematopoietic cells including erythroid progeni- human breast cancer cell lines and tumor tissues, it provides an tors and myeloid cells (4) and is established as a critical regulator essential function protecting breast cancer cells from apoptosis. of the B-cell receptor (BCR) signaling, development, differentia- It has long been appreciated that HER2 is overexpressed or tion, and survival (5–7). Because of its role in BCR signaling– amplified in tumors of about 20% of patients with early-stage induced proliferation, BTK has emerged as a novel target for the breast cancer and confers an increased disease recurrence and a treatment of rheumatoid arthritis and other immune diseases. worse prognosis (16). HER2-directed therapies including trastu- Recent studies have focused on the essential role of BTK in many zumab, pertuzumab, ado-trastuzumab, and lapatinib have been B-cell leukemias and lymphomas (8, 9) which provides a ratio- used in clinic and have significantly improved the outlook for nale for targeting the kinase in these malignancies. BTK inhibitors þ patients with HER2 breast cancer (17). However, a significant including ibrutinib (10), AVL-292 (11), and CGI-1746 (12) proportion of these patients still relapses and succumbs to their developed as immunosuppressants have been used in clinical disease (18). Therefore, new classes of drugs are needed, especially trials for blood malignancies (13). Recently, ibrutinib (Imbru- þ for HER2 advanced-stage breast cancer and those that have vica) gained FDA approval for the treatment of mantle cell developed resistance to current therapies. Here we describe the effects of treating breast cancer cells with fi 1Cancer Research Center and Department of Biomedical Sciences, recently developed BTK inhibitors. We nd that among the new State University of New York, University at Albany, Rensselaer, New inhibitors, ibrutinib is particularly effective at inhibiting breast York. 2Department of Molecular Medicine, Pasteur Institute of Iran, cancer cell growth in vitro and in vivo. Ibrutinib inhibits BTK and all Tehran, Iran. members of the ERBB family of receptor tyrosine kinases making it þ Note: Supplementary data for this article are available at Molecular Cancer especially effective at reducing HER2 breast cancer cell growth Therapeutics Online (http://mct.aacrjournals.org/). and survival. Ibrutinib induces a G1–S arrest and apoptosis in þ Corresponding Author: Douglas S. Conklin, Cancer Research Center, Depart- these cells. We also show that ibrutinib's effects on HER2 breast ment of Biomedical Sciences, University at Albany, State University of New York, cancer cells are not mitigated by NRG1 or EGF stimulation, as CRC Room 308, One Discovery Drive, Rensselaer, NY 12144. Phone: 518-591-7154; occurs with lapatinib. As the expression of BTK-C and HER2 is Fax: 518-591-7151; E-mail: [email protected] positively correlated in surgical specimens of human breast cancer doi: 10.1158/1535-7163.MCT-15-0813 tissues, these results indicate that ibrutinib is a potential therapy 2016 American Association for Cancer Research. for this solid tumor type.

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BTK Inhibitors and Drug Resistance in Breast Cancer

Materials and Methods Immunoblotting Immunoblotting was performed essentially as described pre- Cell culture and chemicals viously. Equal amounts of proteins were used. Antibodies used Breast cancer and MCF-10A cell lines were obtained from the were anti-EGFR (1:1,000), anti-HER2 (1:1,000), anti-ERBB3, ATCC in 2011. All cell lines were cultured in DMEM (Hyclone) ERBB4 antibody, anti-BTK, anti-AKT, ERK, anti-PLCg1, PLCg2 supplemented with 10% FBS (Hyclone) and 100 U/mL of peni- and anti-PARP (1:1,000, Cell Signaling Technology), anti-Flag cillin–streptomycin (CellGro), except for MCF-10A cells cultured antibody (1:1,000, Sigma), anti-rabbit IgG-HRP, and mouse as indicated in ref. (15). All cell lines were authenticated in March IgG-HRP (1:5,000, Jackson ImmunoResearch; ref. 20). 2016 by the SUNY-Albany Center for Functional Genomics Molecular Core Facility using a short tandem repeat method Apoptosis assay (Promega GenePrint 10 system). BTK inhibitors were supplied Apoptotic cells were identified using an Alexa Fluor 488 as follows. Ibrutinib was purchased from ChemieTek, AVL-292 Annexin V Apoptosis Kit (Invitrogen). Cells were treated with was purchased from MedKoo Biosciences, CGI-1764 was pur- lapatinib or ibrutinib for 24 hours. Cells were trypsinized and chased from Axon Medchem. Recombinant human-b1 and Matri- washed with cold PBS and resuspended in 1 Annexin-binding gel were purchased from R&D Systems. Src inhibitor saracatinib buffer to 1 106 cells/mL. Alexa Fluor 488 Annexin V (5 mL) and was purchased from Selleckchem. LY294002 was purchased from 100 mg/mL PI (1 mL) was added to each 100 mL of cell suspension. Cell Signaling Technology. Cells were incubated for 15 minutes at room temperature. After the incubation, 400 mL1 Annexin-binding buffer was added, Cell viability assays mixed gently, and the sample kept on ice. Samples were analyzed For live cell counts, cells were grown on 96-well plates, fixed on a BD LSR II Flow Cytometer (BD Biosciences). The data were with 4% formaldehyde, and counterstained with Hoechst 33342. analyzed using the FlowJo software package (Treestar Inc.). Images of cells were acquired using an In Cell Analyzer 1000 (GE Healthcare) high-content imaging system. At least 30 fields were Animal experiments imaged per well. Statistics were performed using the In Cell NOD/SCID mice were purchased from The Jackson Laboratory. Investigator 3.4 image analysis software (GE Healthcare). All mouse procedures were approved by the Animal Care and Use Three-dimensional (3D) cell culture was performed as Committees of The State University of New York at Albany (SUNY described previously (19). BT474 cells were propagated in DMEM Albany, Albany, NY) and performed in accordance with institu- with 10% FBS. Single cells in medium containing 5% Matrigel tional policies. For xenograft tumor studies, 1 106 SKBR3 cells were seeded at a density of 5 104 cells/cm2 on a Matrigel-coated were suspended in 50-mL Matrigel (BD Biosciences) diluted 1:2 well. The top medium with 5% Matrigel and ibrutinib or lapatinib with DMEM and injected into mammary fat pad. Treatment began was changed every 3 days. 1 mmol/L ethidium bromide was used when tumors were palpable. Ibrutinib was given 6 mg/kg/day or to stain cell death during 3D culture. 12 mg/kg/day orally in a vehicle of 1% DMSO/30% polyethylene Cells were cultured in 6-well plates for indicated number of days. glycol/1% Tween 80. Lapatinib was given 75 mg/kg/day or Cells were fixed with 3.7% paraformaldehyde for 10 minutes and 37.5 mg/kg/day orally. The tumor volume in mm3 was calculated washed with PBS. After washing, cells were stained with 0.05% by the formula: volume ¼ (width)2 length/2 every 7 days. crystal violet for 30 minutes and washed with tap water. Methanol (0.5 mL) was added to the dry plates to solubilize the dye. Crystal violet staining was measured by reading OD 540 nm of each Results fi sample. Tumorspheres were counted under low magni cation. Activity of BTK inhibitors in human breast cancer cells In a functional genomic screen, a novel BTK isoform has been Cell-cycle analysis identified as a gene whose expression protects breast cancer cells Cells were cultured in 6-well plates and treated with vehicle, from apoptosis (15). In addition to genetic evidence, the early BTK lapatinib, or ibrutinib at the concentration as indicated for 16 inhibitor LFM-A13 was shown to increase apoptosis levels in hours. After trypsinization, cells were collected and washed breast cancer cells. The recently developed BTK inhibitors, includ- with cold PBS. Cells were fixed with 70% ethanol, washed, ing ibrutinib, AVL-292, and CGI-1746, are more potent, more and stained with PI/Triton X-100 staining solution (0.1% Triton specific, and more useful clinically compared with LFM-A13 X-100, 2 mg/mL PI, and 0.2 mg/mL DNAse-free RNAse) for (12, 21, 22). In 2013, ibrutinib was approved by the FDA for 30 minutes. Samples were analyzed by flow cytometry. treatment of B cell malignancies (23, 24). As a first step in exploring the potential clinical utility of the recently developed Immunofluorescence BTK inhibitors, we performed cell growth assays to determine the Human breast cancer tissue sections (BR10010b, US Biomax, effect of these inhibitors on breast cancer cells. We find that Inc.) were baked for 1 hour at 62C, subjected to serial alcohol ibrutinib results in decreased cell number in breast cancer cells treatments for rehydration and microwaved in 0.01 mol/L sodi- MCF7, SKBR3, and MDA-MB-231, but not in MCF-10A cells (Fig. um citrate for 20 minutes for antigen retrieval. The sections were 1A). These results are consistent with our previous findings (15). þ serum blocked for 30 minutes, incubated overnight at 4C with Surprisingly, we observed that HER2 breast SKBR3 cancer cells primary antibodies in PBS and subsequently with Cy5-labeled are more sensitive to ibrutinib (1 mmol/L) than nontumorigenic, secondary antibodies for 60 minutes, and nuclei were stained with luminal, or triple-negative cell lines (Fig. 1A). Ibrutinib reduces Hoechst 33342. The stained sections were mounted with anti-fade cell numbers by more than 80% at 3 days. To extend our findings þ solution for microscopy. The two by two tables for human data to another HER2 breast cancer cell, we tested the effect of were analyzed by Fisher exact test. Significance was determined by ibrutinib on BT474 cell survival. The result shows that ibrutinib þ the alpha level of 0.05. decreases HER2 breast cancer cell growth by 20%–30% at a

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þ A Control Ibr 1 Ibr 10 Ibrutinib effects in HER2 breast cancer cells Ibrutinib is a covalent inhibitor of BTK (10) that irreversibly 100 binds to a cysteine residue (Cys-481) near the ATP-binding pocket of BTK. Sequence alignments show that only 10 kinases 80 * in the human genome have a cysteine residue at an analogous * position. They include Blk, Btk, Bmx, EGFR, HER2, ErbB4, Itk, 60 Jak3, Tec, and Txk (25). Given the well-characterized role of the ERBB family in breast cancer, we suspected that, in addition to 40 BTK, ibrutinib may exert its potent antiproliferative affect Live cell percentage through these targets. To compare the inhibitory effect of ibru- 20 þ ** tinib with lapatinib on HER2 breast cancer cells, we first tested 0 their activity in monolayer culture. Lapatinib, a selective, revers- MCF10A MCF7 SKBR3 MDA-MB-231 ible inhibitor of both EGFR and HER2 is currently used to treat þ HER2 breast cancer patients. Treatment with ibrutinib at dif- B μ Control Ibr 0.01 0.05 0.1 mol/L ferent concentrations as indicated for 3 days reduces BT474 cell 100 number. Ibrutinib (0.01 mmol/L) reduces cell populations to 30% of control (P < 0.01). However, lapatinib, at the same 80 * þ * concentration (0.01 mmol/L) fails to reduce HER2 breast cancer 60 cell numbers significantly (Fig. 2A). These results are generally consistent with recently published data also showing ibrutinib ** þ 40 ** inhibits cell viability of HER2 breast cancer cells (26). A number of studies have shown that cells' microenvironment can impact 20 ** þ Live cell percentage ** drug response (27, 28). To test whether HER2 breast cancer cells 0 are still sensitive to ibrutinib in 3D Matrigel culture condition, SKBR3 BT474 treatment of BT474 cells with 0.1 mmol/L or 0.5 mmol/L of C fi 100 ibrutinib for 9 days signi cantly reduces cell number when compared with the control (Fig. 2B). Thus, we find that ibrutinib þ 80 * reduces HER2 breast cancer cell number in both monolayer and * þ * 3D culture, and that ibrutinib has a more potent effect on HER2 60 breast cancer cells than lapatinib. In HER2-overexpressing breast cancer cells, HER2 dimerizes 40 with its partner EGFR or Her3. HER2/Her3 heterodimers directly phosphorylate the p85 regulatory subunit of PI3K activating the 20 **

Live cell percentage PI3K/Akt pathway (29). In parallel, HER2/EGFR heterodimers also activate the MAPK pathway in most cases (30). To examine 0 Con 0.01 0.1 1 Con 0.1 0.5 1 Con 1 5 10 μmol/L the effect of ibrutinib on levels of activated Akt or Erk, we treated BT474 cells with 0.05 mmol/L or 0.1 mmol/L of ibrutinib and Ibrutinib AVL-292 CGI-1746 0.1 mmol/L of AVL-292 for 2 hours. We find that ibrutinib inhibits Figure 1. the phosphorylation of EGFR, HER2, Her3, and ErbB4, which Growth-inhibitory effects of BTK inhibitors on breast cancer cell lines. A, cell results in blocking downstream signaling that requires Akt or Erk counts of MCF-10A, MCF-7, SKBR3, and MDA-MB-231 cells treated with activation. Compared with ibrutinib, AVL-292 does not block vehicle, 1 mmol/L, and 10 mmol/L of ibrutinib (Ibr) for 3 days. B, cell counts of EGFR family signaling pathway activation, even though AVL-292 SKBR3 and BT474 cells treated with vehicle and different concentrations of also covalently binds Cys481 on BTK. Lapatinib, a dual kinase ibrutinib, as indicated for 3 days. C, cell counts of BT474 cells treated with inhibitor of EGFR and HER2, inhibits both AKT and ERK phos- vehicle and ibrutinb, AVL-292, and CGI-1746, at indicated concentrations, for phorylation (Fig. 2D; ref. 31). These results indicate that in breast 3 days. Results are presented as percentage of control (vehicle). Error bars, the SD from three individual experiments; , P < 0.05; , P < 0.01 compared cancer cells, ibrutinib, in addition to inhibiting BTK irreversibly with control. (Supplementary Fig. S2), also serves as a pan-EGFR family inhibitor blocking the activation of each kinase. That ibrutinib is able to block two distinct types of kinases both of which have been concentration of 10 nmol/L (Fig. 1B). The IC50 for ibrutinib's established as important to the growth and survival likely explains þ þ effect on HER2 breast cancer cells measured at 3 days of culture is its greater impact on HER2 breast cancer cells compared with 30 nmol/L. We also explored whether HER2-positive breast cancer other breast cancer cells (Fig. 1). cells are sensitive to other BTK inhibitors. AVL-292 and CGI-1746 þ inhibit BTK-C kinase activity to the same degree as ibrutinib Ibrutinib effects on proliferation and survival of HER2 breast (Supplementary Fig. S1). They are, however, less effective anticancer cells þ proliferative agents than ibrutinib, achieving 30%–40% decreases In HER2 breast cancer cells, the activation of HER2 stimulates þ in cell numbers in HER2 breast cancer cell lines at concentrations both the MAPK and Akt signaling pathways, which results in cell – two orders of magnitude higher than the IC50 of ibrutinib (Fig. proliferation due to increased G1 S phase transition and cell-cycle 1C). These results suggest that ibrutinib not only inhibits BTK-C progression (31). Treatment of HER2 overexpressing BT474 activity, but also affects other targets specifically required for human breast cancer cells for 24 hours with ibrutinib or lapatinib þ – fi HER2 breast cancer cell survival. leads to an appreciable G1 S arrest. A signi cant 50% decrease in

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BTK Inhibitors and Drug Resistance in Breast Cancer

A 0.01 0.03 0.06 0.1 μmol/L

100

80 * * 60 **

Figure 2. 40 ** Effect of lapatinib and ibrutinib on cell growth and signal transduction in 20 BT474 cells. A, cell counts of BT474 Live cell percentage cells treated with vehicle, lapatinib (Lap), and ibrutinib (Ibr) for 3 days. 0 Results are presented as percentage Control Lapatinib Ibrutinib of control (vehicle). Error bars, the SD from three individual experiments; , P < 0.05; , P < 0.01 B D compared with control. B, BT474 cells pEGFR were treated with laptanib and 100 0.1 0.5 μmol/L ibrutinib, as indicated, for 9 days on pHer2 Matrigel. C, alignment of EGFR family 80 members with C481 region of BTK. D, effects of BTK inhibitors on EGFR 60 pERBB3 family activation. BT474 cells were 40 treated with different concentrations pERBB4 of BTK inhibitors ibrutinib and 20 AVL-292 or lapatinib for 2 hours.

Intact structures (%) pAkt Whole-cell lysates were prepared for 0 immunoblotting using antibodies Con Lap Ibr against p-EGFR, p-HER2, p-ERBB3, Akt p-ERBB4, p-Akt, p-ERK proteins. Immunoblotted GAPDH levels provide C 481 pERK loading controls. BTK …MANGCLL… EGFR …MPFGCLL… ERK Her2 …MPYGCLL… ERBB3 …LPLGSLL… GAPDH ERBB4 …MPHGCLL… C 0.1 0.05 0.1 0.05 0.1 μmol/L Ibrutinib AVL292 Lap the number of cells in the S phase of the cell cycle is observed at a produced via autocrine tumor–cell production, paracrine tumor concentration of 0.03 mmol/L for ibrutinib when compared with stroma production, and systemic production (33). An increase in controls (Fig. 3A). Similar results are seen for lapatinib (31). This RTK ligands has been shown to result in the resistance of cancer cell-cycle delay is correlated with an increase in p27, an inhibitor cells to RTK inhibitors which ultimately produces therapeutic þ of cell-cycle progression, and a decrease in cyclin D1 (Fig. 3B). escape (34). In HER2 breast cancer cells, NRG1 is the most An increase in the number of apoptotic cells following treat- broadly active ligand, followed by EGF (35). We hypothesized ment with 0.05 or 0.1 mmol/L ibrutinib or lapatinib for 24 hours is that the greater efficacy of ibrutinib may impact the occurrence of observed. Our results are consistent with the results of earlier growth factor–dependent lapatinib resistance. We first tested the þ studies, which have shown the effect of 0.1 or 0.5 mmol/L effect of exposing HER2 cells to 50 ng/mL of NRG1 with different lapatinib on cell survival (31). Compared with lapatinib, ibruti- concentrations of lapatinib or ibrutinib for 3 days. The results þ nib induces 1.5 fold more apoptosis in HER2 breast cancer cells again show that either lapatinib or ibrutinib potently suppresses (Fig. 3C). The effect of ibrutinib on cell survival regulatory cell growth. However, NRG1 application is able to rescue lapa- þ proteins was also examined. Ibrutinib blocks PLCg1, PLCg2, Akt, tinib-induced growth inhibition in HER2 breast cancer cells and ERK phosphorylation, and increases cleaved PARP (Fig. 3D). allowing resistant cells to emerge from the treatment (Fig. 4A). þ Taken together, these results suggest that ibrutinib decreases cell In contrast, HER2 breast cancer cells cannot be rescued from þ numbers by inducing both a G1–S delay and apoptosis in HER2 ibrutinib-induced growth inhibition by NRG1. Similar results are breast cancer cells. observed using 50 ng/mL of EGF. þ The ability of NRG1 or EGF signaling to rescue HER2 breast BTK inhibition blocks the reactivation of AKT and ERK cancer cells from growth inhibition by HER2 kinase inhibitor þ pathways induced by NRG1 or EGF in lapatinib-treated HER2 lapatinib is consistent with other findings (34). However, the þ breast cancer cells findings that neither NRG1 nor EGF was able to rescue HER2 Cancer cells typically express multiple receptor tyrosine kinases breast cancer cells from the growth inhibition by BTK inhibitor (RTK) which control cell survival signals (32). RTK ligands are ibrutinib were unexpected. To further confirm these findings,

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A B G1 S G2/M

100

80 Ibrutinib Lap C .01.03 .06 0.1 0.1 μmol/L 60 Cyclin D1 Figure 3. 40 Effect of ibrutinib on cell cycle and p27 apoptosis. A, effect of ibrutinib and lapatinib on cell-cycle progression.

Cell-cycle percentage 20 GAPDH BT474 cells were treated with the indicated concentration of ibrutinib and lapatinib for 16 hours. Cells were 0 μ Con 0.01 0.03 0.06 0.1 0.01 0.03 0.06 0.1 mol/L stained with propidium iodide (PI) and Lapatinib Ibrutinib analyzed by ﬂow cytometry. B, BT474 cells were treated with ibrutinib or C lapatinib for 16 hours. Cell lysates were D analyzed by immunoblotting using 35 0.05 0.1 µmol/L anti-cyclin D1 and anti-p27 antibodies. pHer2 ** C, BT474 cells were treated with ibrutinib or lapatinib for 16 hours. 30 γ pPLC 1 (Y783) Apoptotic cells were identiﬁed by Alexa Fluor 488 Annexin V kit. 25 pPLCγ2 (Y759) , P < 0.05; , P < 0.01 compared with control. D, immunoblots PLCγ1 20 ** showing apoptosis in BT474 cells ** after indicated concentration of pAkt ibrutinib treatment for 16 hours. 15 Akt Immunoblotted GAPDH levels provide loading controls. * pERK 10 ERK Apoptotic cells (% of total) 5 Full-length PARP Cleaved PARP 0 Con Lapatinib Ibrutinib GAPDH Ibrutinib (μmol/L) C 5 1 0.1 0.01 0.001

additional long-term cotreatment experiments were performed. in the genesis of lapatinib resistance. That we identiﬁed BTK-C Nine-day lapatinib/NRG1 cotreatment yields cells that exhibit as a critical cell survival gene in a large-scale RNAi screen in lapatinib resistance in both BT474 and SKBR3 cell populations. In these cells was consistent with this notion (15). For these contrast, 9-day ibrutinib/NRG1 cotreatment does not yield ibru- reasons, we hypothesized that BTK-C mediated the reactivation þ tinib-resistant cells in HER2 breast cancer cell lines (Fig. 4B). of AKT induced by NRG1. However, as ibrutinib is as potent as þ Similar results are observed in experiments using EGF where cells an inhibitor of EGFR family activation as lapatinib in HER2 are cotreated with EGF/lapatinib or EGF/ibrutinib (Supplemen- breast cancer cells, we needed to dissociate the effects of EGFR tary Fig. S3). inhibition from the effects of BTK inhibition. To test whether To investigate the signaling pathway which mediates the BTK-C is a secondary kinase that mediates NRG1 rescue in þ þ NRG1- or EGF-induced rescue of HER2 breast cancer cells, we HER2 breast cancer cells, we treated BT474 and SKBR3 cells examined the activation of two downstream survival signaling with lapatinib, lapatinib/NRG1, or lapatinib/NRG1 plus AVL- pathways commonly engaged by RTKs: the PI3K–AKT and MAPK 292, a BTK inhibitor that does not inhibit the EGFR family pathway (35). In the presence of lapatinib or ibrutinib alone, and (Fig. 2). We ﬁnd that NRG1 rescue is blocked by simultaneously the activation of HER2, AKT, and ERK are blocked. When cells are targeting BTK and the EGFR family when cells treated with cotreated with NRG1/lapatinib, NRG1 induces phosphorylation lapatinib and AVL-292 (Fig. 4D). These results provide evi- of AKT while HER2 remains inhibited. Surprisingly, NRG1 fails to dence that BTK-C signaling is involved in the appearance of þ reactivate AKT under conditions of NRG1/ibrutinib cotreatment ligand-dependent lapatinib resistance in treated HER2 breast (Fig. 4C). These results suggest that the NRG1-rescuing effect is cancer cell populations. blocked by ibrutinib and that ibrutinib may target a second þ activated kinase. The results also suggest that lapatinib cannot BTK-C signaling in HER2 breast cancer cells block this second kinase. The BTK signaling pathway has been extensively studied in As ibrutinib was originally designed to block BTK activation hematopoietic cells. Upon antigen binding to the BCR, PI3K is (25) and as BTK has been shown to activate AKT in B cells (36), activated. PI3K activity recruits BTK to the cell membrane through the possibility existed that BTK-C provided a prosurvival signal a PIP3-PH domain interaction, which allows SYK and LYN to fully

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A 1.2 1.2 1 1 0.8 0.8 0.6 0.6 (drug/control) 0.4 (drug/control) 0.4 Lap Percentage of live cells 0.2 0.2 Ibr Lap + NRG1 Percentage of live cells Ibr + NRG1 0 0 10-4 10-3 10-2 10-1 100 101 10-4 10-3 10-2 10-1 100 101 Lapatinib (μmol/L) Ibrutinib (μmol/L) Figure 4. Inhibition of prosurvival pathway B reactivation by ibrutinib. A, live cell Control Lap Lap+NRG Ibr Ibr+NRG count assay showing the NRG effects BT474 on drug-treated BT474 cells (72 hours), complete rescue, lapatinib (Lap) with NRG treatment; no rescue lapatinib, ibrutinib (Ibr), and ibrutinib with NRG treatment. B, crystal violet Day 9 0.06 0.37 0.06 0.06 0.08 cell staining of BT474 or SKBR3 cells treated with lapatinib (1 mmol/L) with SKBR3 or without NRG1 (50 ng/mL), ibrutinib (1 mmol/L) with or without NRG (50 ng/mL). C, immunoblots showing effects of NRG (50 ng/mL) on p-AKT and p-ERK after cells treated Day 9 0.1 0.03 0.56 0.07 0.08 0.09 with lapatinib or ibrutinib (2 hours) in BT474 and SKBR3 cells. Immunoblotted GAPDH levels provide C D 1.2 loading controls. D, live cell count BT474 assay showing the NRG effects on 1 drug-treated BT474 cells (72 hours), 0.8 Ibr Ibr+NRG1 Ibr+NRG1

complete rescue, lapatinib with NRG1 Lap+ NRG1 Control Ibr Lap+ NRG1 Lap Control Lap treatment; no rescue, lapatinib alone, 0.6 pHer2 or lapatinib and AVL with NRG1 0.4 Lap treatment. 0.2 Lap+NRG1 Lap+NRG1+AVL pAKT 0 1.2 SKBR3 pERK 1 0.8 ERK 0.6 0.4 Lap GAPDH 0.2 Lap + NRG1 Percentage of live cells (drug/control) Lap + NRG1+AVL BT474 SKBR3 0 10-4 10-3 10-2 10-1 100 101 Lapatinib (μmol/L) activate BTK (37–39). In our previous studies, we showed that a centration of LY294002 may not block BTK-C activation, we novel isoform of BTK (BTK-C) is expressed in human breast increased the concentration of LY294002 to 50 mmol/L and cancer cell lines and tissues. To explore the signaling activation repeated the test. The results show that LY294002 at 50 mmol/L of BTK-C in breast cancer cells, we assessed two potential completely blocks AKT activation, but not BTK-C activation upstream regulatory molecules of BTK-C: PI3K and Src (40). (Fig. 5B). Collectively, these results suggest that Src, or a closely First, we treated the SKBR3-BTK-C cells for 24 hours with related kinase or kinases, is a signiﬁcant player in the upstream þ established concentrations of the PI3K inhibitor LY294002 signaling pathway of BTK-C activation in HER2 breast cancer (5 or 10 mmol/L) or the Src inhibitor saracatinib (5 or cells. The Src/FAK signaling pathway is involved in the lapati- 10 mmol/L). The phosphorylation of BTK-C is appreciably nib-induced kinome reprogramming (42) that contributes to þ decreased by saracatinib at 10 mmol/L (41). The phosphoryla- drug resistance in HER2 breast cancer. Inhibition of Src/FAK tion of AKT, as a downstream target of BTK-C, also decreases. In signaling enhances lapatinib growth inhibition in these cells. contrast, 10 mmol/L LY294002 does not suppress BTK-C acti- Consistent with the notion that saracatinib inhibits BTK-C vation (Fig. 5A). As the possibility exists that this lower con- activation we ﬁnd that blocks the NRG1-mediated rescue of

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A Ctr Ibr LY294002 Saracatinib tumor growth when administered to animals between 6 mg/ 0.1 5 10 5 10 μmol/L kg/day and 12 mg/kg/day. At day 28, tumor volumes in mice that received 12 mg/kg are 45% smaller than the volumes in BTK-C-Flag mice that receive a vehicle control (P < 0.01; Fig. 6A). To determine whether inhibition of tumor growth was correlated p-BTKC with inhibition of the target molecules, HER2 and BTK phosphorylation were examined in tumor tissues by immunofluo- p-AKT rescence staining as well as the phosphorylation of downstream targetsAKTandERK.PhosphorylationofHER2,BTK,AKT,and p-ERK ERK are inhibited by ibrutinib treatment (Fig. 6B). In addition, changes in proliferation and apoptosis markers in tumor tissues GAPDH reflect the effect that ibrutinib has in xenografts of SKBR3. The ability of ibrutinib to target both HER2 and BTK-C in these B xenografts confirms the in vitro findings and provides a strong þ pEGFR rationale for the use of ibrutinib in HER2 breast cancer EGFR chemotherapy.

þ pHer2 The expression of BTK-C in HER2 breast cancer tissue The above results suggest that simultaneously targeting HER2 pBTK and BTK represents an improved approach to the treatment of þ BTK-C-Flag HER2 breast cancer. We identified the BTK-C isoform as a gene pAkt whose function is critical for breast cancer cell survival even though its expression levels are quite low. For this gene product pERK to be a useful target, it is necessary to determine the frequency þ of its expression in HER2 breast cancer. Although currently ERK routine for many genes, establishing BTK-C expression patterns GAPDH in tumors by querying available databases is not possible. The C 0.1 10 20 30 50 μmol/L BTK-C isoform has only recently been described and little is Ibr LY294002 C understood regarding its expression. This is due in part to the Control Lap Lap+NRG Lap+NRG+Saracatinib fact that Affymetrix probes for this region have only been included in exon microarrays very recently. In addition, the BTK-C isoform encodes the entire B-cell version sequence (BTK- A) and is annotated as a 50 UTR splice variant of BTK-A (15). To provide further evidence about the expression of BTK-C in þ breast cancer tissues, especially HER2 breast cancer tissues, Day 9 0.09 0.04 0.43 0.06 0.11 0.04 we examined the coexpression of BTK-C and HER2 in surgical specimens of human breast cancer tumor tissues by immuno- Figure 5. fluorescence. Consistent with our previous studies (15), we BTK-C activation by Src in breast cancer cells. A, SKBR3-BTKC cells were treated find that 30% of breast cancer tumor tissues stain positively for with ibrutinib, LY294002, and saracatinib at indicated concentrations for þ BTK (Fig. 7). Among 23 HER2 human breast cancer tissues, 24 hours. Cell lysates were probed for p-BTK, p-AKT, and p-ERK. B, fi SKBR3-btkc cells were treated with ibrutinib and different concentrations of 43% are positive for BTK. There is a statistically signi cant LY294002 for 24 hours. Cell extraction were tested for phosphorylation of the association of expression of BTK with HER2 expression (P < indicated protein. Anti-ERK was used as a loading control. C, crystal violet cell 0.01; Fig. 7B). These findings suggest that the expression of staining of BT474 cells treated with lapatinib (1 mmol/L) with or without NRG1 BTK-C and HER2 are positively correlated in human breast (50 ng/mL), and lapatinib with NRG (50 ng/mL) and saracatinib (5 mmol/L). cancer tissues. This increases the likelihood that ibrutinib has Immunoblotted GAPDH levels provide loading controls. breast cancer therapeutic utility as the opportunity to target both kinases simultaneously should occur in a significant þ þ percentage of HER2 breast cancers. lapatinib sensitivity in HER2 breast cancer cells (Fig. 5C). These findings suggest that BTK-C is a downstream target of Src or a closely related kinase or kinases and that this signaling Discussion þ contributes to NRG1-mediated drug resistance in HER2 breast Our previous findings showed that a novel isoform of BTK cancer cells. (BTK-C) is frequently expressed in human breast cancer cellsandtissuesandthatthisisoformplaysacrucialrolein þ Effects of ibrutinib treatment on HER2 breast tumor xenograft cell survival. Consistent with these findings, we report that growth in vivo several recently developed BTK inhibitors reduce breast cancer Results from molecular experiments carried out in vitro cell number in a variety of cell types including MCF7, MDA- pointed to the possibility that ibrutinib treatment might be MB-231 and SKBR3 cells. Importantly, we found that HER2- useful in inhibiting HER2-positive tumor progression. To test positive breast cancer cells are highly sensitive to ibrutinib. this possibility, we assessed the effect of ibrutinib on xenografts Ibrutinib, with an IC50 for these cells of 0.03 mmol/L, is of SKBR3 in NOD/SCID mice. Ibrutinib treatment inhibits much better at decreasing cell numbers than the other BTK

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BTK Inhibitors and Drug Resistance in Breast Cancer

A 2,000 Control Control 1,800 ) Ibrutinib 6 mg/kg 3 1,600 Ibrutinib 12 mg/kg Lapatinib 37.5 mg/kg 1,400 Lapatinib Lapatinib 75 mg/kg 37.5 mg/kg 1,200 Lapatinib 1,000 75 mg/kg 800 600 Ibrutinib volume (mm Tumor Figure 6. 6 mg/kg 400 Ibrutinib inhibits Her2þ breast cancer cells growth in vivo. A, images of 200 tumors formed in animals after Ibrutinib 0 mammary fat pad injection of SKBR3 12 mg/kg cancer cells. Animals were treated 0 1 2 3 4 with vehicle, lapatinib (37.5 mg/kg or Days of treatment (week) 75 mg/kg) or ibrutinib (6 mg/kg or 12 mg/kg) for 4 weeks. B, tumor Control Ibrutinib 12 mg/kg Control Ibrutinib 12 mg/kg growth curves obtained following fat B pad injection of SKBR3 cells. The

data represent the mean SE (n ¼ 5; pHH3 , P < 0.01). C, histologic analysis of pHer2 tumors from SKBR3 cells treated with ibrutinib (12 mg/kg) or vehicle. Shown are pHH3, caspase 3, pHER2, pBTK, pAkt, and pErk staining. Caspase 3 AtpErk pAkt pBTK

inhibitors, AVL-292 and CGI-1746, and compares favorably resistance to lapatinib. As we find that the expression of BTK-C with lapatinib. In vitro, enzymatic activity assays show that IC50 and HER2 are positively correlated in human breast cancer of lapatinib for EGFR and HER2 are10.8 and 9.2 nmol/L tissue with 40% of HER2-positive breast cancer tissues also respectively. Our results indicate that the sensitivity of being BTK-C positive, the potential utility of the drug is þ HER2 breast cancer cells to ibrutinib is due to the drug's significant. ability to simultaneously suppress the activation of both BTK-C In accord with the in vitro studies, our results show that as well as the EGFR family. Consistent with previous studies, ibrutinib treatment at 12 mg/kg/day causes a significant 45% ibrutinib treatment results in induction of a pronounced G1–S inhibition of HER2-positive tumor growth in vivo. Animals tol- þ delay and increased apoptosis in HER2 breast cancer cells erate ibrutinib at this dose, although ibrutinib–related toxicity (31). We also find that BTK-C plays an essential role in the was observed. Consistent with ibrutinib's antitumor effects, stain- þ ability of HER2 breast cancer cells to develop resistance to ing of the proliferation marker, phosphorylated histone H3, lapatinib under conditions when the growth factors NRG1 or decreased in treated xenografts tumor tissues compared with EGF are present. NRG1 or EGF are unable to reactivate PI3K/Akt untreated control tumor tissues. Moreover, we observed focal or MAPK signaling pathways and allow cells to escape the apoptotic lesions caused by ibrutinib treatment, as evidenced by inhibitory effects of lapatinib when BTK-C is blocked by caspase-3 staining in xenografts tumor tissues. In these experi- AVL-292, which is incapable of inhibiting the EGFR family. ments, lapatinib treatment at 75 mg/kg/day was less potent in Ibrutinib inhibits the activity of the EGFR family and inhibits SKBR3 tumor xenograft models than ibrutinib treatment at 12 the BTK-dependent reactivation of the PI3K/Akt or MAPK mg/kg/day. This is likely due to the ability of ibrutinib to target signaling pathways. It is, therefore, a strong candidate to replace irreversibly both BTK and EGFR family members and block lapatinib as a combination therapy with trastuzumab for reactivation of PI3K/AKT or MAPK caused by growth factors such þ HER2 breast cancer patients who might otherwise develop as NRG1 in vivo. Taken together, our findings show that ibrutinib

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Wang et al.

A Case 1 2 3 leads to constitutive signaling of downstream pathways: PI3K/ Akt and the Ras/Raf/MEK/MAPK, which are involved in regu- lating cell growth, survival, differentiation, migration, and metastasis. Reactivation of these pathways allows cells to escape from the antiproliferative and antiapoptotic effects of the HER2 inhibitors. Ibrutinib was the first reported covalent inhibitor of BTK (25). As an orally bioavailable, selective and irreversible inhibitor, ibrutinib has been undergoing multiple clinical trials targeting treatment of various B-cell malignancies and has shown prom- Her2 ising clinical efficacy (10). On the basis of sequence alignments, 10 kinases in the human genome have an orthologous cysteine residue (25). The EGFR family includes EGFR/Her1, HER2, Her3, and Her4 all of which are involved in several aspects of tumor- igenesis. These kinases include EGFR, HER2, and Her4 covalently bind to the thiol group of Cys481 in the ATP pocket region of BTK B (position Cys515 of BTK-C). In vitro, enzymatic activity assays Her2 expression P show that and have IC50 of ibrutinib for each enzyme in the Posive Negave Total nanomolar range BTK, EGFR, HER2 and Her4 are 0.5, 5.6, 9.4, and 0.6 nmol/L respectively. In addition, ibrutinib also inhibits ITK, Posive 10 8 18 an essential enzyme in Th2 T cells, which shifts the balance BTK-C between Th1 and Th2 T cells and potentially enhances antitumor Negave 13 19 32 immune responses (43). Given the relevance of this spectrum of Total 23 27 50 <0.01 targets, it is therefore not surprising that ibrutinib is effective in þ decreasing the proliferation and increasing apoptosis in HER2 Figure 7. breast cancer cells. Positive correlation between BTK and HER2 expression in surgical specimens of In summary, this work demonstrates that BTK-C is a novel human breast cancer tissue. A, Her2þ and negative surgical specimens of human therapeutic target for breast cancer, and that current recently breast tumors (BR10010b) were subject to stain BTK and HER2. The data developed BTK inhibitors may be useful in the treatment of þ represent 50 of breast tumor specimens. Case 1 and case 2 represent positive HER2 breast cancers. On the basis of its efficacy in breast cancer staining BTK-C, case 1 and case 3 represent positive staining HER2. B, summary of the positive correlation. The correlation between the overexpression of BTK-C cells, its ability to reduce xenograft growth and its effects on EGFR and HER2 was analyzed by Fisher exact test. family members, tests of ibrutinib used in a neoadjuvant or adjuvant setting with trastuzumab or other chemotherapeutic þ compounds for HER2 breast cancer is warranted. Moreover, as actively blocks BTK and EGFR family activation efficiently inhi- BTK inhibition abolishes the ability of either NRG1 or EGF to þ þ biting HER2 breast cancer growth in vivo. As a result, this study reactivate Akt or Erk in HER2 breast cancer cells, drugs such as þ supports the use of ibrutinib as a HER2 breast cancer treatment AVL-292 might also be useful in suppressing therapeutic escape in and indicates that targeting BTK-C with recently developed BTK these cancers. inhibitors such as ibrutinib or AVL-292 may avert the development of drug resistance in breast cancer patients. Disclosure of Potential Conflicts of Interest fi The advent of HER2-directed therapy has signi cantly X. Wang, L. Kokabee, and D.S. Conklin are currently inventors on the patents improved the prognosis of patients with metastatic and early but do not have ownership interest at this juncture. No potential conflicts of þ HER2 breast cancer. Currently, there are two classes of HER2- interest were disclosed by the other authors. targeting drugs used in the clinic. Trastuzumab, trastuzumab- DM1, and pertuzumab are antibody-based drugs, whereas Disclaimer lapatinib is a small molecule that reversibly inhibits both Some of the data in this study have been used to support one or more patent EGFR and HER2, blocking the PI3K/Akt and MAPK pathways. þ applications. Lapatinib is the only small compound used to treat HER2 breast cancer that has metastasized to the brain due to its Authors' Contributions ability to cross the blood–brain barrier. Despite this recent Conception and design: X. Wang, D.S. Conklin progress, acquired resistance to HER2-directed therapy still þ Development of methodology: X. Wang, J. Wong, L. Kokabee results in relapse and progression of HER2 disease. The Acquisition of data (provided animals, acquired and managed patients, primary mechanism of resistance to lapatinib stems from provided facilities, etc.): X. Wang, J. Wong, L. Kokabee, D.S. Conklin increased levels of EGFR or HER3 ligands such as NRG1 or Analysis and interpretation of data (e.g., statistical analysis, biostatistics, EGF in the tumor microenvironment which reactivate the computational analysis): X. Wang, L. Kokabee, Y. Sun, D.S. Conklin PI3K/Akt and MAPK signaling pathways that are blocked by Writing, review, and/or revision of the manuscript: X. Wang, J. Wong, F. Khan, Y. Sun, D.S. Conklin the drug (34). The activation of the signaling pathway down- Administrative, technical, or material support (i.e., reporting or organizing stream of EGFR family members other than HER2 is dependent data, constructing databases): X. Wang, J. Wong, L. Kokabee, F. Khan, Y. Sun, on heterodimerization of the EGFR family member triggered D.S. Conklin by NRG1 or EGF binding to the extracellular ligand-binding Study supervision: X. Wang, D.S. Conklin domain. Increased expression of HER2 at the cell membrane Other (conception and design of IHC experiment): C.J. Sevinsky

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BTK Inhibitors and Drug Resistance in Breast Cancer

Acknowledgments The costs of publication of this article were defrayed in part by the The authors thank members of the Conklin laboratory and the University at payment of page charges. This article must therefore be hereby marked advertisement Albany Laboratory Animal Care Facility for help with experimentation and in accordance with 18 U.S.C. Section 1734 solely to indicate critical reading of the manuscript. this fact. Grant Support This work was supported by NCI R01CA136658 and a Technology Accel- Received October 8, 2015; revised April 12, 2016; accepted May 19, 2016; erator Fund Award from The Research Foundation for the State University of published OnlineFirst June 2, 2016. New York (to D.S. Conklin).

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2208 Mol Cancer Ther; 15(9) September 2016 Molecular Cancer Therapeutics

Bruton's Tyrosine Kinase Inhibitors Prevent Therapeutic Escape in Breast Cancer Cells

Xianhui Wang, Jason Wong, Christopher J. Sevinsky, et al.

Mol Cancer Ther 2016;15:2198-2208. Published OnlineFirst June 2, 2016.

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