Defective Cyclin B1 Induction in Trastuzumab-Emtansine (T-DM1) Acquired Resistance in HER2-Positive Breast Cancer

Published OnlineFirst August 18, 2017; DOI: 10.1158/1078-0432.CCR-17-0696

Cancer Therapy: Preclinical Clinical Cancer Research Defective Cyclin B1 Induction in Trastuzumab- emtansine (T-DM1) Acquired Resistance in HER2-positive Breast Cancer Mohammad A. Sabbaghi1, Gabriel Gil-Gomez 1, Cristina Guardia1, Sonia Servitja1,2, Oriol Arpí1, Sara García-Alonso3, Silvia Menendez1, Montserrat Arumi-Uria4, Laia Serrano4, Marta Salido1,4, Aura Muntasell5, Maria Martínez-García1,2, Sandra Zazo6, Cristina Chamizo6, Paula Gonzalez-Alonso 6, Juan Madoz-Gurpide 6, Pilar Eroles7, Joaquin Arribas8,9,10, Ignasi Tusquets1,2, Ana Lluch11, Atanasio Pandiella3, Federico Rojo6, Ana Rovira1,2, and Joan Albanell1,2,12

Abstract

Purpose: Trastuzumab-emtansine (T-DM1) is a standard treat- SKBR3/TDR cells). HER2 remained ampliﬁed in the resistant cells. ment in advanced HER2-positive breast cancer. However, resis- Binding to HER2 and intracellular uptake of T-DM1 were main- tance inevitably occurs. We aimed to identify mechanisms of tained in resistant cells. T-DM1 induced cyclin B1 accumulation in acquired T-DM1 resistance. sensitive but not resistant cells. Cyclin B1 knockdown by siRNA Experimental Design: HER2-positive breast cancer cells in parental cells induced T-DM1 resistance, while increased (HCC1954, HCC1419, SKBR3, and BT474) were treated in a levels of cyclin B1 by silencing cdc20 partially sensitized resis- pulse-fashion with T-DM1 to induce a resistant phenotype. Cel- tant cells. In a series of 18 HER2-positive breast cancer fresh lular and molecular effects of T-DM1 in parental versus resistant explants, T-DM1 effects on proliferation and apoptosis paral- cells were compared. CDK1 kinase activity and cyclin B1 expres- leled cyclin B1 accumulation. sion were assayed under various conditions. Genetic modiﬁca- Conclusions: Defective cyclin B1 induction by T-DM1 med- tions to up- or downregulate cyclin B1 were conducted. Effects of iates acquired resistance in HER2-positive breast cancer cells. T-DM1 on cyclin B1 levels, proliferation, and apoptosis were These results support the testing of cyclin B1 induction upon assayed in human HER2-positive breast cancer explants. T-DM1 treatment as a pharmacodynamic predictor in HER2- Results: We obtained three cell lines with different levels of positive breast cancer. Clin Cancer Res; 23(22); 1–14. 2017 AACR. acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and

Introduction covalently linked to the antimitotic agent DM1 through a stable linker that potently inhibits growth of both trastuzumab-sensitive Trastuzumab-emtansine (T-DM1) is an antibody–drug conju- and -resistant HER2-ampliﬁed cancer cells (1). T-DM1 has gate (ADC) consisting of the anti-HER2 antibody trastuzumab mechanisms of action containing of the antitumor effects related to trastuzumab and those associated with intracellular DM1 1Cancer Research Program, IMIM (Hospital del Mar Research Institute), Barce- catabolites (2, 3). DM1 is a derivative of maytansine, a highly lona, Spain. 2Medical Oncology Department, Hospital del Mar- CIBERONC, potent antimitotic drug (4). Once bound to HER2, T-DM1 enters Barcelona, Spain. 3Centro de Investigacion del Cancer, Universidad de Sala- in the cell by receptor-mediated endocytosis and the HER2–T- manca-CSIC-CIBERONC, Salamanca, Spain. 4Pathology Department, Hospital DM1 complex is processed via degradation of trastuzumab in 5 del Mar, Barcelona, Spain. Immunity and infection Laboratory, IMIM (Hospital lysosomes giving rise to the intracellular release of the active del Mar Research Institute), Barcelona, Spain. 6Pathology Department, IIS- catabolite Lys-MCC-DM1. In the cytoplasm, DM1 exerts its func- Fundacion Jimenez Díaz- CIBERONC, Madrid, Spain. 7INCLIVA Biomedical Research Institute, Valencia, Spain. 8Preclinical Research Program, Vall d'Hebron tions through binding to the beta subunit of tubulin and mod- Institute of Oncology (VHIO)-CIBERONC, Barcelona, Spain. 9Autonomous Uni- ifying its assembly properties. By doing so, DM1 is able to disrupt versity of Barcelona, Barcelona, Spain. 10Institucio Catalana de Recerca I Estudis the formation of the mitotic spindle necessary for accurate chro- Avancats¸ (ICREA), Barcelona, Spain. 11Oncology and Hematology Department, mosome segregation along mitotic process. Overall, DM1 as other 12 Hospital Clínico Universitario-CIBERONC, Valencia, Spain. Universitat Pompeu antimitotic drugs elicits a mitotic arrest and cells therefore fail Fabra, Barcelona, Spain. to complete a normal mitosis. This prolonged cell-cycle delay Note: Supplementary data for this article are available at Clinical Cancer eventually culminates in cell death by mitotic catastrophe, necro- Research Online (http://clincancerres.aacrjournals.org/). sis, or apoptosis (5). In addition to these DM1-related actions, Corresponding Author: Joan Albanell, Medical Oncology Department, Hospital T-DM1 maintains properties of trastuzumab such as inhibition of del Mar, Passeig Maritim 25-29, Barcelona 08003, Spain. Phone: 349-3248-3137; HER2-directed signal transduction and activation of antibody- Fax: 349-3248-3366; E-mail: [email protected] dependent cell-mediated cytotoxicity (2, 3). doi: 10.1158/1078-0432.CCR-17-0696 T-DM1 is a standard second-line treatment for HER2- 2017 American Association for Cancer Research. positive metastatic breast cancer patients based on the results

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action of T-DM1, we focused on resistance potentially related to Translational Relevance the cell-cycle–modulating effects of DM1. We identified that the – Trastuzumab-emtansine (T-DM1) is an antibody drug con- G2–M arrest induced by T-DM1 in sensitive HER2 breast cancer jugate constituted by trastuzumab linked to DM1 (a tubulin cells did not occur in their resistant counterparts in a CDK1/cyclin polymerization inhibitor). T-DM1 is a standard treatment in B1–dependent manner. In fresh HER2-positive breast cancer advanced HER2-positive metastatic breast cancer. However, explants, lack of induction of cyclin B1 correlated with T-DM1 resistance inevitably occurs. To date, no clinical biomarker of failure to induce apoptosis. T-DM1 resistance has been identified. In this study, we in obtained three T-DM1 acquired resistance breast cancer Materials and Methods vitro models. We report that cyclin B1 induction is a hallmark of T-DM1 activity in both trastuzumab primary sensitive and Cell lines and reagents resistant cells. In HER2-positive breast cancer cells with Breast cancer cell lines BT474, SKBR3, AU565, EFM-192A, acquired T-DM1 resistance, the drug failed to induce cyclin HCC1954, and HCC1419 were obtained from the ATCC. B1, while reducing cyclin B1 degradation in these cells Authenticity of the cells was tested by STR DNA Profiling partially restores sensitivity. In fresh human HER2-positive analysis at the ATCC (June 2013 and December 2014) before breast cancer explants, the induction (but not the baseline starting the generation of resistant cells. The number of pas- levels) of cyclin B1 by T-DM1 correlated with apoptosis, sages between thawing and use in the described experiments suggesting that a pharmacodynamic assay to test cyclin B1 was five or less. T-DM1 (trastuzumab emtansine, Kadcyla) was induction in breast cancer patients treated with T-DM1 may provided by Genentech under MTA agreement (Sliwkowski MX, help to identify early the patients more likely to benefitfrom Lewis Phillips GD) and trastuzumab by Hospital del Mar that drug treatment. pharmacy (Barcelona, Spain).

Cell proliferation assays Cells were plated in duplicate into 12-well culture plates at a density of 8–12 103 cells per well and left overnight and then on day zero, treatment was initiated. At this time T-DM1 (0.1–1 mg/mL) of a phase III clinical trial (EMILIA trial), that compared the was added. On days 3, 7, and 10, cells were washed with PBS, efficacy and toxicity of T-DM1 versus the combination of trypsinized, resuspended in media, and counted with Scepter Auto- lapatinib and capecitabine in patients previously treated with mated Cell Counter (Millipore). trastuzumab and chemotherapy. T-DM1 offered superior activity, tolerability, and survival than lapatinib and capecitabine Generation of cell lines with acquired T-DM1 resistance (6). However, resistance to T-DM1 occurs (3, 7, 8). To date, the T-DM1–resistant cell lines were derived from original paren- main T-DM1 clinical resistance mechanisms studies included tal cell lines by exposure to stepwise increasing concentrations an exposure–response and exploratory biomarker analysis, of T-DM1 in a pulse fashion (13). The protocol is summarized both based on patients from the EMILIA and TH3RESA trial in Fig. 1A. Three T-DM1 acquired resistance sublines were (9–11). In the exposure–response analysis, the data showed collected named SKBR3/TDR, HCC1954/TDR, and HCC1419/ that higher T-DM1 exposure was associated with improved TDR. In addition, vehicle-treated parental cell lines were kept in efficacy. This analysis suggested that there is an opportunity to culture during this period as control cell lines. We established an optimize T-DM1 dose in the patient subgroup with low expo- exposure to 0.1 mg/mL of T-DM1 for 3 days as a reference sure (10). With regards to the exploratory biomarker study of schedule to define resistance in our model. This schedule was the EMILIA trial, focusing only in the T-DM1 group, univariate selected on the basis of experiments performed in MCF7 cells analysis of the data suggested that there were no evident that do not overexpress HER2, whose growth was unaffected differences in progression-free survival (PFS) related to EGFR, until higher concentrations and longer exposure to T-DM1 and HER3 median mRNA concentration ratios, PIK3CA muta- were used. tions status, or PTEN expression; patients with HER2 mRNA above the median had better outcomes on T-DM1 than those HER2 equal or below the median (11). In the TH3RESA phase III FISH for fi fi trial, performed in heavily pretreated HER2-positive advanced Formalin- xed and paraf n-embedded cell pellets were pre- – breast cancer, patients were randomized to T-DM1 or treat- pared from parental and T-DM1 resistant cells to assess the status HER2 fi ment of physician's choice. An exploratory biomarker analysis of gene by scoring its ampli cation following ASCP/CAP HER2 included HER2 and HER3 mRNA expression, PIK3CA mutation guidelines (14). The commercial PathVysion DNA probe status, and PTEN protein expression. T-DM1 prolonged median was used. PFS in all the subgroups analyzed. A numerically greater benefit was reported in patients with tumors expressing HER2 mRNA T-DM1/HER2 receptor binding above the median (9). More recently, in the ZEPHIR trial, the T-DM1 binding to the cell surface was evaluated by indirect use of molecular imaging of HER2 by HER2-PET/CT with immunofluorescence staining and flow cytometry. Cells (2–5 (89)Zr-trastuzumab combined with early metabolic response 105) were incubated with T-DM1 (7.5 mg/mL) for 30 minutes assessment by FDG-PET/CT, discriminated patients with short followed by R-Phycoerythrin AffiniPure F(ab0)₂ Fragment Goat versus long time to T-DM1 treatment failure (12). Anti-Human IgG, Fcg Fragment Specific (Jackson ImmunoRe- The biomarker analyses discussed above were focused on the search; 1:500 dilution) for 30 minutes at 4C. Trastuzumab HER2 signaling pathway. However, given the dual mechanism of (7.5 mg/mL) and rituximab (MabTera, Roche; 7.5 mg/mL) were

OF2 Clin Cancer Res; 23(22) November 15, 2017 Clinical Cancer Research

Cyclin B1 and T-DM1 Acquired Resistance

A T-DM1 [1 μg/mL] T-DM1 [2 μg/mL] T-DM1 [4 μg/mL] 18 days 18 days 18 days Parental T-DM1 Acquired cells resistant cells 3 days 3 days 3 days 3 days 3 days 3 days 3 days 3 days 3 days on/off on/off on/off on/off on/off on/off on/off on/off on/off

120 *** 120 120

80 *** 80 ** 80 *** *** 40 40 40 Cell number (% of control) 0 0 0 00.11 00.11 00.11 T-DM1 [μg/mL] T-DM1 [μg/mL] T-DM1 [μg/mL] HCC1954 HCC1954/TDR HCC1419 HCC1419/TDR SKBR3 SKBR3/TDR

C ** *** 120 120 120 Control 80 80 80 Trastuzumab

40 40 40 Cell number (% of control) 0 0 0 HCC1954 HCC1954/TDR HCC1419 HCC1419/TDR SKBR3 SKBR3/TDR

Figure 1. Generation and growth characteristics of HER2-positive breast cancer cells with T-DM1 acquired resistance. A, Pulsed treatment strategy for the in vitro establishment of acquired resistant cells to T-DM1. The procedure consisted of three consecutive cycles of 3 days on treatment followed by 3 days off treatment for each T-DM1 concentration of 1, 2, and 4 mg/mL. The entire 54-day protocol resulted in the generation of cells with varying levels of T-DM1 resistance. B, Growth characteristics of T-DM1–resistant cells. Each resistant cell line was compared with the corresponding age and passage-matched parental cell line. HCC1954- HCC1954/TDR, HCC1419-HCC1419/TDR, and SKBR3-SKBR3/TDR pairs of sensitive and resistant cells were seeded in duplicate in 12-well plates at a density of 8–12 103 cells per well and allowed to adhere overnight. Then, cells were treated without (white columns) or with two concentrations of T-DM1 (black columns) for 3 days. Cells were collected by trypsinization and counted using a Scepter cell counter (Millipore). Experiments were performed in triplicate and data represent the mean SD cell number relative to control. Error bars, SD. C, T-DM1–resistant cells retain a similar sensitivity to trastuzumab. Each paired sensitive/ resistant cell lines were seeded as in B and treated in duplicate with or without 15 mg/mL trastuzumab for 7 days. Medium changes were performed every 3 days. Cells were trypsinized, collected, and counted using Scepter. The results are presented as mean percent of viability in treated versus nontreated controls SD. used as positive and negative controls, respectively. Live/dead gate Cell-cycle assay was set with DAPI counterstaining. Samples were acquired on LSR Cells were seeded on 6-well plates and treated with compounds Fortessa flow cytometer (BD Biosciences), and data analyzed with for 24 hours by T-DM1 (0.1 mg/mL). Thereafter, cells were incu- FlowJo software (TreeStar). bated with 30 mmol/L bromodeoxyuridine (BrdUrd, B9285, Sigma) for 30 minutes at 37C, and then subsequently trypsi- T-DM1 internalization assay nized, washed with PBS, and fixed in ice-cold ethanol for at least T-DM1 internalization was evaluated by immunofluores- one hour at 20 C. Cells were digested in prewarmed 1 mg/mL cence staining. Cells (1.5 105) were seeded on coverslips and pepsin in 30 mmol/L HCl (pH 1.5) for 30 minutes at 37 C with treated with 1.5 mg/mL T-DM1 for 15 minutes. After washing gentle shaking, and then incubated in 2 mol/L HCl for 20 minutes out the drug, cells were cultured for 24 hours with or without at room temperature. Next, cells were washed with PBS and 5 mmol/L chloroquine (a drug that induced changes in lyso- antibody buffer (0.5% w/v BSA, 0.5% v/v Tween-20 in PBS) and somal pH) to accumulate intracellular T-DM1. Anti-human incubated with mouse primary antibody against BrdUrd (555627, Cy3-conjugated antibody was used to detect T-DM1, phalloi- BD Pharmingen) in antibody buffer for one hour at room tem- din-FITC (P5282 Sigma) was used for actin staining, and perature. After washing with PBS, samples were incubated with nuclei were counterstained with DAPI. Sample processing was secondary goat anti-mouse IgG FITC-conjugated antibody for 30 performed as reported previously (15). minutes at room temperature in the dark. Finally, cells were

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washed one more time with PBS and then stained with 25 mg/mL specific optimized program for siRNA delivery with the Nucleo- propidium iodide. Flow cytometry was performed using a Becton fector (SKBR3 E-009). Dickinson FACScan operated by the CELLQuest software. Exposure of fresh human breast cancer explants to T-DM1 ex vivo Cdc2/CDK1 activity assay Cdc2/CDK1 kinase activity was measured with the nonradio- The study was approved by the ethics committee of the Hospital active MESACUP Cdc2/CDK1 Kinase Assay Kit (MBL, Interna- del Mar and conducted following institutional guidelines. Fresh tional Corporation) following the manufacturer's instructions. tumor specimens from women with HER2-positive breast cancer The method is based on an ELISA assay that utilizes a biotinylated undergoing routine cancer surgery, which were not needed for ex vivo synthetic peptide as a substrate for the Cdc2/CDK1 kinases diagnostic purposes, were collected to add T-DM1 and present in the samples and a mAb conjugated to horseradish assess its molecular effects according to our experience (16). peroxidase (HRP) recognizing the phosphorylated form of the Samples were sliced and cultured in RPMI1640 medium supple- peptide. The HRP substrate is then added and the intensity of the mented with 10% FBS, 2 mmol/L L-glutamine, and 100 U/mL – color was measured at 492 nm. The results are reported as fold penicillin streptomycin for 120 hours in the absence (control) or fi induction of OD 492 nm in arbitrary units (RLA) of treated presence of T-DM1 (0.1 mg/mL). Specimens were xed in 10% fi sample using untreated condition as reference set at 1. neutral-buffered formalin for 16 hours and embedded in paraf n then assayed by IHC.

Immunocytochemistry IHC Cells were seeded on glass coverslips and cultured as indicated Three-micrometer–thick paraffin sections from tissue blocks of in the figure legends. Cells washed by PBS, and then fixed in 4% the tumors were stained for HER2 (Herceptest P980018/S010, paraformaldehyde for 10 minutes at room temperature. Then the Dako), cyclin B1(sc-245, Santa Cruz Biotechnology), Ki-67 samples permeabilized by PBS containing 0.25% Triton X-100, (GA62661-2, MIB1 clone, Dako), phosphorylated (Ser10) His- and then, incubated with 1% BSA in PBST for 30 minutes tone 3 (9701, Cell Signaling Technology), and active caspase-3 for blocking unspecific binding of the antibodies. In the next (9664, Cell Signaling Technology) followed by incubation with step, samples incubated for 1 hour with anti-a-tubulin FITC- an anti-rabbit Ig dextran polymer (Flexþ, Dako) and 3,30-diami- conjugated antibody (F2168, Sigma). Cell nuclei were stained nobenzidine as chromogen in a Dako Link platform. HER2 with DAPI (1:1,000; Sigma) for 15 minutes at room tempera- staining was scored following ASCP/CAP guidelines (14). For the ture. After washing, coverslips were mounted with a drop of other markers, the percentage of positive tumor cells was scored. mounting medium and viewed using a Leica SP5 upright confocal microscope. Statistical analysis Data are presented as mean SD. The GraphPad Prism soft- Immunoblotting ware was used to construct graphs and statistical analysis. Statis- Cellular protein lysates were prepared in lysis buffer [50 mmol/L tical significance was determined using Student t test or one-way Tris-HCl, pH 8.0, 100 mmol/L NaCl, 1% (v/v) Nonidet P-40, 0.1% ANOVA. P 0.05 was considered as significant. (w/v) SDS] containing protease inhibitors and quantified by use of the Bradford assay (Bio-Rad Laboratories). Equivalent protein Results amounts of each sample were analyzed. Protein detection on Western blots was performed according to standard protocols. Generation of T-DM1–resistant HER2-positive breast cancer The antibodies used were: HER2 (Biogenex), cyclin B1 (sc-245), cells and CDC2 p34 (CDK1; sc-54) purchased from Santa Cruz Bio- We assessed the half maximal inhibitory concentration (EC50) technology and b-actin (A-5316) was purchased from Sigma. values of T-DM1 in a panel of HER2-positive breast cancer cells. The median EC50 values for BT474, SKBR3, AU565, EFM-192A, HCC1954, and HCC1419 cell lines at 72 hours were 0.025, 0.002, Apoptosis and cell death analysis 0.005, 0.009, 0.020, and 0.018 mg/mL, respectively. Four cell For measuring apoptosis, the Annexin V and Dead Cell Assay lines, two sensitive to trastuzumab (SKBR3 and BT474) and Kit (Millipore) was used according to the manufacturer's instruc- two with primary resistance to trastuzumab (HCC1954 and fl tions. Brie y, after treatment, the cells were incubated with HCC1419) were selected to generate T-DM1 resistance (TDR) by Annexin V and Dead Cell Reagent (7-AAD) for 20 minutes at applying a pulsed administration strategy of the drug, often used room temperature in the dark, and the events for dead, late for the development of chemotherapy drug resistance (Fig. 1A; apoptotic, early apoptotic, and live cells were counted with the ref. 13). This protocol encompasses short pulses of drug treatment Muse Cell Analyzer (Millipore) and analyzed with MuseSoft followed by rest periods in drug-free media to allow the cells to 1.4.0.0 (Millipore). recover from toxicity between treatments until a stable resistant phenotype is observed. Specifically, the procedure consisted of Cyclin B1 and cdc20 silencing three consecutive cycles of 3 days on treatment followed by 3 days For transient transfection experiments, cells were transfected by off treatment for each T-DM1 concentration of 1, 2, and 4 mg/mL. use of the Amaxa 4D-Nucleofector device, according to manu- The entire 54-day protocol resulted in the generation of cells with facturer's instruction. For each electroporation reaction, 100 mLof varying levels of T-DM1 resistance. complete Nucleofector solution combined with 300 nmol/L We established an exposure to 0.1 mg/mL of T-DM1 for 3 days as siRNA against Cyclin B1, cdc20, and scrambled siRNA as a control a reference schedule to define resistance in our model. This (duplex siRNAs were purchased from GE Dharmacon) under a schedule was selected on the basis of experiments performed in

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Cyclin B1 and T-DM1 Acquired Resistance

A B Parental Resistant HER2

β-Actin

D HCC1954 HCC1954/TDR T-DM1 T-DM1 T-DM1 + T-DM1 T-DM1 T-DM1 + 0h 24h Chloroquine-24h 0h 24h Chloroquine-24h HCC1419 SKBR3 T-DM1

10μm Phalloidin HCC1954

C DAPI Merged SKBR3 SKBR3/TDR T-DM1 T-DM1 T-DM1 + T-DM1 T-DM1 T-DM1 + 0h 24h Chloroquine-24h 0h 24h Chloroquine-24h HCC1954 % of Max % of T-DM1 Phalloidin % of Max % of HCC1419 DAPI Merged HCC1419 HCC1419/TDR T-DM1 T-DM1 T-DM1 + T-DM1 T-DM1 T-DM1 + 0h 24h Chloroquine-24h 0h 24h Chloroquine-24h SKBR3 % of Max % of T-DM1

T-DM1 Trastuzumab Phalloidin

Resistant (+T-DM1) Resistant (+Trastuzumab) Parental (+T-DM1) Parental (+Trastuzumab)

Resistant (rituximab) Resistant (rituximab) DAPI Parental (rituximab) Parental (rituximab) Merged

Figure 2. Pharmacodynamic characterization of T-DM1–resistant HER2-positive breast cancer cells. A, FISH assay for HER2 gene amplification. Paraffin-embedded cell pellets were prepared from each resistant cell line (HCC1419/TDR, SKBR3/TDR, and HCC1954/TDR; right) and the corresponding age and passage-matched parental cells (HCC1419, SKBR3, and HCC1954; left). The level of HER2 amplification in cell nuclei was determined as the ratio HER2/CEP17. Nuclei are highlighted by the blue fluorescent counter stain [40, 6-diamidino-2-phenylindole (DAPI)]. Nuclei with high levels of HER2 gene amplification (red signal) are shown. Green signal (CEP17): centromere of chromosome 17. (scale bar: 10 mm). B, HER2 protein expression in parental and T-DM1–resistant cells. Expression of HER2 in whole-cell lysates from the cell pairs under basal growth. b-Actin served as internal loading control. Western blot is from a representative experiment. C, Flow cytometric analysis of T-DM1-receptor binding in parental and T-DM1–resistant cells. Cell line pairs were incubated with 7.5 mg/mL rituximab (a humanized anti-CD20 IgG1 mAb, as a negative isotype control) or 7.5 mg/mL of T-DM1, for 30 minutes on ice. Trastuzumab at 7.5 mg/mL was used as positive control. Phycoerythrin (PE)- conjugated goat F(ab0)2 anti-human immunoglobulin was used as a secondary reagent. Representative flow cytometry histograms are shown. D, T-DM1 internalization analysis. Cells were incubated with 1.5 mg/mL T-DM1 at 37C for 15 minutes. In some conditions, cells were cotreated with chloroquine (50 mmol/L) to accumulate T-DM1 intracellularly. Cells were fixed and stained with Cy3-conjugated anti-human (for T-DM1 detection; red), FITC-phalloidin (for distribution of the F actin filaments; green), and DAPI (a nuclear DNA stain; blue). Scale bar, 7.5 mm.

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A B HCC1954 HCC1954/TDR 120 800 Parental Resistant Control Control 100 Multinucleated cells T-DM1 T-DM1 *** − M (%) 600

80 − 2 G2 M 60 S 400

% Cells 40 G1

% Cells 20 Sub-G1 0 200 l Control ro 1 rol 1 Fold change in G nt M nt M 0 o D o D HCC1954 HCC1954/TDR C T- /C T- R R/ 800 D HCC1419 HCC1419/TDR 120 T TD

Control Control 100 − M (%) 600 T-DM1 Multinucleated cells 2 T-DM1 80 − HCC1954 G2 M 400 60 S

% Cells 40 * G1

% Cells 200

20 T-DM1 Sub-G1

0 Fold change in G l l 0 o tr HCC1419 HCC1419/TDR n M1 o D C T- /Contro /T-DM1 R R 800 SKBR3/TDR D SKBR3 TD T Control Control 120 − M (%) 600 ** T-DM1 T-DM1 2 100 Multinucleated cells 80 − 400 G2 M 60 S *** % Cells % Cells 200 % Cells 40 G1 20 Fold change in G Sub-G Control 1 0 0 SKBR3 SKBR3/TDR l o 1 ol 1 r tr nt M n M Control T-DM1 DNA content DNA content o -D o D C T /C /T- R R D TD T C HCC1419 100 HCC1954 HCC1954/TDR 120 T-DM1 Control T-DM1 Control T-DM1 100 80 80 * Dead cells 60 60 Late apoptosis/Dead

% Cells 40 Early apoptosis 40

20 Cell death (%) Live 20 Propidium Iodide 0 l l 1 1 0 ro tro nt n M HCC1954 HCC1954/TDR o DM D C - - T /Co /T 100 R R HCC1419 HCC1419/TDR D D T T 120 80 Control T-DM1 Control T-DM1 100 Control 80 Dead cells 60 60 Late apoptosis/Dead 40

% Cells 40 *

Early apoptosis Cell death (%)

Propidium Iodide 20 Live 20

0 SKBR3 0 l 1 ol HCC1419 HCC1419/TDR tro tr M n on -D o C T SKBR3 SKBR3/TDR /C /T-DM1 100 R R D Control T-DM1 Control T-DM1 T TD T-DM1 120 80 ***

100 Dead cells 60 80 Late apoptosis/Dead * 60 Early apoptosis 40 Cell death (%) Propidium Iodide % Cells 40 Live 20 20 AnnexinV AnnexinV AnnexinV AnnexinV 0 0 SKBR3 SKBR3/TDR l 1 1 ntro M M Control T-DM1 o -D ontrol -D C T /C /T R R D TD T

Figure 3. Cellular effects of T-DM1 in parental and resistant cells. A, The action of T-DM1 on cell-cycle distribution. Cell-cycle status was analyzed using BrdUrd incorporation and propidium iodide (PI) to assess DNA content by flow cytometry. Sensitive (HCC1954 HCC1419 and SKBR3) and T-DM1–resistant (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR) cells were seeded into 6-well plates at a density of 2 105 cells/well for 24 hours. Cells were then treated without (Control) or with T-DM1 (0.1 mg/mL) for 24 hours and pulsed for an hour with BrdUrd prior to cell harvest and analysis. Left, representative flow cytometry plots from an experiment performed in triplicate that is consistent with other biological replicates. Right, the bar chart shows the percentage of cells in the G0–G1,S, and G2–M phases of the cell cycle following T-DM1 treatment. A bar chart with statistics for each pair of cell lines showing the percentage of change in G2–M population after T-DM1 treatment assigning a value of 100% to the untreated condition is shown. , P < 0.05; , P < 0.01; , P < 0.001. B, Effects of T-DM1 in microtubules. The three pairs of sensitive and T-DM1–resistant cells were grown at low density on cover slips, and incubated with T-DM1 (0.1 mg/mL) for 24 hours. Cells were washed, fixed, and permeabilized before incubation with anti-a tubulin FITC-conjugated antibody. The nuclei were counterstained with DAPI. Representative confocal images of cells visualizing the organization of tubulin filaments (green) are shown. Right, a magnification of the picture shown and a phase contrast image of the same area. Scale bar, 24 mm. C, T-DM1 is able to induce apoptosis in responsive cells. The three pairs of sensitive and resistant cells were treated with or without T-DM1 (0.1 mg/mL) for 48 hours. The cells were harvested, washed with PBS, and subsequently stained with Annexin-V conjugated to FITC. Viable (Annexin-V/PI) preapoptotic (Annexin-Vþ/PI), apoptotic (Annexin-Vþ/PIþ), and the residual damaged (Annexin-V/PIþ) cells were quantified by flow cytometric analysis using the Muse Cell Analyzer. Representative flow cytometry dot plots of three independent experiments are shown. Annexin V in combination with PI can discriminate between among early apoptotic cells, late apoptotic cells, and cells that are in either the very late stages of apoptosis or necrosis. Graphs show the average percentage of indicated subsets of cells from three experiments. A bar chart with the average percentage of total cell death from three experiments with statistics is shown. , P < 0.05; , P < 0.001.

MCF7 cells that do not overexpress HER2, whose growth was TDR cells decreased the sensitivity to the antiproliferative effects unaffected until higher concentrations and longer exposure to T- of T-DM1 by 20%–30% at 0.1 mg/mL after 3 days. This level of DM1 were used. After approximately 2 months, three cell lines resistance, albeit modest, has been helpful to identify mechan- developed resistance to T-DM1 (called HCC1954/TDR, isms of resistance to several agents (13). T-DM1–resistant cells HCC1419/TDR and SKBR3/TDR; Fig. 1B). We were unsuccessful had a growth rate similar to parental cells in vitro, as assessed by to generate BT474-resistant cells even after repeated attempts. cell counting after 7 days of culture under the same conditions. HCC1954/TDR cells were completely resistant after 10 days of The relative cell numbers of resistant versus parental cells were exposure to T-DM1 at 0.1 mg/mL. HCC1419/TDR and SKBR3/ 110% 8.3% in HCC1954/TDR, 93% 4% in HCC1419/TDR

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Cyclin B1 and T-DM1 Acquired Resistance

A BT474 SKBR3 AU565 EFM-192A HCC1954 HCC1419 T-DM1 ++ + + + +

Cyclin B1

CDK1

β-Actin

B Control 3 3 3 3 * * T-DM1

) ** **

492 * * 2 2 2 2 *

1 1 1 1 (relative to control) CDK Activity (A Activity CDK

0 0 0 0 BT474 BT474/TR AU565 AU565/TR SKBR3 SKBR3/TR EFM-192A EFM-192A/TR

BT474 BT474/TR AU565 AU565/TR SKBR3 SKBR3/TR EFM-192A EFM-192A/TR T-DM1 ++ ++ ++ ++

Cyclin B1

CDK1

β-Actin

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HCC1954 HCC1419 SKBR3 Control T-DM1 Control T-DM1 Control T-DM1 P TDR P TDR P TDR P TDR P TDR P TDR Cyclin B1

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Figure 4. Effects of T-DM1 on CDK1/cyclin B1 kinase activity and expression in different HER2-positive breast cancer preclinical models. A, T-DM1 is able to upregulate the expression of cyclin B1 in a number of T-DM1 sensitive HER2-positive breast cancer cell lines. Cells were treated with T-DM1 (0.1 mg/mL) for 24 hours. Protein expression levels of cyclin B1 and CDK1 were evaluated by Western blot analysis using 25 mg of protein cell lysate. The anti-b-actin antibody was used to verify equal protein loading. Representative image of three separate experiments is shown. B, Effects of T-DM1 on CDK1/cyclin B1 kinase activity and expression in HER2-positive breast cancer cell with acquired resistance to trastuzumab. (Continued on the following page.)

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Sabbaghi et al.

and 103% 7% in SKBR3 (no statistical differences). The mor- was internalized into both parental and resistant cells. The mag- phology of parental versus resistant cells was similar by optic nitude of T-DM1 internalization and intracellular pattern were microscopic observation. All resistant cell lines exhibited resis- similar in parental and resistant SKBR3 and HCC1419 cells. In tance features for at least one year after their generation. Of note, HCC1954/TDR cells we also detected T-DM1 intracellularly, but SKBR3/TDR cells retained a similar antiproliferative response to to a lesser extent than in their parental HER2-positive counterpart, trastuzumab alone than parental cells (50% growth inhibition), in agreement with their lower HER2 ampliﬁcation level and the suggesting that the resistance was associated to DM1 (Fig. 1C). surface expression. Both parental and resistant cells showed similar EC50 values

for paclitaxel [HCC1954 (4.5 nmol/L), HCC1954/TDR (6.5 Effects of T-DM1 on G2–M arrest and mitotic catastrophe in nmol/L), HCC1419 (6.0 nmol/L), HCC1419/TDR (5.6 nmol/L), parental versus resistant cells SKBR3 (3.2 nmol/L), SKBR3/TDR (3.1 nmol/L)]. T-DM1 increased significantly the percentage of cells in G2– M phase while it induced a decrease in the S and G0–G1 phases T-DM1 binds to cell surface HER2 and is internalized in in the three parental cell lines (Fig. 3A). In the resistant cells, the – resistant cells effects of T-DM1 on G2 M cell-cycle arrest were less pro- HCC1419/TDR and SKBR3/TDR cells retained the same level of nounced than in parental cells. In HCC1954/TDR and – HER2 amplification and protein expression than parental cells HCC1419/TDR, there was no increase in G2 M. In SKBR3 there (Fig. 2A and B). However, HCC1954/TDR cells displayed was a significant increase following T-DM1 but to a much lesser decreased amplification of HER2 and reduced HER2 protein (Fig. degree than in parental cells (Fig. 3A). Next, we evaluated the 2A and B), and mRNA levels (6% of relative mRNA expression) effects of T-DM1 on microtubule arrangement. In parental cells, compared with parental cells. Parental HCC1954 cells had a mean T-DM1 caused the formation of multinucleated giant cells with of 40 HER2 copies but they contained two additional subpopula- severely defective microtubule arrangements (Fig. 3B). Previ- tions; 17% of the cells had less than 6 copies (some scored as ously, it has been reported that T-DM1 causes tumor growth amplified for having only one copy of CEP17) and 11.4% had inhibition by mitotic catastrophe (17). These morphologic 6–10 HER2 copies. During the generation of the resistant cells, alterations suggestive of mitotic catastrophe were undetected T-DM1 eradicated the cells with strong HER2 amplification after a in T-DM1–resistant cells. As most cells undergoing mitotic short exposure, allowing the emergence of subdominant clones catastrophe are destined to die by apoptosis, we evaluated the with low HER2 amplification (average number of 8 HER2 copies, effects of T-DM1 on apoptosis induction. T-DM1 induced HER2 1þ by IHC). The emergence of HER2 low resistant cells in apoptosis in HCC1954 and SKBR3 parental cells at 48 hours HCC1954 was confirmed in three independent experiments. but the effect on the resistant counterparts was much less Despite this important change in the population, the resistant pronounced under the same conditions (Fig. 3C). cells still met the criteria for scoring them as HER2 amplified as they have more than 6 HER2 copies (14). In addition, other genes T-DM1–resistant cells failed to induce CDK1/cyclin B1 such as ORMDL3, STARD3, PPP1R1B, MIEN1 located in the We hypothesized that resistant cells may have a defective cell- minimal common region of 17q12 amplification were also down- cycle–regulatory machinery that does not allow T-DM1 to induce modulated as assayed by expression arrays. SKBR3/TDR and G2–M arrest and consequently, mitotic catastrophe. We focused HCC1419/TDR did not have any significant differences in the on the potential involvement of cyclin dependent kinase 1 levels of HER2, ORMDL3, STARD3, PPP1R1B and MIEN1 mRNA (CDK1) and cyclin B1 in T-DM1 resistance. Activation of the in comparison with parental cells, as assayed by qRT-PCR. We next CDK1–cyclin B1 complexes are essential for progression into M- assayed the ability of T-DM1 to bind to cell surface HER2 by flow phase, but prolonged mitotic arrest (for example because of the cytometric analysis. Although surface HER2 levels might vary in inability of forming the mitotic spindle in cells treated with HCC1954/TDR compared with parental cells, T-DM1-HER2 microtubule-affecting agents such as DM1) leads to mitotic catas- binding was preserved in all TDR cells (Fig. 2C). trophe (18). The activity of CDK1–cyclin B1 complex is regulated We assayed whether T-DM1 was capable of inducing internal- mainly by the expression of cyclin B1 and by the phosphorylation ization upon binding to HER2 in paired sensitive and resistant status of the catalytic subunit CDK1. Degradation of cyclin B1 by cells (15). Cells were treated with 10 nmol/L T-DM1 for 15 the proteasome after ubiquitination by the multi-subunit ubiqui- minutes at 37C. After washing out the drug, cells were cultured tin E3-ligase APC/Ccdc20 is essential for exiting mitosis. T-DM1 for 24 hours with or without 50 mmol/L chloroquine to accumu- exposure augmented cyclin B1 expression in a panel of HER2- late T-DM1 intracellularly. As shown in Fig. 2D, T-DM1 (red dots) positive parental breast cancer cells (except BT-474; Fig. 4A).

(Continued.) Acquired trastuzumab-resistant cells (BT474/TR, SKBR3/TR, AU565/TR, and EFM-192A/TR) have been generated by our group and used in these experiments together with the corresponding age and passage-matched parental cell line (19). Cells were treated with T-DM1 (0.1 mg/mL) and harvested at 24 hours. Whole-cell extracts were prepared from each experimental condition and CDK1/cyclin B1 kinase activity was measured with the nonradioactive MESACUP Cdc2/CDK1 Kinase Assay. The results are reported as fold induction of OD 492 nm in arbitrary units (RLA) of treated sample using untreated condition as reference set at 1. Data are presented as mean SD for three experiments. Cells treated as in the CDK1/cyclin B1 kinase activity assay experiment were lysed and Western blots were performed with equal amounts of cell lysate (25 mg protein). Expression of cyclin B1 and CDK1 was evaluated. b-Actin was used as an internal control. Shown are representative images from one of experiments. , P < 0.05; , P < 0.01. C, Trastuzumab is not able to upregulate the expression of cyclin B1 in a number of T-DM1–sensitive HER2-positive breast cancer cell lines. Cells were treated with trastuzumab (15 mg/mL) and T-DM1 (0.1 mg/mL) for 24 hours and analyzed as in A. Shown are representative images from one of experiments. D, Effects of T-DM1 on CDK1/cyclin B1 kinase activity and expression in pairs of HER2-positive sensitive and T-DM1–resistant breast cancer cells. HCC1954 and HCC1954/TDR, HCC1419-HCC1419/TDR, and SKBR3-SKBR3/TDR cells were treated with T-DM1 (0.1 mg/mL) for 24 hours and analyzed for CDK1 kinase activity using the MESACUP Cdc2/CDK1 Kinase Assay Kit and cyclin B1 levels by Western blot analysis as in Fig. 3C. Data are expressed as fold induction versus control arbitrarily set at 1. , P < 0.05; , P < 0.001.

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Cyclin B1 and T-DM1 Acquired Resistance

We analyzed the effects of T-DM1 on the activity of CDK1. Induction of cyclin B1 by T-DM1 in HER2-positive human We first tested this effect in four parental HER2 positive cell breast cancer explants associates with apoptosis lines and in their trastuzumab resistant (TR) counterparts that To overcome, at least in part, the issue of the limited number of we recently reported (19). These TR cell lines were included in cell lines, we tested the association between cyclin B1 induction this analysis to confirm whether acquired trastuzumab resis- and T-DM1 antiproliferative effect in a panel of 18 fresh human tance (that corresponds to the clinical setting in which T-DM1 HER2-positive breast cancer explants. The results confirmed such is approved) affects T-DM1 ability to induce CDK1 activity. The association. We assayed the basal levels of cyclin B1 in 18 HER2- results showed a significant increase in CDK1 Kinase activity 24 positive breast cancers according to our experience in fresh hours posttreatment with T-DM1 (0.1 mg/mL) in both sensitive explants (16). In 7 cases, cyclin B1 was undetected and in the and trastuzumab-resistant cells by using the MESACUP Cdc2/ remaining 11, the percentage of tumor cells with detected cyclin Cdk1 Kinase Assay Kit with exception of the BT-474 cell pairs B1 staining ranged from 2% to 10% (Fig. 6A). A fraction of these (Fig. 4B). In all the cell lines with acquired trastuzumab tissues were cultured ex vivo for 5 days in the presence of T-DM1 or resistance, T-DM1 exposure decreased viable cells (cell number control and assayed for modulation in cyclin B1 expression. There relative to control at 3 days of 20% 3.2% in AU565/TR, 40% were two main observations; first, cyclin B1 was induced in the 6% in EFM-192A, 17% 3% in SKBR3/TR and 72% 3% in majority of explants exposed to T-DM1 (12 of 18, 66.6% of the BT474/TR) and cyclin B1 was induced following drug exposure tumors), including those with undetected baseline levels; second, (Fig.4B).Trastuzumabalonedid not induce cyclin B1 in these the percentage of tumor cells that stained positive for cyclin B1 cell lines (Fig. 4C). was dramatically increased in many specimens (61.1% of cases), a We then assessed T-DM1 effects on the CDK1/cyclin B1 finding consistent with the induction of a G2–M arrest by T-DM1 complex kinase activity and cyclin B1 levels in the paired cell (Fig. 6A and B). These findings support the effect of T-DM1 on lines parental and resistant to T-DM1. We performed a time– inducing a persistent accumulation of cyclin B1 in tumor, which course study the HCC1954 and HCC1954/TDR cells. The was a hallmark of T-DM1 effects in our panel of sensitive breast activation of the mitotic kinase CDK1/cyclin B1 was detected cancer cells. Of note, in 4 explants with cyclin B1 detected at asearlyas12hourspost-T-DM1inparentalHCC1954cells(2- baseline, T-DM1 exposure did not further increase the percentage fold) but not in HCC1954/TDR cells (P < 0.05). Protein levels of expressing cells. We also assayed induction of apoptosis by of total CDK1 were unchanged in parental and resistant cells. expression of active caspase-3, proliferation (Ki67), and activa- Cyclin B1 protein levels were moderately increased (2-fold) at tion of the M-phase checkpoint kinases (phospho-Histone H3, p- 12 hours following T-DM1 treatment in parental cells in par- H3; Fig. 6A). With regards to proliferation, Ki67 staining was not allel with CDK1 activity and they were maintained elevated up modulated by T-DM1 after 5 days while reduction in p-H3 to 24 hours afterwards. This increase in the cyclin B1 levels was staining paralleled the induction of apoptosis by T-DM1. The undetected in resistant cells (Fig. 4D). A similar pattern of lack of an effect on Ki67 is consistent with the fact that cells cyclin B1 expression and appearance of CDK1/cyclin B1 kinase arrested in G2–M also stain positive to Ki67 (Fig. 6A; ref. 21). We activity were observed with the other T-DM1–sensitive/resistant found two main patterns of cyclin B1 and apoptosis changes cell pairs after 24 hours of T-DM1 exposure (Fig. 4C). The following T-DM1 exposure: no/very low cyclin B1 accumulation analysis of the levels of cyclin B1 mRNA by qRT-PCR showed a and very low upregulation of apoptosis versus strong cyclin B1 nonsignificant increase (Supplementary Fig. S1), suggesting (accumulation) and high induction of apoptosis. Trastuzumab postranscriptional regulatory mechanisms. alone did not induce cyclin B1 in ex vivo explants (Fig. 6B). The tumor cell areas with cyclin B1 staining and the ones with caspase- Cyclin B1 mediates T-DM1 resistance 3 active staining did not frankly overlapped in the tissue sections, We tested whether cyclin B1 knockdown could mediate suggesting different stages of T-DM1 effects. resistance to T-DM1 in parental cells. Cell lines were transfected Fifteen explants were from diagnostic specimens derived with siRNA directed against cyclin B1 mRNA or with a suitable from patients that received neoadjuvant treatment without T- control. Cyclin B1 levels were measured 24 and 48 hours after DM1.Theotherthreewerefrommetastaticpatients.Two the transfection by Western blot confirming that they were received T-DM1 and had cyclin B1 and apoptosis induction significantly lower in the siRNA-transfected cells than in the ex vivo. One had de novo metastatic disease, the explant was from siControl (Fig. 5A). The paired cell lines transfected with the the diagnostic breast cancer biopsy, and received T-DM1 as cyclin B1 siRNA or the siControl were exposed to T-DM1 and second line achieving a partial response. The second patient cell viability was assayed after 48 hours. Silencing of cyclin B1 had bone and liver disease and after several lines of treatment induced a significant resistance to T-DM1 in the three parental received T-DM1. The explant was obtained from a liver metas- cell lines (Fig. 5A). tasis just before T-DM1 (Supplementary Fig. S2) and subse- We next tested whether increasing the levels of cyclin B1 in quently had a partial response. resistant cells might sensitize them to T-DM1. Cdc20 is a regulatory subunit of the multi-subunit ubiquitin E3-ligase APC/C that is responsible of cyclin B1 degradation at the end of mitosis. Discussion We silenced cdc20 to promote cyclin B1 accumulation (20). As We have generated three different HER2-positive breast cancer shown in Fig. 5B, cyclin B1 protein level were increased following cell lines with various levels of acquired T-DM1 resistance by cdc20 silencing in two of the three resistant cells. In HCC1954/ applying a pulsed administration strategy of the drug, an TDR cells, no differences in T-DM1 sensitivity between control approach commonly used for the development of chemotherapy (scrambled siRNA) and cdc20-silenced cells were observed. How- drug-resistant cancer cells (13). The resistant phenotype was ever, T-DM1 resistance was partially reverted in cdc20-silenced achieved within the first two months of drug exposure, suggesting SKBR3/TDR and HCC1419/TDR cells (Fig. 5B). the emergence of a chemotherapy-driven mechanism. We chose

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A HCC1954 HCC1419 SKBR3 24h 48h 24h 48h 24h 48h Figure 5. Genetic modulation of cyclin B1 affects the viability of cancer cells treated with T-DM1. A, Depletion of cyclin B1 in the parental drug-sensitive cells Cyclin B1 reduced T-DM1 sensitivity. Parental T-DM1–sensitive cells were transfected with target-speciﬁc-cyclin β-Actin B1 siRNA and scrambled control siRNA by using a Nucleofector. After transfection, cells were recovered and used for experiments. Viability HCC1954 120 HCC1419 SKBR3 experiments were performed on an 120 120 aliquot of transfected cells after *** ** siControl siCyclin B1 seeding in 12-well plates at a 80 concentration of 10–15 103 cells/well 80 80 *** and allowed to adhere overnight. Cells were then incubated with T-DM1 (0.1 40 40 mg/mL). Duplicated samples were

Cell number 40 (% of control) harvested and counted at 48 hours with the Scepter automated cell 0 counter. The results are presented as 00.1 0 0 00.1 00.1 the mean SD from experiments T-DM1 [μg/mL] T-DM1 [μg/mL] T-DM1 [μg/mL] replicated five times (, P < 0.01; , P < 0.001). The remaining B HCC1954/TDR HCC1419/TDR SKBR3/TDR transfected cells were analyzed by 48h 72h 48h 72h 48h 72h Western blot analysis. Total cellular protein was extracted from cells at 48 hours after siRNA transfection. Western blots confirmed that cyclin B1 was downregulated. b-Actin was blotted for loading control. B, Increase cdc20 of cyclin B1 in the drug-resistant cells augments their response to T-DM1. Cyclin B1 T-DM1–resistant cells were transfected with target-specific cdc20 siRNA and β-Actin scrambled control siRNA by using a Nucleofector. After transfection, cells were recovered and used for cell viability and Western blot SKBR3/TDR HCC1954/TDR HCC1419/TDR experiments. Cells were incubated 120 120 120 siControl sicdc20 with T-DM1 (0.1 mg/mL). Cell number * was determined after 72 hours * 80 80 80 posttransfection with Scepter. The results are presented as the mean SD from triplicate experiments 40 40 40 (, P 0.05; , P 0.001). Lysates Cell number < < (% of control) were prepared 48 and 72 hours posttransfection and analyzed for the 0 0 0 levels of cyclin B1 and cdc20 by 00.1 00.1 00.1 Western blot. T-DM1 [μg/mL] T-DM1 [μg/mL] T-DM1 [μg/mL]

initially four cells lines to develop resistance, but in one of them subpopulation (93% of cells) with high Her2 amplification (BT474) we were unsuccessful. The three remaining cell lines, and a minority subpopulation (7%) with low, but amplified albeit not sufficient to establish the generalizability of the find- HER2 gene. An early clonal selection of the subpopulation with ings, were sufficient to test mechanistically the role of cyclin B1. lower HER2 amplification following T-DM1 exposure appears to Similarly, the degree of acquired resistance varied in the 3 cell lines contribute to resistance. Regardless of this, the rapid emergence but a role of cyclin B1 in T-DM1 resistance was observed in all of of resistance, also in cell lines that retain the same level of HER2 them. The modest resistance observed in two of the cell lines amplification, suggests a mechanistic link with the cytotoxic clearly suggested that cyclin B1-independent mechanisms of DM1 component rather than to trastuzumab, by as yet unknown action of T-DM1 remain active. mechanisms such as epigenetic and/or cellular pathway rewir- The generation of resistant cell lines in a short timeframe may ing. SKBR3 T-DM1–resistant cells retained sensitivity to trastu- be caused by several mechanisms and may vary between cell zumab, whereas the other two remained trastuzumab resistant. lines. In HCC1954, a specific finding was a marked reduction of This suggests that multiple mechanisms of resistance may coex- HER2 gene amplification after the first round of exposure to T- ist in the same cell line. Additional studies are needed to DM1. In parental HCC1954 cells, there was a predominant understand if these mechanisms coexist in a single cell or

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Cyclin B1 and T-DM1 Acquired Resistance

A Cyclin B1 c-casp3 Ki-67 p-H3

100 40 60 60 80

60 40 40 20 cells 40 20 20 20 Percentage of tumor 0 0 0 0 Baseline T-DM1 Baseline T-DM1 Baseline T-DM1 Baseline T-DM1 B C Control Trastuzumab T-DM1 Cyclin B1 c-casp3 c-casp3 Ki67 Ki67 Annotation 4 16

Cyclin B1 Cyclin 1 17 18 Case 4 6 2

c-casp3 7 50 μm 5 8 10 12 9 14

Cyclin B1 Cyclin 3 13 11 Case 11 Case 19 c-casp3 50 μm −5 +50

Figure 6. Effects of T-DM1 added ex vivo to fresh human breast cancer explants. Formalin-fixed paraffin-embedded (FFPE) blocks were prepared for control and treated breast tumor at five days after the T-DM1 treatment. A, Graphs showing the percentage of cells positive for each marker. Cyclin B1, active caspase-3, Ki67, and p-H3 were determined in basal control condition and after treatment with T-DM1 for 5 days. B, Representative IHC images of control and T-DM1–treated tumors. Two representative examples of staining for cyclin B1 and caspase-3 active (apoptosis) are shown. Scale bar, 50 mm. These tumors were also treated with trastuzumab (15 mg/mL) and the results are also shown. C, Hierarchical cluster analysis of T-DM1 effects on cyclin B1 and active caspase-3 in a total of 18 human breast cancers. Each row represents an effect on expression of the paired control- and T-DM1–treated samples, and each column, a single IHC marker, including cyclin B1 and active caspase-3. Upregulation of expression is displayed in red, and no effect in white. whether this represents tumor cell heterogeneity (as in mechanism, such as caveolae membranes composed mainly by HCC1954 relative to HER2 amplification). Nevertheless, the caveolin-1 has also been demonstrated (22). Furthermore, it has antimitotic essence of DM1 should be considered as a source been shown that the high endocytic activity of stem cell–like of heterogeneity generation. breast cancer cells make them particularly sensitive to T-DM1 The cytotoxic effect of T-DM1 might be impaired by inefficient (23). We have shown that T-DM1 was internalized and exhibited a internalization or enhanced percentage of HER2–T-DM1 complex similar intracellular pattern in parental and resistant cells, albeit that is recycled back to the cell surface (3). It is believed that the HCC1954/TDR cells had less T-DM1 detected intracellularly. HER2/T-DM1 conjugate enters cancer cells via the clathrin-depen- Overall, it appeared that the pathway mediating HER2-T-DM1 dent endocytosis pathway. However, a clathrin-independent endocytosis was intact in resistant cell lines.

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In parental cells, T-DM1 induced G2–M arrest and mitotic ptotic or antiproliferative effects. Instead, we found that the catastrophe. The phenomenon of "mitotic catastrophe" consid- induction of cyclin B1 following T-DM1 exposure ex vivo was ered a mode of cell death per se resulting from prolonged mitotic highly associated to induction of apoptosis and reduced tumor arrest is now believed to represent a prestage of apoptosis or even cell proliferation. necrosis or senescence (5). In two of the three cell lines sensitive to Interestingly, two patients with metastastic breast cancer had a T-DM1, the mitotic arrest induced by T-DM1 preceded apoptosis. response to T-DM1 and in both of them cyclin B1 was upregulated Mitotic catastrophe did not occur in resistant cells suggesting that by T-DM1 ex vivo. These anecdotal data suggest that cyclin B1 the machinery for inducing cell-cycle arrest at the G2–M phase was induction in explants may be associated to clinical response. On disrupted. the other hand, the lack of a predictive effect of baseline cyclin B1 This led us to hypothesize a potential involvement of CDK1 expression is in line with an observation in 7 HER2-positive breast and cyclin B1, the members of mitotic promoting factor complex, cancer patients that had been treated at Hospital del Mar (Barce- in T-DM1 resistance (18). Entry into mitosis is initiated by CDK1 lona, Spain) with T-DM1 as part of a neoadjuvant clinical trial. and binding of CDK1 to cyclin B1 is essential for its activation. Four of them had detectable levels of cyclin B1, of whom 3 CDK1/cyclin B1 complex and the kinase activity of CDK1 is achieved a pathologic complete response (pCR). In the 3 patients controlled by cyclin B1 accumulation. During mitosis, chromo- with undetected baseline cyclin B1, one pCR was achieved. Albeit some segregation is facilitated by the kinetochore, an assembly of patients received additional anti-HER2 agents or systemic che- proteins built on centromeric DNA that attach chromosomes to motherapy as part of their neoadjuvant treatment, these few cases spindle microtubules. After the last unattached kinetochore is suggest that baseline cyclin B1 expression is not a prerequisite to attached to microtubules and the chromosomes are properly achieve a pCR with a neoadjuvant regimen including T-DM1. We aligned, the mitotic checkpoint is switched off. CDK1 is then plan to prospectively test cyclin B1 induction as a pharmacody- inactivated as cyclin B is rapidly degraded, and cells progress namics assay (i.e., serial biopsies comparing baseline cyclin B1 through anaphase, undergo cytokinesis, and exit mitosis (24). expression with expression at an early time point after T-DM1 However, in the presence of an antimitotic agents (such as DM1), treatment) to predict T-DM1 clinical benefit in a GEICAM (Span- the impossibility of assembling the mitotic spindle activates the ish Breast Cancer Research Group) study in the near future. mitotic checkpoint arresting the cells in mitosis for a prolonged A few additional mechanisms of T-DM1 resistance have period and they may undergo mitotic catastrophe followed by cell been proposed. For instance, those factors that reduce the death (25). intracellular DM1 load per cell, namely the overexpression of In the three parental HER2-positive breast cancer cells, cyclin B1 multidrug resistance (MDR) proteins or an impaired lysosom- induction and CDK1/cyclin B1 activation were observed follow- al degradation of trastuzumab that might limit the subsequent ing T-DM1 treatment and were maintained elevated for up to 24 release of DM1 into the cytoplasm (3, 33). Although we cannot hours. However, T-DM1 failed to raise cyclin B1 levels and, rule out a potential role of these mechanisms in our models, consequently, CDK1/Cyclin B1 activity in T-DM1–resistant cells. we believe that MDR did not play a significant role since Furthermore, silencing of cyclin B1 induced resistance to T-DM1 in paclitaxel, a microtubule-stabilizing agents and a substrate of parental cell lines while increasing the levels of cyclin B1 in MDR, exhibited the same cytotoxicity in parental and in resistant cells sensitized them to T-DM1 in two out of the three resistant cells. cell lines. The failure to generate T-DM1 BT474–resistant cells With regards to alterations in the endosome pathways, we under our experimental protocol may be related, at least in part, to believe that the detection of intracellular T-DM1 in the resistant the inability of T-DM1 to upregulate cyclin B1. cells, as well as the effects of cdc20 silencing on restoring T-DM1 Other CDK–cyclin complexes have been previously reported as response in resistant cells, limits the potential role of this putative implicated in resistance to anti-HER2 therapies. For instance, mechanism of resistance. Nonetheless, a biparatopic HER2-tar- cyclin E has a role in trastuzumab resistance and treatment with geting ADC containing the tubulysin variant AZ13599185 dem- CDK2 inhibitors has been proposed for tumors displaying cyclin onstrated superior antitumor activity over T-DM1 in various E amplification/overexpression (26, 27). A relationship of CDK4/ tumor models including T-DM1 refractory. The new ADC by cyclin D1 activity in resistance to trastuzumab and lapatinib has targeting two nonoverlapping epitopes on HER2 can induce been also reported and high levels of cyclin D1 predicted poor HER2 receptor clustering, which in turn promotes robust inter- response to trastuzumab (28). Preclinical studies have also shown nalization, lysosomal trafficking, and degradation (34). Also, it that residual cells surviving within 48 hours following T-DM1 has been suggested a combination of inhibitors of the chaperone treatment began to reenter the cell cycle and the sequential HSP90 that promote HER2 targeting to lysosomes and its degra- treatment with CDK4/6 inhibitors suppressed the proliferation dation suggested as a new strategy to improve therapy with T-DM1 of these residual/resistant clones (29). A clinical trial with the (35). In addition to this, other novel anti-HER2 antibody–drug CDK4/6 inhibitor palbociclib in combination with T-DM1 conjugate offer promising activity in T-DM1–pretreated breast (NCT01976169) in HER2-positive patients is underway. On the cancer (36, 37). Other potential mechanisms of resistance that other hand, our results showing that CDK1/Cyclin B1 activity is come from the trastuzumab part of the T-DM1 molecule have not needed for T-DM1 action suggests a note of caution regarding proven its clinical utility when assayed in patient samples from possible combinations of T-DM1 with pan-CDKs inhibitors or clinical trials (9, 11). A mechanism of T-DM1 resistance that has selective CDK1 inhibitors (30). been reported preclinically is the presence of the HER3 ligand, In human breast cancer, cyclin B1 expression has been associ- heregulin, that reduced the activity of T-DM1 in breast cancer cells ated with poor survival (31) and is a prognostic proliferation by causing HER2/HER3 dimerization thus strongly activating the marker in lymph node–negative breast cancer cohorts (32). In our PI3K pathway; and this effect was reversed by the addition of series of fresh HER2-positive breast tumor explants, the basal pertuzumab, a HER2–HER3 dimerization inhibitor. However, the levels of cyclin B1 were not significantly related to T-DM1 apo- combination of T-DM1 and pertuzumab in the clinic has not

OF12 Clin Cancer Res; 23(22) November 15, 2017 Clinical Cancer Research

Cyclin B1 and T-DM1 Acquired Resistance

proven sufficiently superior to T-DM1 alone, suggesting this Analysis and interpretation of data (e.g., statistical analysis, biostatistics, mechanism operates at most in a small proportion of patients computational analysis): M.A. Sabbaghi, G. Gil-Gomez, C. Guardia, S. Servitja, S. García-Alonso, S. Menendez, M. Salido, A. Muntasell, P. Gonzalez-Alonso, (38). Finally, T-DM1 is able to alter genes related with immune fl J. Madoz-Gurpide, I. Tusquets, A. Pandiella, F. Rojo, J. Albanell response and this can in uence the response in patients (39). Writing, review, and/or revision of the manuscript: M.A. Sabbaghi, G. Gil- In short, the studies presented here show that cyclin B1 induc- Gomez, S. Servitja, S. García-Alonso, L. Serrano, A. Muntasell, M. Martínez- tion allowed to differentiate T-DM1 sensitive parental cells from García, P. Eroles, J. Arribas, I. Tusquets, A. Lluch, A. Pandiella, F. Rojo, A. Rovira, their counterparts with acquired resistance. The induction of J. Albanell cyclin B1 in T-DM1–sensitive cells was independent of their prior Administrative, technical, or material support (i.e., reporting or organizing trastuzumab sensitivity. The mitotic arrest consequence of the data, constructing databases): O. Arpi, J. Madoz-Gurpide, F. Rojo, J. Albanell Study supervision: I. Tusquets, F. Rojo, A. Rovira, J. Albanell failure in assembling the mitotic spindle leads to sustained CDK1/ Cyclin B1 kinase activity, a hallmark of mitotic catastrophe that is Acknowledgments resolved by apoptosis. In fresh human HER2-positive breast The authors would like to thank Marc Bataller and Raul Pena~ for technical cancer explants, the induction of cyclin B1 by T-DM1 correlates assistance and comments. Flow cytometry analysis was technically supported by with apoptosis, suggesting that a pharmacodynamic assay to test Oscar Fornas, head of Universitat Pompeu Fabra (UPF) flow cytometry core cyclin B1 induction in breast cancer patients treated with T-DM1 facility. We thank Fundacio Cellex (Barcelona) for a generous donation to the may help to identify early the patients more likely to benefit from Hospital del Mar Medical Oncology Service. We also thank the patients for their generous participation in the study. this drug. Grant Support fl Disclosure of Potential Con icts of Interest This work was supported by ISCiii (CIBERONC CB16/12/00481, RD12/ J. Albanell reports receiving speakers bureau honoraria from and is a 0036/0051, RD12/0036/0070, RD12/0036/0003, PIE15/00008, PI13/00864, fl consultant/advisory board member for Roche. No potential con icts of interest PI15/00146, PI15/00934, PI15/01617, PT13/0010/0005), Generalitat de Cat- were disclosed by the other authors. alunya (2014 SGR 740), and the "Xarxa de Bancs de tumors sponsored by Pla Director d'Oncologia de Catalunya (XBTC). MINECO through BFU2015- Authors' Contributions 71371-R grant supported work in A. Pandiella's laboratory. Our work was Conception and design: M.A. Sabbaghi, G. Gil-Gomez, P. Eroles, J. Arribas, supported by the EU through the regional funding development program F. Rojo, A. Rovira, J. Albanell (FEDER). P. Gonzalez-Alonso was supported by Fundacion Conchita Rabago Development of methodology: M.A. Sabbaghi, G. Gil-Gomez, C. Guardia, de Jimenez Díaz grant. O. Arpi, S. Menendez, S. Zazo, C. Chamizo, P. Gonzalez-Alonso, F. Rojo, The costs of publication of this article were defrayed in part by the payment of advertisement A. Rovira, J. Albanell page charges. This article must therefore be hereby marked in Acquisition of data (provided animals, acquired and managed patients, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. provided facilities, etc.): M.A. Sabbaghi, G. Gil-Gomez, S. Servitja, O. Arpi, S. García-Alonso, S. Menendez, M. Arumi-Uria, L. Serrano, A. Muntasell, Received March 10, 2017; revised June 29, 2017; accepted August 11, 2017; M. Martínez-García, A. Lluch, F. Rojo published OnlineFirst August 30, 2017.

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OF14 Clin Cancer Res; 23(22) November 15, 2017 Clinical Cancer Research

Defective Cyclin B1 Induction in Trastuzumab-emtansine (T-DM1) Acquired Resistance in HER2-positive Breast Cancer

Mohammad A. Sabbaghi, Gabriel Gil-Gómez, Cristina Guardia, et al.

Clin Cancer Res Published OnlineFirst August 18, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-0696

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