Use of Ex Vivo Patient-Derived Tumor Organotypic Spheroids to Identify

Published OnlineFirst February 7, 2020; DOI: 10.1158/1078-0432.CCR-19-1844

CLINICAL CANCER RESEARCH | TRANSLATIONALCANCER MECHANISMS AND THERAPY

Use of Ex Vivo Patient-Derived Tumor Organotypic Spheroids to Identify Combination Therapies for HER2 Mutant Non–Small Cell Lung Cancer Elena Ivanova1,2, Mari Kuraguchi1,2, Man Xu1,2, Andrew J. Portell1,2, Luke Taus2, Irmina Diala3, Alshad S. Lalani3, Jihyun Choi1, Emily S. Chambers1, Shuai Li1, Shengwu Liu1, Ting Chen1, Thanh U. Barbie4, Geoffrey R. Oxnard1,5, Jacob J. Haworth1, Kwok-Kin Wong6, Suzanne E. Dahlberg7, Amir A. Aref1, David A. Barbie1,2,5, Magda Bahcall1,2, Cloud P. Paweletz1,2, and Pasi A. Janne€ 1,2,5

ABSTRACT ◥ Purpose: Evaluating drug responses using primary patient- validated in vivo using the DFCI359 and DFCI315 PDXs and a derived cells ex vivo represents a potentially rapid and efficient HER2YVMA genetically engineered mouse model. approach to screening for new treatment approaches. Here, Results: Both neratinib and afatinib, but not gefitinib, induced we sought to identify neratinib combinations in HER2 mutant cell death in DFCI359 XDOTS. The combinations of neratinib/ non–small cell lung cancer (NSCLC) patient xenograft-derived trastuzumab and neratinib/temsirolimus enhanced the therapeu- organotypic spheroids (XDOTS) using a short-term ex vivo tic benefit of neratinib alone in DFCI315 and DFCI359. The system. combination of neratinib and trastuzumab in vivo was more Experimental Design: We generated two HER2-mutant effective compared with single-agent neratinib or trastuzumab NSCLC PDX models [DFCI359 (HER2 exon19 755_757LREde- and was associated with more robust inhibition of HER2 and linsRP) and DFCI315 (HER2 exon20 V777_G778insGSP)] and downstream signaling. used the PDX tumors to generate XDOTS. Tumor spheroids were Conclusions: TheXDOTSplatformcanbeusedtoevaluate growninamicrofluidicdeviceandtreatedex vivo with neratinib- therapies and therapeutic combinations ex vivo using PDX based drug combinations. Live/dead quantification was per- tumors. This approach may accelerate the identification and formed by dual-labeling deconvolution fluorescence microscopy. clinical development of therapies for targets with no or few The most efficacious ex vivo combination was subsequently existing models and/or therapies.

Introduction correlation with responses to tyrosine kinase inhibitors (TKI; refs. 2–6). Nevertheless, similar success is yet to be seen in a number of other Tumor genotyping has expanded the repertoire of biomarkers in NSCLC genotypes, including in-frame exon 20 insertions in HER2. non-small cell lung cancers (NSCLC) and as such has become the The irreversible dual EGFR/HER2 inhibitors afatinib (NCT00796549), primary diagnostic tool in predicting responses to targeted thera- dacomitinib (NCT00548093), and neratinib (NCT01827267) showed pies (1). Notably, expression of mutant EGFR, or oncogenic fusions little clinical beneﬁt as monotherapies in HER2-mutant lung cancer, all such as those of anaplastic lymphoma kinase (ALK), ROS1, RET,or with response rates of <10% (7–9). Although the combination of NTRK1 receptor tyrosine kinases (RTK) has demonstrated good afatinib and rapamycin demonstrated promising combination efﬁcacy in a preclinical in vivo study using a genetically engineered mouse model (GEMM) of HER2-mutant NSCLC, the subsequent clinical trial 1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, (NCT01822767) evaluating the combination of neratinib and temsir- Massachusetts. 2Belfer Center for Applied Cancer Science, Dana Farber Cancer Institute, Boston, Massachusetts. 3Puma Biotechnology Inc., Los Angeles, olimus resulted in only a minimal improvement in response rate (14% California. 4Department of Surgery, Brigham and Women's Hospital, Boston, vs. 0%) compared with single-agent neratinib (10, 11). More recently, Massachusetts. 5Lowe Center for Thoracic Oncology, Dana Farber Cancer the trastuzumab-based antibody drug conjugate (ADC), ado- Institute, Boston, Massachusetts. 6Department of Medicine, Division of Hema- trastuzumab (T-DM1), has demonstrated some encouraging activity tology and Medical Oncology, New York University Langone Medical Center, in HER2 mutant NSCLC and next-generation ADCs (DS-8201a; New York, New York. 7Department of Biostatistics and Computational Biology, NCT03505710) are undergoing clinical evaluation (12, 13). On the Dana-Farber Cancer Institute, Boston, Massachusetts. basis of the current landscape of available HER2-directed therapies, Note: Supplementary data for this article are available at Clinical Cancer and the lack of approved therapies for HER2 mutant NSCLC, addi- Research Online (http://clincancerres.aacrjournals.org/). tional studies are needed to identify therapeutic strategies for this Prior presentation: Part of the work was previously presented at the 2017 World subset of patients with NSCLC. Congress of Lung Cancer and the 2018 American Association for Cancer Models generated directly from cancer patients’ tumors including Research Annual Meeting. cell lines, patient-derived xenografts (PDX), or organoids are increas- € Corresponding Authors: Pasi A. Janne, Dana-Farber Cancer Institute, 450 ingly being used to screen for novel treatment approaches (14–16). Brookline Avenue, LC4114, Boston, MA 02215. Phone: 617-632-6036; Fax: 617- However, the process of cell line development and the cells’ adaptation 582-7683; E-mail: [email protected]; and Cloud P. Paweletz, [email protected] to growth in a two-dimensional environment can render some tumors that were drug-sensitive in vivo resistant in vitro. PDX experiments can Clin Cancer Res 2020;26:2393–403 often take months to complete due to slow tumor growth kinetics, and doi: 10.1158/1078-0432.CCR-19-1844 are impractical to perform in scale requisite for more comprehensive 2020 American Association for Cancer Research. drug combination screens. In addition to these intrinsic limitations of

AACRJournals.org | 2393

Ivanova et al.

Generation of HER2-mutant Ba/F3 and NIH-3T3 cells Translational Relevance The HER2 755_757LREdelinsRP mutation was introduced via site The development of anticancer therapies requires appropriate directed mutagenesis into the pDNR-Dual Donor Vector (Clontech) preclinical models in which to test treatments. HER2-mutant non– containing wild type HER2 using the QuikChange II XL Site-Directed small cell lung cancer (NSCLC) occurs in 3% of patients with lung Mutagenesis Kit (Agilent Tech) according to the manufacturer's adenocarcinoma, and very few models of HER2-mutant NSCLC instructions. Two rounds of mutagenesis were necessary to create the are available for preclinical therapeutic development. We gener- HER2 755_757LRE delinsPR mutation. The first round introduced the ated 2 patient-derived HER2-mutant xenograft (PDX) models and HER2_L755P substitution mutation using primers F-50-AAATTC- developed a platform to rapidly test HER2-targeted therapies CAGTGGCCATCAAAGTCCCGAGGGAAAACACATCCCCC-30 ex vivo using the PDX tumors. Our methods may accelerate the and R-50-GGGGGATGTGTTTTCCCTCGGGACTTTGATGGC- identification and prioritization of promising anticancer therapies CACTGGAATTT-30. The second round introduced the E757 dele- to pursue further in clinical trials. tion using primers F-50-GCCATCAAAGTCCCGAGdelAAACA- CATC CCCCAAAG-30 and R-50-CTTTGGGGGATGTGTTT- delCTCGGGACTTTGATGGC-30. All constructs were confirmed by DNA sequencing. The constructs were shuttled into the retro- the preclinical models, the identification of effective HER2-directed viral vector JP1540 or lentiviral vector JP1698 using the BD Creator treatments is further complicated by the commercial availability of System (BD Biosciences) and Ba/F3 and NIH-3T3 cells were only one HER2-mutant NSCLC cell line, H1781 (17). infected with lentivirus according to standard protocols and as We recently described a novel ex vivo system of organotypic described previously (23). Stable clones were obtained by selection tumor spheroids growth in a 3-dimensional microfluidic device in puromycin (1 mg/mL). (DOTS; refs. 18, 19). The device is designed to support short-term (≤7 days) culture of freshly harvested primary tumor cells and Focus formation assay associated immune cells resuspended in collagen for the duration of NIH/3T3 cells expressing WT HER2 or the HER2 755_757LRE- a screen, and allows for conventional immunofluorescence and delinsRP mutation were cultured in DMEM with 10% bovine calf microscopy based analysis. Thanks to its scalability, the DOTS serum. Once cells reached confluency, serum was reduced to 5%. Plates system allows for rapid evaluation of multiple different therapies were stained with 0.5% crystal violet solution two weeks later. using biopsies derived from mouse models; primary murine models (MDOTS) or patient derived xenografts (XDOTS) or Patient-derived lung tumor xenografts and genetic engineered directly from patients (PDOTS) for which preclinical models mouse models cannot be established or do not exist. In the current study, we PDX models were generated from core needle biopsies, surgical have adopted this system to study new therapeutic approaches for biopsies, or pleural effusions from lung cancer patients at the Dana- HER2 mutant NSCLC using tumors derived from HER2-mutant Farber Cancer Institute (DFCI, Boston, MA) at the Belfer Center for PDXs. Applied Cancer Science under IRB-approved protocols. All patients provided written informed consent and the studies were performed in accordance to the Declaration of Helsinki. Tumor cells isolated from Materials and Methods pleural effusions and surgical tumor tissue (3 mm 3mm 3mm) Cell lines and drug compounds were implanted subcutaneously, while tumors from core biopsies were The EGFR-mutant (HCC827 GR and PC9 GR) and ALK rear- implanted under the sub-renal capsule. Female NOD.Cg-Prkdcscid ranged cell lines (DFCI 32) have been previously characterized and Il2rgtm1Wjl/SzJ (NSG) mice were purchased from The Jackson Labo- described previously (20–22). Ba/F3 cells were a generous gift from ratory. Mice were 7 weeks old or older when tumor cells or tumor the laboratory of Dr. David Weinstock (in 2014) and cultured as tissues were implanted. described previously (21, 23). NIH-3T3 cells were purchased from The HER2YVMA GEMM SH26 was established in the Wong Lab at American Type Culture Collection (ATCC). All human cancer cells DFCI and previously published (10). Six breeders of the tetO-HER2– were authenticated in May 2017 using the Promega GenePrint 10 mutant mice and CCSP-rtTA mice were maintained to generate System at the RTSF Research Technology Support Facility in the inducible lung-specific bitransgenic hHER2mt/CCSP-rtTA cohorts. Genomic Core Laboratory, Michigan State University. All murine Pups were genotyped and those that were positive were fed on a regular mutant Ba/F3 and NIH-3T3 cells were not authenticated because diet for approximately 7 to 8 weeks after which 2,000 ppm doxycycline their short tandem repeat profile has not been made publicly chow was used to begin tumor induction. Tumors were grown for available but were sequenced to ensure they possess the correct approximately 4 weeks until they reached approximately 300 mm3. mutations. All cell lines tested negative for Mycoplasma using the MRI imaging of tumors before and during treatment was performed as Mycoplasma Plus PCR Primer Set (Agilent). All cell lines were previously described (10, 21). All breeding, mouse husbandry, and passaged and used for no longer than 4 weeks before new cells with in vivo treatment experiments were performed with the approval of the similar passage numbers were thawed for all described experiments. DFCI Animal Care and Use Committee. Gefitnib, afatinib, poziotinib, crizotinib, alpelisib, osimertinib, and AZD8055 were purchased from Selleck Chemicals. Stock solutions PDX-derived organotypic tumor spheroids (XDOTS) of all drugs were prepared in DMSO and stored at 80C. Tras- Tumor spheroid culture was carried out as previously described tuzumab and TDM1 were purchased from the DFCI pharmacy. with slight modifications (18). Following harvsesting, fresh tumor Neratinib was obtained from Puma Biotechnology, Inc. The con- specimens were maintained on ice in RPMI supplemented with 10% centrations of each of the drugs used in the in vitro and in vivo FBS (Gemini Bio-Products) and penicillin–streptomycin (Thermo studies were based on prior studies whereby the specificagentshave Fisher Scientific). Tumors were minced using sterile scalpel and razor demonstrated effective target inhibition (24–30). blades in prewarmed to 37C full media þ 100 U/mL collagenase type

2394 Clin Cancer Res; 26(10) May 15, 2020 CLINICAL CANCER RESEARCH

Preclinical HER2 Combination Therapy

IV (Life Technologies) and 50 mg/mL DNase I (Roche) for approxi- of propagating the tumor tissue. Of the 15 potential models, only two, mately 20 minutes. Digested samples were strained sequentially DFCI315 and DFCI359, were successfully established as stable PDX through a 100-mm and 40-mm filter and spheroids smaller than 100mm models (Fig. 1A; Supplementary Table S1 and Supplementary but greater than 40 mm were transferred to an ultralow-attachment Fig. S1A). The growth kinetics (passage 2) were faster for DFCI 315 tissue culture plate (Corning) and grown in RPMI media overnight at compared to DFCI 359 (Fig. 1A). Both models were histologically 37C. Cells were resuspended in type I rat tail collagen (Corning) at a characterized as adenocarcinomas with moderate (2þ) expression of concentration of 2.8 mg/mL and loaded into the gel ports of the DAX-1 HER2 as detected by IHC (Fig. 1B). DFCI315 harbored a known 3-D cell culture chip (AIM Biotech) at a concentration of 30 to 50 and previously characterized HER2 exon 20 insertion mutation, spheroids/device, hydrated with full media with appropriate drugs or V777_G778insGSP (also referred to as G778_P780dup), while drug combinations, and incubated in humidity chambers at 37C, 5% DFCI359 carried a novel HER2 exon 19 deletion mutation CO2 for 72 to 96 hours. Baseline viability determination were per- (755_757LREdelinsRP; Supplementary Fig. S1B). In addition, the DFCI formed on a separate device prior to drug treatment. 359 tumor also contains a mutation in IDH (IDH R132C) and TP53 (G187_splice and loss of heterozygosity (LOH)) while the DFCI 315 Viability and immunofluorescence tumor contains a high copy number gain (as measured by NGS) in For immunofluorescence studies, XDOTS were blocked with FcR CDK4 and MDM2 (data not shown). By sequence homology, the HER2 blocking reagent (BioLegend) for 30 minutes at room temperature. mutation present in DFCI359 corresponds to a common EGFR exon 19 Baseline viability was defined by the presence of calcein AM (Invitro- deletion mutation (Supplementary Fig. S1C), suggesting that this gen) staining. Tumor content was assessed by an anti-human EpCAM mutant is likely the oncogenic driver in DFCI359. As the HER2 antibody (clone 9C4, BioLegend). For the samples not expressing mutation in DFCI359 had not been previously characterized, we EpCAM, tumor content was defined by the morphology of Hoechst evaluated its oncogenic potential using two independent assays. Expres- 33342 (Invitrogen)-stained nuclei and negative expression of a cocktail sion of the HER2 755_757LREdelinsRP mutant resulted in IL-3 inde- of anti-human CD90 (clone 5E10, BioLegend), and anti-mouse mar- pendent cell growth in Ba/F3 cells, and colony formation in NIH/3T3 kers CD45 to identify mouse immune cells (clone 30-F11, BioLegend), cells (Fig. 1C; Supplementary Fig. S1D). Ba/F3 cells expressing HER2 and CD47 for mouse fibroblast and endothelial cells (clone miap301, 755_757LREdelinsRP were sensitive to covalent HER2 kinase inhibitors BioLegend) in another channel. neratinib, poziotinib, and afatinib but not to the EGFR inhibitor The assessment of live/dead end-point was performed using either gefitinib (Fig. 1D). Of note, we were not able to establish a stable PDX ViaStain AO/PI Staining (Nexcelom) for 20 minutes or by 10 mg/mL from a NSCLC patient harboring the common HER2 YVMA mutation solution of Hoechst 33342 (Life Technologies) and 1 mg/mL solution of despite several attempts. In addition, neither DFCI315, nor DFCI359 propidium iodide (Thermo Fisher Scientific) in full media (H/PI) for was able to grow in 2D culture as a cell line (data not shown). We also 45 minutes. established an EGFR-mutant (L858R/T790M) PDX (DFCI 282) from a patient who had developed acquired resistance to erlotinib (Supple- Image analysis mentary Table S1) for use in our XDOT validation studies. TheentiredevicewasimagedonaNikonEclipse80ifluorescence microscope equipped with automated motorized stage (Proscan), Z- Establishment of XDOT platform to assess drug response stack (Prior) and Zyla 5.5 sCMOS camera (Andor). Image capture Given our inability to establish DFCI315 and DFCI359 as stable cell and analysis were performed using the NIS-Elements AR software lines, we sought to use an alternative ex vivo system where we could package. Dead cell quantitation was performed by measuring total efficiently evaluate new therapeutic strategies for HER2 mutant PI positive cell area; live cell assessment was done either by NSCLC. To that end, we optimized our recently developed DOTS measuring total AO positive cell area or by subtracting dead cell system, which had previously been used to assess the efficacy of area from total area of Hoechst 33342 staining. AO/PI and H/PI immune checkpoint inhibition (18, 19). The central component of produced similar results in the samples where tumor cells had low this platform is a microfluidic device which supports the survival and cytoplasmic to nucleus ratio (data not shown), but in the samples growth of both, established and primary tumor, stromal and immune with large cytoplasm AO tended to overestimate live cell area and cells ex vivo for up to one week (18, 19). Previously established methods H/PI staining was chosen instead. were adapted to facilitate the dissociation of tumors isolated from Tumor and stromal viability was evaluated separately in the samples stable PDX models into tumor spheroids 40 to 100 mm in diameter. In with low tumor content (<80%) by live/dead four-color fluorescent addition, and in contrast to the prior studies, we included a baseline staining. At least 5 fields of view from 2 to 3 wells were captured for quality control step using 4-color immunofluorescence. For samples each treatment condition at 200 total magnification and viability of with aggregate viability >20%, the remaining tumor material was tumor and nontumor cells was analyzed semi quantitively using the resuspended in collagen, loaded in the device, and cultured as spher- NIS-Elements AR software package. oids (30–50 per condition, in triplicates) in the presence of drug or vehicle for up to 96 hours. For samples with baseline tumor content (TC) > 80%, drug efficacy was assessed by dual-color fluorescent Results staining with propidium iodide (PI) marking dead cells combined with Establishment of patient-derived HER2-mutant NSCLC either the live-cell stain acridine orange (AO) or general nuclear xenograft models counterstain Hoechst 33342 (Hoechst). For samples with < 80% TC, þ To develop new HER2-mutant models we obtained research biop- efficacy was quantified by counting calcein AM-positive hEpCAM / sies, under an IRB approved protocol, from NSCLC patients with hCD90 /mCD45 /mCD47 cells. Off-target drug toxicity was mon- known HER2-mutant NSCLC. Between August 2010 and June 2017, itored by measuring viability of individual stromal cells (i.e., we obtained 15 biopsies from HER2-mutant NSCLC patients and hEpCAM ). Cell death quantification was performed independently placed them either subcutaneously (n ¼ 9) or in the subrenal capsule of cell proliferation rate, and as such wasn't vulnerable to the growth (n ¼ 6) of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, with the goal rate variability inherent to ex vivo models (Fig. 2).

AACRJournals.org Clin Cancer Res; 26(10) May 15, 2020 2395

Ivanova et al.

Figure 1. Characterization of patient-derived HER2-mutant NSCLC xenograft models. A, Comparative growth kinetics of DFCI 315 and DFCI 359. Each curve represents a separate mouse. B, Representative H&E and HER2 IHC stains. C, Expression of the HER2 755_757LREdelinsRP mutant in Ba/F3 cells results in IL3-indepdent cell growth. D, HER2 755_757LREdelinsRP Ba/F3 cells are sensitive to HER2 kinase inhibitors. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls.

Validation of XDOTS system using characterized NSCLC models through EGFR and HER2 co-activation, we demonstrated its sen- To evaluate whether cells grown in our three-dimensional device sitivity to the combination of 0.1 mmol/L crizotinib with 1 mmol/L recapitulate drug responses seen in previously reported acquired gefitinib (63% death) while being resistant to either of the single resistance models, we generated spheroids using cell lines (CLS; cell agents (crizotinib: 3% death; gefitinib 4% death; Fig. 3D). Using line spheroids) with previously characterized resistance mechan- CellTiter-Glo as a cross-validation of the DOTS assay, we estab- fi – fi isms (21). In a dose-escalated CLS assay, we show that the ge tinib lished that the IC50 for the crizotinib ge tinib combination in this resistant EGFR mutant PC9 GR4 (EGFR E746_A750/T790M) is model was 25 nmol/L, translating to more than 1,000-fold shift in highly sensitive to osimertinib (IC50 of 12 nmol/L), an irreversible sensitivity relative to the single agent treatments (Supplementary third-generation EGFR inhibitor that targets EGFR T790M Fig. S2B). We next evaluated the gefitinib resistant EGFR mutant (Fig. 3A). Similar results were obtained when the PC9 GR4 cells HCC827GR6 cell line (EGFR Del E746_A750/MET amplified) were let adhere to flat-bottom plates or cultured in ultralow grown as spheroids in our microfluidic device and demonstrated attachment plates for the assay (Supplementary Fig. S2A). We next its sensitivity to the combination of gefitinib and crizotinib (62% established XDOTS using an EGFR-mutant, erlotinib-resistant, death; Fig. 3E) and relative resistance to either drug as a single osimertinib-sensitive PDX model, DFCI282 (EGFR L858R/ agent, consistent with prior reports (20). As an orthogonal assay, we T790M; Supplementary Table S1; Fig. 3B). The DFCI282 XDOTS monitored apoptosis in the HCC827GR6 cells in real time following were assessed for sensitivity to erlotinib or osimertinib in a dose- treatment with gefitinib alone, crizotinib alone or with the combi- escalated assay and were remarkably sensitive to osimertinib, with nation of both drugs, and demonstrated a marked increase in close to 100% inhibition at ≥100 nmol/L (Fig. 3C). apoptosis induction only in the cells treated with the combination We further assayed cell lines or PDXs with described resistance (Supplementary Fig. S2C). Similarly in DFCI358 XDOTS, which mechanisms that respond only to a combination of therapies and were generated from a crizotinib-resistant KRAS amplified MET which we had previously orthogonally validated (20, 22, 31). Ana- ex14 mutant (MET c.2903_3028þ67del193insA) PDX, the crizoti- lyzing CLS derived from DFCI32, an EML4-ALK driven cell line nib/alpelisib combination previously published to be effective in that we previously reported to be resistant to single-agent crizotinib this model resulted in 90% death in the XDOT system

2396 Clin Cancer Res; 26(10) May 15, 2020 CLINICAL CANCER RESEARCH

Preclinical HER2 Combination Therapy

Figure 2. PDX (XDOT) organotypic spheroid and PDX spheroids cell line spheroid (CLS) workflow. Tumors are harvested from PDXs and XDOTS allowed to recover overnight in ultralow Baseline IF HER2 Hoechst EpCAM attachment (ULA) plates. Cell lines are mCD45+mCD47 used the same day. After dissociation and filtration isolation, spheroids that are between 100 mm and 40 mm are seeded and evaluated for tumor content (TC; EpCAMþ, CD45/CD47/CD90)and viability (calcein/propidium iodide; PI) CLS by immunofluorescence. If TC > 80% and spheroids viability > 20%, live/dead ratios (AO/PI) Cell line–derived are obtained. If TC < 80% and viability Device loading > 20%, endpoint viability is quantified by Viab. <20% þ individual cell count in hEpCAM / TC >80% Live/Dead mCD45/mCD47/mCD90 tumor and QC hEpCAM stromal cells. Samples with TC <80% IF Live/Dead viability < 20% are excluded from the analysis.

72- to 96-h drug treatment

Live/Dead IF Live/Dead mCD45/mCD47/hCD90 Calcein AM Hoechst PI

(Fig. 3F) (31). Collectively, these findings suggest that sensitivity to thermore, in HER2-amplified breast cancer models, treatment with a either single-agent or to combination targeted therapy can be HER2 TKI can lead to an increase in cell surface levels of HER2 accurately and efficiently assessed using the DOTS system. enhancing the effect of trastuzumab (33–35). However, this strategy has not been tested in HER2-mutant NSCLC. In DFCI359 XDOTS, Screening of drug combinations in HER2-mutated XDOTS single agents AZD8055 and trastuzumab were ineffective at clinically Having established that our DOTS system can generate highly achievable concentrations (Fig. 4C). In contrast, the combination of reproducible data corroborated by standard methods, we set out to trastuzumab and neratinib was significantly more effective, inducing use the platform to screen for effective targeted therapies for HER2- approximately 50% cell death (Fig. 4C). AZD8055 in combination mutant lung cancer, a genotype with minimal therapeutic options with neratinib was also more effective than either single-agent due to the lack of stable patient-derived mutant models. Using the treatment (Fig. 4C). We also evaluated these agents in the DFCI315 system, we specifically sought to identify drugs that would enhance model. While neratinib alone was already relatively efficacious in this the effects of neratinib, a panHERTKI, when used in combination model (48% cell death vs. 18% cell death in DFCI359) the antitumor against HER2-mutant patient-derived material. We first generated efficacy was further enhanced by the addition of trastuzumab (65% XDOTS from the DFCI359 PDX tumors and treated them with 0.5 cell death; Fig. 4D). The combination of neratinib and trastuzumab mmol/L gefitinib, neratinib, or afatinib for 3 days as single agents. is likely additive in both the DFCI 359 and 315 models. No formal Both neratinib and afatinib (covalent panERBB inhibitors), but not analyses of synergy were performed. gefitinib (ATP competitive inhibitor), induced tumor cell death, which is the expected finding for this particular HER2 genotype In vivo evaluation of combination therapy in HER2-mutant (Fig. 4A). Using fluorescence microscopy, we further affirmed that NSCLC the effects of neratinib were specific to the tumor cells (Fig. 4B). To determine whether the findings made using the XDOTS Encouraged by these results we set out to test combinations of system are predictive of in vivo efficacy, we tested the different neratinib, the TORC1/TORC2 kinase inhibitor AZD8055 and the drug combinations in the corresponding HER2 mutant PDX models anti-HER2 directed antibody trastuzumab in DFCI359 XDOTS. in vivo. DFCI359 PDX-bearing animals were treated for 28 days Preclinically, coinihibition of mTOR and HER2 signaling had with neratinib alone (40 mg/kg; oral gavage daily for 28 days), emerged as a promising therapeutic strategy, to which end the mTOR trastuzumab alone (20 mg/kg; intraperitoneal injection; twice week- inhibitortemserolimuswascombined with neratinib in a clinical ly for 4 weeks), temsirolimus alone (20 mg/kg; intravenous injec- trial, serving as a rationale for our drug candidate selection (10, 11). tion, every 3 days for 5 doses) or with the combinations of neratinib/ In place of neratinib, trastuzumab is also sometimes used in com- temsirolimus or neratinib/trastuzumab (n ¼ 8 mice/cohort). The bination with chemotherapy for HER2-mutant NSCLC (32). Fur- most efficacious treatment was neratinib/trastuzumab, which

AACRJournals.org Clin Cancer Res; 26(10) May 15, 2020 2397

Ivanova et al.

Figure 3. CLS and XDOTS recapitulate responses observed in previously characterized models of drug resistance. A, Osimertinib and gefitinib dose response in PC9GR CLS. Live (AO ¼ green)/dead (PI ¼ red) are normalized to cell area. B, In vivo efficacy study of osimertinib or vehicle in DFCI 282 (EGFR L858R/T790M) PDX tumor model (mean tumor volume SEM; n ¼ 3 each). C, Life/dead analysis of DFCI282 XDOTS following ex vivo treatement with osimertinib or erlotinib. D, Live (AO ¼ green)/ dead (PI ¼ red) analysis of DFCI 32 CLS treated with crizotinib (0.1 mmol/L), gefitinib (1 mmol/L), and crizotinib/gefitinib combination (0.1 mmol/L/1 mmol/L). E, HCC827 GR6 CLS treated with crizotinib (0.1 mmol/L), gefitinib (1 mmol/L), and crizotinib/gefitinib combination (0.1 mmol/L/1 mmol/L). F, Live/dead analysis of DFCI 358 XDOTS treated with the indicated concentrations of crizotinib, alpelisib, or the combination of both agents. All comparisons were performed by two-way ANOVA with Tukey multiple comparison tests (, P < 0.05; , P < 0.01; , P < 0.001).

consistently resulted in tumor regressions (Fig. 5A and B). Max- We employed a similar study design assessing the efficacy of HER2- imum tumor regression was 48% and 22% in mice treated with targeted therapies in the DFCI315 PDX model. The same order of neratinib/trastuzumab and neratinib/temsirolimus, respectively, efficacy was observed in vivo as in the XDOTS system with the while only minimal tumor growth inhibition (TGI) was observed combination of neratinib/trastuzumab exhibiting comparable degree following treatment with neratinib (18% TGI) or trastuzumab alone of growth inhibition as neratinib alone (Fig. 5D and E). We did not (28% TGI; Fig. 5B). Eight mice treated with neratinib/trastuzumab include temsirolimus alone or in combination with neratinib in this combination therapy showed significant tumor regression (>30%) model given the superiority of neratinib/trastuzumab to the neratinib/ with mice achieving up to a 75% decrease in tumor volume temsirolimus combination in the DFCI359 model. The effects of compared with baseline. In comparison, no mice showed >30% neratinib and/or trastuzumab inhibition were further assessed in a tumor regression in the vehicle or neratinib cohort (Fig. 5B). The pharmacodynamic study, evaluating on-target activity of these agents order of most to least efficacious treatment in vivo was similar to against HER2 and downstream signaling at the molecular level that observed in the XDOTS system (compared Figs. 4C and 5B). A (Fig. 5F). DFCI315 tumor bearing animals were treated with neratinib pharmacodynamic study, performed in parallel with the efficacy alone (daily), trastuzumab alone (once) or with the combination of study for 4 days (daily neratinib alone or in combination with one both agents for 4 days and harvested 2 hours after the last treatment. dose of trastuzumab), revealed effective inhibtion of pHER2 with Tumor sample analysis revealed that neratinib effectively reduced more modest impact on downstream signaling (Fig. 5C). HER2 phosphorylation and inhibited major downstream signaling

2398 Clin Cancer Res; 26(10) May 15, 2020 CLINICAL CANCER RESEARCH

Preclinical HER2 Combination Therapy

Figure 4. Efficacy of HER2-targeted therapies in DFCI 359 and DFCI 315 XDOTS. A, Live (AO ¼ green)/dead (PI ¼ red) analysis of DFCI 359 following ex vivo treatment with gefitinib (0.5 mmol/L), afatinib (0.5 mmol/L), or neratinib (0.5 mmol/L); n ¼ 3, pooled biological replicates. B, Deconvolution fluorescence microscopy (green ¼ EpCAM, red ¼ PI) following 3 days of neratinib treatment demonstrates that the therapeutic effect is specific to the tumor cells and not stroma (right). C, Percent live (AO ¼ green)/dead (PI ¼ red) of DFCI 359 XDOTS (HER 2 755_757LREdelinsRP) following a 72-hour ex vivo treatment with neratinib, trastuzumab, AZD8055, or the combination of the different drugs. D, Percent live/dead of XDOTS 315 (HER2 exon20 V777_G778insGSP) treated with neratinib (0.5 mmol/L), trastuzumab (10 mg/mL), or the combination of both agents. All comparisons were performed by two-way ANOVA with Tukey multiple comparison tests (, P < 0.05; , P < 0.01; , P < 0.001). targets including pAKT and pERK. Notably, the combination of regression, achieving up to a 65% decrease in tumor volume neratinib and trastuzumab resulted in a greater and nearly complete (Fig. 5G). In contrast, vehicle-treated mice developed progressive inhibition of pHER2, pHER3, and pAKT compared with the single disease by the 4-week time point. Mirroring our XDOTS efficacy data, agents (Fig. 5F). neratinib or trastuzumab administered as monotherapy exhibited only As neither the DFCI315 nor the DFCI359 model contain the a limited antitumor activity. To further test whether the combination common HER2 YVMA mutation, we evaluated the efficacy of ner- therapy could maintain a durable antitumor response, we continued atinib alone, trastuzumab alone or the combination of both agents in a monitoring the mice for survival upon treatment cessation (Fig. 5H). previously reported HER2YVMA GEMM (10). Mice were evaluated for The HER2YVMA mice treated with the neratinib and trastuzumab tumor formation using serial MRI and those with established tumors combination therapy showed a significantly longer overall survival were treated for 6 weeks and monitored by serial MRI. The HER2YVMA (P ¼ 0.0005; logrank) compared with those treated with other regi- model responded dramatically to the combination therapy with mens or vehicle (Fig. 5H). The median OS for mice in the vehicle group neratinib and trastuzumab: all mice (n ¼ 6) treated with the nerati- was 38 days [95% confidence interval (CI), 21–NA]; for mice treated nib/trastuzumab combination therapy showed significant tumor with neratinib, it was 62.5 days (40–NA), and for mice treated with

AACRJournals.org Clin Cancer Res; 26(10) May 15, 2020 2399

Ivanova et al.

A B C 450 300 Vehicle VN TN+T ) 400 3 Neratinib 350 Vehicle pHER2 Trastuzumab 300 Neratinib 200 Neratinib/Trastuzumab HER2 Temsirolimus 250 Trastuzumab pEGFR Neratinib/Temsirolimus 200 Temsirolimus 100 EGFR 150 Neratinib/Temsirolimus pHER3 100 Neratinib/Trastuzumab 50 0 HER3 Tumor volume (mm volume Tumor 0 pAKT 0 10 20 30 volume tumor in Change % AKT Days −100 pERK1/2

ERK 1/2

GAPDH

D E F 1,500

) 1,000

3 T 100 V N N+T 500 75 Vehicle pHER2 Vehicle Neratinib 400 Neratinib 50 Trastuzumab HER2 300 Trastuzumab 25 Neratinib/Trastuzumab Neratinib/Trastuzumab pEGFR 200 0 −25 EGFR 100 Tumor volume (mm volume Tumor −50 pHER3 0 −75 0 10 20 30 40 change) (% volume Tumor HER3 Days −100 pAKT

AKT

pERK1/2

ERK 1/2

G H GAPDH

75 100 Vehicle Neratinib 50 Vehicle Trastuzumab Neratinib 75 Neratinib/Trastuzumab 25 Trastuzumab 0 Neratinib/Trastuzumab 50 −25 25

−50 Percent survival Percent

Tumor volume (% change) (% volume Tumor −75 0 0 50 100 150 200 Days

Figure 5. Evaluation of the neratinib/trastuzumab combination in vivo. A, Tumor volumes (TV; mean SEM) over time of PDX DFCI 359 dosed with neratinib (40 mg/kg), trastuzumab (20 mg/kg), temsirolimus (20 mg/kg), and their combinations (n ¼ 8 mice/cohort). B, Waterfall plot of PDX DFCI 359 (% change in TV) following 4 weeks of treatment with neratinib, trastuzumab, temsirolimus, or their combinations. C, Pharmacodynamic study in DFCI 359 following treatment with vehicle (V), neratinib (N), trastuzumab (T), or the combination of neratinib and trastuzumab (N þ T). Cell extracts were immunoblotted to detect the indicated proteins. D, Tumor volumes (mean SEM) over time of PDX DFCI 315 treated in vivo with neratinib (40 mg/kg), trastuzumab (20 mg/kg), or the combination of both agents (n ¼ 8 mice/ cohort). E, Waterfall plot of PDX 315 following 4 weeks of treatment using drugs in C. F, Pharmacodynamic study in DFCI 315 following treatment with vehicle (V), neratinib (N), trastuzumab (T), or the combination of neratinib and trastuzumab (N þ T). Cell extracts were immunoblotted to detect the indicated proteins. G, Waterfall plot of HER2 YVMA GEMs following 2 weeks of treatment with vehicle (n ¼ 4), neratinib (40 mg/kg; n ¼ 4), trastuzumab (20 mg/kg; n ¼ 4), or the combination of both drugs (n ¼ 6). H, Kaplan–Meier survival curve of mice in F treated with the indicated drugs.

trastuzumab it was 93 days (77–NA). Mice treated with the combi- architecture, fidelity to original tumor material, and promise to nation therapy had a median OS of 127 days (95% CI, 89–NA). No faster translate targeted therapies for genotype defined cancers, they significant weight loss was observed with the combination therapy in are impractical for drug combination screens as they are costly and the HER2YVMA mice (Supplementary Fig. S4). time consuming (15, 36). Our ex vivo system bypasses most of these limitations and enables expeditious screening of drug combinations, aiding in prioritization of promising candidates for in vivo evalu- Discussion ation. In addition, the DOTS system circumvents the necessity of The development of new therapeutic approaches requires clin- establishing cell lines and/or organoids, which can be both time ically reflective pre-clinical models in which to test such consuming and artifact-prone due to the inevitable loss of tumor approaches. Although all pre-clinical models have some advantages heterogeneity. Although grown in a matrix like organoids, there are and disadvantages, some are more suited for drug screening. some significant differences between the XDOTS and organoids. Although PDXs have been shown to maintain intratumor clonal Organoids are similarly generated from individual patient biopsies,

2400 Clin Cancer Res; 26(10) May 15, 2020 CLINICAL CANCER RESEARCH

Preclinical HER2 Combination Therapy

are grown in 3D, and have been shown to preserve molecular, either model (Supplementary Fig. S3). It is possible that the level of phenotypic and genotypic characteristics of the original tumor, includ- HER2 expression (2þ for both models) accounts for the lack of ing oncogenic drivers (36, 37). However, organoids can take months to efficacy of TDM1. generate, typically lack stromal cells, and require a culture medium that The dual targeting strategy, combining a tyrosine kinase inhibitor facilitates the propagation of progenitor cells for their establishment. and a therapeutic antibody, has been used to inhibit EGFR combining The culture conditions, for example, the presence of EGF, may affect afatinib and cetuximab both preclinically and in a phase II clinical drug response, when studying e.g., EGFR inhibitors. Also, a challenge trial (44, 45). In addition, a clinical trial of osimertinib and necitu- specific to organoids is the outgrowth of normal epithelial cells in mumab is currently underway (NCT02496663). Intriguingly, the culture, affecting the generation of lung cancer organoids in particular, afatinib/cetuximab combination resulted in a downregulation of total due to the commonly seen phenomenon of field cancerization of the EGFR in the genetically engineered murine model of EGFR-mutant normal lung epithelium (37). NSCLC, which may explain the therapeutic efficacy of the combina- Somatic mutations in HER2 are as common as anaplastic lympho- tion (44). Similarly, we observed an increased downregulation of total ma kinase (ALK) rearrangements in NSCLC but unlike for ALK HER2 and a more extensive inhibition of HER2 and downstream rearranged cancers, no targeted therapies are approved for HER2 signaling in vivo (DFCI 315 only) with the combination of neratinib mutant NSCLC (1). Single-agent pan-ERBB kinase inhibitors have and trastuzimab compared with treatment with each of the single limited anti-tumor activity and most currently used treatments are agents, which could underlie the increased efficacy of the combination repurposed therapies indicated for HER2-positive breast cancer and as (Fig. 5F). In contrast to the efficacy, pharmacodynamic studies in the such are not specifically tailored towards the unique HER2 mutations DFCI 359 model revealed little total HER2 downregulation or impact found in NSCLC. (11, 12, 38). To that end, it is imperative to focus on on downstream signaling (Fig. 5C). Whether this disconnect stems the development of therapeutic approaches for this “orphan” NSCLC from the timing of the pharmacodynamic studies (4 days) or pathway genotype. Unfortunately, the lack of available preclinical models that rewiring following drug treatment will require additional assessment. could more authentically recapitulate real human cancers has signif- Nonetheless, based on our preclinical efficacy findings, the combina- icantly hindered this effort. For instance, the HER2 inhibitor neratinib tion of neratinib and trastuzumab is being actively investigated in is quite effective in Ba/F3 cell lines engineered to express various HER2 patients with HER2-mutant lung cancer in the ongoing SUMMIT < “ ” mutations (IC50s 20 nmol/L), yet is ineffective as a single agent in basket trial (NCT01953926). patients with lung cancer whose tumors harbor the same mutation (9, 39). These findings suggest that either, drug exposures required Disclosure of Potential Conflicts of Interest to inhibit mutant HER2 cannot be achieved in patients, or that human I. Diala is an employee/paid consultant for Puma Biotechnology, Inc. A.S. Lalani is cancers are more complex than can be modeled in simplified drug- an employee/paid consultant for and holds ownership interest (including patents) in screening systems, such as Ba/F3 cell models. Consistent with the Puma Biotechnology, Inc. T.U. Barbie is an employee/paid consultant for Qiagen. G. fi R. Oxnard reports receiving speakers bureau honoraria from Foundation Medicine clinical ndings, neratinib treatment alone of both the DFCI 315 and and Guardant, and is an advisory board member/unpaid consultant for AstraZeneca 359 PDX models is predominately associated with tumor stasis and and Takeda. D.A. Barbie is an employee/paid consultant for N of One/Qiagen and minimal tumor regression (Fig. 5). Tango Bioscences; reports receiving commercial research grants from Novartis, Ultimately, combination therapies will likely be more successful Bristol-Myers Squibb, Gilead Sciences, and Eli Lilly, and speakers bureau honoraria clinically than single agent treatments. There are two major cate- from Esai/H3 Biomedicine; and holds ownership interest (including patents) in gories of combination therapies: dual inhibition of the same target, Xsphera Biosciences. C.P. Paweletz holds ownership interest (including patents) in and is an advisory board member/unpaid consultant for Xsphera Biosciences. P.A. or inhibition of the target and a critical downstream signaling J€anne is an employee/paid consultant for AstraZeneca, Boehringer Ingelheim, Pfizer, pathway. Both strategies can achieve a more durable target and/ Roche/Genentech, ACEA Biosciences, Ignyta, LOXO Oncology, Eli Lilly, Araxes or pathway inhibition which can translate into an improved clinical Biosciences, SFJ Pharmarceuticals, Daiichi Sankyo, Biocartis, Voronoi, Sanofi Oncol- outcome. Dual target inhibition has been successful in HER2 ogy, Mirati Therapeutics, Novartis, and Takeda Oncology; reports receiving com- positive breast cancer where the combination of trastuzumab and mercial research grants from AstraZeneca, Boehringer Ingelheim, Astellas, Takeda pertuzumab with docetaxel lead to an increase in survival compared Oncology, PUMA, Daiichi Sankyo, Revolution Medicines, and Eli Lilly; and holds ownership interest (including patents) in Gatekeeper Pharmaceuticals, LOXO Oncol- with trastuzumab and chemotherapy (40). The combination of ogy, and Lab Corp. No potential conflicts of interest were disclosed by the other BRAF inhibitor dabrafenib and the MEK inhibitor trametinib is authors. also associated with an improvement in survival in patients with BRAF V600E–mutant melanoma compared with the BRAF inhib- Authors’ Contributions itor vemurafenib alone (41). In HER2-mutant lung cancer, the Conception and design: E. Ivanova, M. Kuraguchi, A.S. Lalani, A.A. Aref, combination of neratinib and temsirolimus has been evaluated as C.P. Paweletz, P.A. J€anne a strategy to achieve a nearly complete pathway inhibition based on Development of methodology: E. Ivanova, M. Kuraguchi, A.J. Portell, A.S. Lalani, preclinical data and a phase I clinical trial (10, 42). However, in a T.U. Barbie, A.A. Aref, D.A. Barbie, C.P. Paweletz randomized phase II clinical trial, the combination only led to Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E. Ivanova, M. Kuraguchi, M. Xu, A.J. Portell, L. Taus, S. Li, S. Liu, incremental improvement in response rates and progression-free T. Chen, G.R. Oxnard, K.-K. Wong, M. Bahcall, P.A. J€anne survival (11). In the current study, we evaluated the concept of dual Analysis and interpretation of data (e.g., statistical analysis, biostatistics, HER2 inhibition using a HER2 kinase inhibitor (neratinib) and an computational analysis): E. Ivanova, M. Kuraguchi, M. Xu, A.J. Portell, anti-HER2 antibody (trastuzumab). Intriguingly, despite the clin- J.J. Haworth, K.-K. Wong, S.E. Dahlberg, A.A. Aref, C.P. Paweletz, P.A. J€anne ical efficacy of ado-trastuzumab emtasine (TDM1) in HER2-mutant Writing, review, and/or revision of the manuscript: E. Ivanova, M. Kuraguchi, NSCLC (12), it was ineffective in the DFCI359 model, only mod- L. Taus, I. Diala, A.S. Lalani, G.R. Oxnard, D.A. Barbie, M. Bahcall, C.P. Paweletz, P.A. J€anne erately effective in the DFCI315 in model and less effective than Administrative, technical, or material support (i.e., reporting or organizing data, neratinib alone in both models (Supplementary Fig. S3). Further- constructing databases): A.S. Lalani, E.S. Chambers, S. Liu, J.J. Haworth more, and in contrast to recent studies with poziotinib (43), there Study supervision: C.P. Paweletz, P.A. J€anne was no added benefit when TDM1 was combined with neratinib in Other (cell line establishment): J. Choi

AACRJournals.org Clin Cancer Res; 26(10) May 15, 2020 2401

Ivanova et al.

Acknowledgments The costs of publication of this article were defrayed in part by the payment of page This work was supported in part by the NCI at the NIH [R35 CA220497 (P.A. charges. This article must therefore be hereby marked advertisement in accordance J€anne)], The American Cancer Society [CRP-17-111-01-CDD (P.A. J€anne)] the with 18 U.S.C. Section 1734 solely to indicate this fact. Expect Miracles Foundation, the Robert and Rene Belfer Foundation, and sponsored Received June 5, 2019; revised December 30, 2019; accepted February 4, 2020; research funding from PUMA Biotech. published ﬁrst February 7, 2020.

References 1. Kris MG, Johnson BE, Berry LD, Kwiatkowski DJ, Iafrate AJ, Wistuba II, et al. 20. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, et al. MET Using multiplexed assays of oncogenic drivers in lung cancers to select targeted amplification leads to gefitinib resistance in lung cancer by activating ERBB3 drugs. JAMA 2014;311:1998–2006. signaling. Science 2007;316:1039–43. 2. Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, et al. Gefitinib or 21. Zhou W, Ercan D, Chen L, Yun CH, Li D, Capelletti M, et al. Novel mutant- carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361: selective EGFR kinase inhibitors against EGFR T790M. Nature 2009;462:1070–4. 947–57. 22. Koivunen JP, Mermel C, Zejnullahu K, Murphy C, Lifshits E, Holmes AJ, et al. 3. Soria JC, Ohe Y, Vansteenkiste J, Reungwetwattana T, Chewaskulyong B, Lee EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. KH, et al. Osimertinib in untreated EGFR-mutated advanced non-small-cell Clin Cancer Res 2008;14:4275–83. lung cancer. N Engl J Med 2018;378:113–25. 23. Ercan D, Choi HG, Yun CH, Capelletti M, Xie T, Eck MJ, et al. EGFR mutations 4. Shaw AT, Ou SH, Bang YJ, Camidge DR, Solomon BJ, Salgia R, et al. Crizotinib in and resistance to irreversible pyrimidine-based EGFR inhibitors. Clin Cancer ROS1-rearranged non-small-cell lung cancer. N Engl J Med 2014;371:1963–71. Res 2015;21:3913–23. 5. Drilon A, Laetsch TW, Kummar S, DuBois SG, Lassen UN, Demetri GD, et al. 24. Shimamura T, Ji H, Minami Y, Thomas RK, Lowell AM, Shah K, et al. Non- Efficacy of larotrectinib in TRK fusion-positive cancers in adults and children. small-cell lung cancer and Ba/F3 transformed cells harboring the ERBB2 N Engl J Med 2018;378:731–9. G776insV_G/C mutation are sensitive to the dual-specific epidermal growth 6. Subbiah V, Velcheti V, Tuch BB, Ebata K, Busaidy NL, Cabanillas ME, et al. factor receptor and ERBB2 inhibitor HKI-272. Cancer Res 2006;66:6487–91. Selective RET kinase inhibition for patients with RET-altered cancers. 25. Li D, Ambrogio L, Shimamura T, Kubo S, Takahashi M, Chirieac LR, et al. Ann Oncol 2018;29:1869–76. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical 7. De Greve J, Moran T, Graas MP, Galdermans D, Vuylsteke P, Canon JL, et al. lung cancer models. Oncogene 2008;27:4702–11. Phase II study of afatinib, an irreversible ErbB family blocker, in demo- 26. Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL. Herceptin- graphically and genotypically defined lung adenocarcinoma. Lung Cancer induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for 2015;88:63–9. antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res 8. Kris MG, Herbst R, Rischin D, LoRusso P, Baselga J, Hammond L, et al. Objective 2002;62:4132–41. regressions in non-small cell lung cancer patients treated in Phase I trials of oral 27. Petigny-Lechartier C, Duboc C, Jebahi A, Louis MH, Abeilard E, Denoyelle C, ZD 1839 ('Iressa'), a selective tyrosine kinase inhibitor that blocks the epidermal et al. The mTORC1/2 Inhibitor AZD8055 Strengthens the Efficiency of the MEK growth factor receptor (EGFR). Lung Cancer 2000;29(Suppl 1):72. Inhibitor Trametinib to Reduce the Mcl-1/[Bim and Puma] ratio and to Sensitize 9. Hyman DM, Piha-Paul SA, Won H, Rodon J, Saura C, Shapiro GI, et al. HER Ovarian Carcinoma Cells to ABT-737. Mol Cancer Ther 2017;16:102–15. kinase inhibition in patients with HER2- and HER3-mutant cancers. Nature 28. Schwab CL, English DP, Black J, Bellone S, Lopez S, Cocco E, et al. Neratinib 2018;554:189–94. shows efficacy in the treatment of HER2 amplified carcinosarcoma in vitro and 10. Perera SA, Li D, Shimamura T, Raso MG, Ji H, Chen L, et al. HER2YVMA drives in vivo. Gynecol Oncol 2015;139:112–7. rapid development of adenosquamous lung tumors in mice that are sensitive to 29. Francia G, Man S, Lee CJ, Lee CR, Xu P, Mossoba ME, et al. Comparative impact BIBW2992 and rapamycin combination therapy. Proc Natl Acad Sci U S A 2009; of trastuzumab and cyclophosphamide on HER-2-positive human breast cancer 106:474–9. xenografts. Clin Cancer Res 2009;15:6358–66. 11. Besse B, Soria J, Yao B, Kris M, Chao B, Cortot A, et al. Neratinib (N) with or 30. Geoerger B, Kerr K, Tang CB, Fung KM, Powell B, Sutton LN, et al. Antitumor without temsirolimus (TEM) in patients (pts) with non-small cell lung cancer activity of the rapamycin analog CCI-779 in human primitive neuroectodermal (NSCLC) carrying HER2 somatic mutations: an international randomized phase tumor/medulloblastoma models as single agent and in combination chemo- II study. Ann Oncol 2014;25:v1–41. therapy. Cancer Res 2001;61:1527–32. 12. Li BT, Shen R, Buonocore D, Olah ZT, Ni A, Ginsberg MS, et al. Ado- 31. Bahcall M, Awad MM, Sholl LM, Wilson FH, Xu M, Wang S, et al. Amplification trastuzumab emtansine for patients with HER2-mutant lung cancers: results of wild-type KRAS imparts resistance to crizotinib in MET exon 14 mutant non- from a phase II basket trial. J Clin Oncol 2018;36:2532–7. small cell lung cancer. Clin Cancer Res 2018;24:5963–76. 13. Peters S, Stahel R, Bubendorf L, Bonomi P, Villegas A, Kowalski DM, et al. 32. Mazieres J, Peters S, Lepage B, Cortot AB, Barlesi F, Beau-Faller M, et al. Lung Trastuzumab emtansine (T-DM1) in patients with previously treated HER2- cancer that harbors an HER2 mutation: epidemiologic characteristics and overexpressing metastatic non-small cell lung cancer: efficacy, safety, and therapeutic perspectives. J Clin Oncol 2013;31:1997–2003. biomarkers. Clin Cancer Res 2019;25:64–72. 33. Scaltriti M, Verma C, Guzman M, Jimenez J, Parra JL, Pedersen K, et al. 14. Tiriac H, Belleau P, Engle DD, Plenker D, Deschenes A, Somerville TDD, et al. Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumu- Organoid profiling identifies common responders to chemotherapy in pancre- lation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity. Onco- atic cancer. Cancer Discov 2018;8:1112–29. gene 2009;28:803–14. 15. Liu JF, Palakurthi S, Zeng Q, Zhou S, Ivanova E, Huang W, et al. Establishment of 34. Sanchez-Martin M, Pandiella A. Differential action of small molecule HER patient-derived tumor xenograft models of epithelial ovarian cancer for pre- kinase inhibitors on receptor heterodimerization: therapeutic implications. Int J clinical evaluation of novel therapeutics. Clin Cancer Res 2017;23:1263–73. Cancer 2012;131:244–52. 16. Nikolic MZ, Caritg O, Jeng Q, Johnson JA, Sun D, Howell KJ, et al. Human 35. Collins DM, Gately K, Hughes C, Edwards C, Davies A, Madden SF, et al. embryonic lung epithelial tips are multipotent progenitors that can be expanded Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody- in vitro as long-term self-renewing organoids. Elife 2017;6.pii:e26575. dependent cell-mediated cytotoxicity in breast cancer cell lines. Cell Immunol 17. Shigematsu H, Takahashi T, Nomura M, Majmudar K, Suzuki M, Lee H, et al. 2017;319:35–42. Somatic mutations of the HER2 kinase domain in lung adenocarcinomas. 36. Bruna A, Rueda OM, Greenwood W, Batra AS, Callari M, Batra RN, et al. A Cancer Res 2005;65:1642–6. Biobank of Breast Cancer Explants with Preserved Intra-tumor Heterogeneity to 18. Jenkins RW, Aref AR, Lizotte PH, Ivanova E, Stinson S, Zhou CW, et al. Ex Vivo Screen Anticancer Compounds. Cell 2016;167:260–74. Profiling of PD-1 blockade using organotypic tumor spheroids. Cancer Discov 37. Karthaus WR, Iaquinta PJ, Drost J, Gracanin A, van Boxtel R, Wongvipat J, et al. 2018;8:196–215. Identification of multipotent luminal progenitor cells in human prostate orga- 19. Aref AR, Campisi M, Ivanova E, Portell A, Larios D, Piel BP, et al. 3D microfluidic noid cultures. Cell 2014;159:163–75. ex vivo culture of organotypic tumor spheroids to model immune checkpoint 38. Kris MG, Camidge DR, Giaccone G, Hida T, Li BT, O'Connell J, et al. Targeting blockade. Lab Chip 2018;18:3129–43. HER2 aberrations as actionable drivers in lung cancers: phase II trial of the pan-

2402 Clin Cancer Res; 26(10) May 15, 2020 CLINICAL CANCER RESEARCH

Preclinical HER2 Combination Therapy

HER tyrosine kinase inhibitor dacomitinib in patients with HER2-mutant or epidermal growth factor receptor 2-dependent and other solid tumors. J Clin ampliﬁed tumors. Ann Oncol 2015;26:1421–7. Oncol 2014;32:68–75. 39. Kosaka T, Tanizaki J, Paranal RM, Endoh H, Lydon C, Capelletti M, et al. 43. Robichaux JP, Elamin YY, Vijayan RSK, Nilsson MB, Hu L, He J, et al. Pan- Response heterogeneity of EGFR and HER2 exon 20 insertions to covalent EGFR cancer landscape and analysis of ERBB2 mutations identiﬁes poziotinib as a and HER2 inhibitors. Cancer Res 2017;77:2712–21. clinically active inhibitor and enhancer of T-DM1 activity. Cancer Cell 2019; 40. Swain SM, Baselga J, Kim SB, Ro J, Semiglazov V, Campone M, et al. Pertuzumab, 36:444–57. trastuzumab, and docetaxel in HER2-positive metastatic breast cancer. N Engl J 44. Regales L, Gong Y, Shen R, de Stanchina E, Vivanco I, Goel A, et al. Dual targeting Med 2015;372:724–34. of EGFR can overcome a major drug resistance mutation in mouse models of 41. Robert C, Karaszewska B, Schachter J, Rutkowski P, Mackiewicz A, Stroiakovski EGFR mutant lung cancer. J Clin Invest 2009;119:3000–10. D, et al. Improved overall survival in melanoma with combined dabrafenib and 45. Janjigian YY, Smit EF, Groen HJ, Horn L, Gettinger S, Camidge DR, et al. Dual trametinib. N Engl J Med 2015;372:30–9. inhibition of EGFR with afatinib and cetuximab in kinase inhibitor-resistant 42. Gandhi L, Bahleda R, Tolaney SM, Kwak EL, Cleary JM, Pandya SS, et al. Phase I EGFR-mutant lung cancer with and without T790M mutations. Cancer Discov study of neratinib in combination with temsirolimus in patients with human 2014;4:1036–45.

AACRJournals.org Clin Cancer Res; 26(10) May 15, 2020 2403

Use of Ex Vivo Patient-Derived Tumor Organotypic Spheroids to Identify Combination Therapies for HER2 Mutant Non−Small Cell Lung Cancer

Elena Ivanova, Mari Kuraguchi, Man Xu, et al.

Clin Cancer Res 2020;26:2393-2403. Published OnlineFirst February 7, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-1844

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2020/02/07/1078-0432.CCR-19-1844.DC1

Cited articles This article cites 45 articles, 19 of which you can access for free at: http://clincancerres.aacrjournals.org/content/26/10/2393.full#ref-list-1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/26/10/2393. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.