CANCER RESEARCH | TRANSLATIONAL SCIENCE Therapy-Induced Senescence Enhances the Efficacy of HER2-Targeted Antibody–Drug Conjugates in Breast Cancer Santiago Duro-Sánchez1,2,3,4, Mercedes Nadal-Serrano1,2, Marta Lalinde-Gutiérrez1,2,3,4, Enrique Javier Arenas1,2, Cristina Bernadó Morales1,2, Beatriz Morancho1,2, Marta Escorihuela1, Sandra Pérez-Ramos1, Santiago Escrivá-de-Romaní5, Lucía Gandullo-Sánchez2,5, Atanasio Pandiella2,6, Anna Esteve-Codina7,8, Vero nica Rodilla1, Fred A. Dijcks9, Wim H.A. Dokter9, Javier Cortés1, Cristina Saura , and Joaquín Arribas1,2,4,8,10 4 Downloaded from http://aacrjournals.org/cancerres/article-pdf/82/24/4670/3230524/4670.pdf by Autonomous University of Barcelona user on 06 February 2023 ABSTRACT ◥ Antibody–drug conjugates (ADC) are antineoplastic agents cancer cell lines. While senescence-inducing drugs did not recently introduced into the antitumor arsenal. T-DM1, a tras- increase the cytotoxic effect of ADCs on target cells, the bystander tuzumab-based ADC that relies on lysosomal processing to release effect was enhanced when HER2-negative cells were cocultured the payload, is approved for HER2-positive breast cancer. Next- with HER2-low cells. Knockdown experiments demonstrated the generation ADCs targeting HER2, such as [vic-]trastuzumab importance of cathepsin B in the enhanced bystander effect, duocarmazine (SYD985), bear linkers cleavable by lysosomal suggesting that cathepsin B mediates linker cleavage. In breast proteases and membrane-permeable drugs, mediating a bystander cancer patient-derived xenografts, a combination treatment of effect by which neighboring antigen-negative cells are eliminated. CDK4/6 inhibitor and SYD985 showed improved antitumor Many antitumor therapies, like DNA-damaging agents or CDK4/ effects over either treatment alone. These data support the 6 inhibitors, can induce senescence, a cellular state characterized strategy of combining next-generation ADCs targeting HER2 by stable cell-cycle arrest. Another hallmark of cellular senescence with senescence-inducing therapies for tumors with heterogenous is the enlargement of the lysosomal compartment. Given the and low HER2 expression. relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would Significance: Combining ADCs against HER2-positive breast potentiate the efficacy of HER2-targeting ADCs. Treatment with cancers with therapies that induce cellular senescence may improve the DNA-damaging agent doxorubicin and CDK4/6 inhibitor their therapeutic efficacy by facilitating a bystander effect against induced lysosomal enlargement and senescence in several breast antigen-negative tumor cells. Introduction DM1, Kadcyla), an ADC based on trastuzumab, is a therapeutic mAb that targets the tyrosine kinase receptor HER2. This was the Antibody–drug conjugates (ADC) are being rapidly incorporat- first ADC approved for use against a solid tumor, namely HER2- ed into the antitumor armory. Ado-trastuzumab emtansine (T- positive breast cancer. As the design of ADCs refined over time, their antitumor activity has become markedly enhanced (reviewed 1 in ref. 1). Recently, the next-generation ADC trastuzumab der- Preclinical and Translational Research Program, Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain. 2Centro de Investigacio n Biomedica en uxtecan (T-DXd, ENHERTU) received accelerated approval for the Red de C ancer (CIBERONC), Barcelona, Spain. 3Department of Biochemistry and treatment of patients with metastatic HER2-positive breast cancer Molecular Biology, Universitat Auto noma de Barcelona, Bellaterra, Spain. who had received two or more prior anti–HER2-based regi- 4 Cancer Research Program, Hospital del Mar Medical Research Institute (IMIM), mens (2). Results reported at ESMO 2021 also showed a marked Barcelona, Spain. 5Breast Cancer and Melanoma Group, Vall d’Hebron Institute increase in progression-free survival (PFS) in patients in early, of Oncology (VHIO), Vall d’Hebron University Hospital, Barcelona, Spain. second, and subsequent lines of treatment (3). Additional ADCs 6 Instituto de Biología Molecular y Celular del Cancer, CSIC, Salamanca, Spain. 7 targeting HER2 are currently in advanced stages of clinical devel- NAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology, Barcelona, Spain. 8Universitat Pompeu Fabra (UPF), Barcelona, opment, such as [vic-]trastuzumab duocarmazine (SYD985), Spain. 9Byondis B.V., Nijmegen, the Netherlands. 10Institucio  Catalana de RC48, and ARX788 (1). Recerca i Estudis Avançats (ICREA), Barcelona, Spain. HER2 is generally associated with breast and gastric cancers because S. Duro-S anchez and M. Nadal-Serrano contributed equally to this article. the corresponding gene (ERBB2) is amplified in approximately 15% of these tumors [source, cBioportal (4)]. However, it is also amplified in Corresponding Author: Joaquín Arribas, Cancer Research Program, Hospital del Mar Medical Research Institute (IMIM), Doctor Aiguader 88, Barcelona 08003, additional tumors. These include approximately 15% of urothelial and Spain. Phone: 349-3274-6026; E-mail: jarribas@imim.es esophageal carcinomas; approximately 10% of pancreatic cancers and uterine carcinosarcomas; approximately 5% of prostate cancers, cer- Cancer Res 2022;82:4670–9 vical squamous cell carcinomas, bladder urothelial carcinomas, uterine doi: 10.1158/0008-5472.CAN-22-0787 corpus endometrial carcinoma; and 3% of pediatric neuroblastomas, This open access article is distributed under the Creative Commons Attribution- colorectal adenocarcinomas, bladder cancers, acral melanomas, and NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license. lung squamous carcinomas (4). Overall, it is estimated that 3% of all 2022 The Authors; Published by the American Association for Cancer Research patients with cancer have tumors with amplifications of ERBB2 and AACRJournals.org | 4670 Effect of Therapy-Induced Senescence on ADCs Targeting HER2 may in turn potentially benefit from HER2-targeting therapies like described previously. HCC1954 (RRID:CVCL_1259) and T47D ADCs (4). (RRID:CVCL_0553) cells were maintained in RPMI medium (Gibco). In first-generation ADCs, such as T-DM1, the chemotherapeutic All media were supplemented with 10% FBS, 2 mmol/L L-glutamine, agent was bound to the antibody via an uncleavable linker (5). Thus, and 1% penicillin-streptomycin. Mycoplasma contamination was after binding to cell surface HER2 and subsequent internalization, tested monthly using the MycoAlert Mycoplasma Kit (Lonza). Cells T-DM1 required complete degradation in the lysosome to release its were not authenticated in-house and were not passaged more than antimicrotubule payload, emtansine (6). Because emtansine cannot 30 times. cross lipidic membranes, in principle, it only acts on the cell that has Knockdown of CTSB or CTSL was performed with short hairpin internalized T-DM1 (6). SYD985 is a next-generation ADC bearing a RNAs (shRNA) from lentiviral MISSION shRNA Library (SIGMA). potent duocarmycin-hydroxybenzamide-azaindole (DUBA) payload For detailed explanation, see Supplementary Materials and Methods. that alkylates DNA by binding to its minor groove. This ADC was the focus of the phase III TULIP clinical trial reported at ESMO 2021 that Reagents Downloaded from http://aacrjournals.org/cancerres/article-pdf/82/24/4670/3230524/4670.pdf by Autonomous University of Barcelona user on 06 February 2023 showed that SYD985 increased PFS compared with standard “Physi- Doxorubicin (Aurovitas), trastuzumab (Roche), T-DM1 (Roche), cian’s Choice” treatments for patients with HER2-positive metastatic and T-DXd (Daichii Sankyo) were obtained from a local pharmacy. breast cancer (7). Similar to other next-generation ADCs, SYD985 has SYD985 was obtained from Byondis B.V. Cdk4/6i palbociclib (LC a cleavable linker. This results in a payload that is more easily released Laboratories) and Ca-074Me (SIGMA) were purchased. from the targeting antibody, even outside the tumor cell (8). Most next- generation ADCs bear comparable peptide linkers designed to be Senescence induction and cell count cleaved by lysosomal proteases such as cathepsins (9). In addition, Cells were counted, plated in 10 cm plates, and treated the next day DUBA is able to permeate the plasma membrane upon release from the with varying doses of doxorubicin (4 days) or Cdk4/6i palbociclib antibody, enabling it to act on neighboring cells that are negative for (5 days). In oncogene-induced senescence, p95HER2 expression was the target antigen. This so-called “bystander effect” increases the induced via doxycycline removal for 5 days. At days 4 and/or 5, when efficacy of ADCs such as SYD985, especially on tumors with hetero- cells were fully senescent, cells were detached to remove nonviable cells geneous expression of the target (1). and were counted again. Collected cells were seeded in two plates, one The term cellular senescence refers to a broad range of heteroge- to analyze senescence-associated b-galactosidase (SA-b-gal) and the neous phenotypes that include cell-cycle arrest and an inflammation- other to test sensitivity to ADCs. Only cultures with >80% of cells promoting secretory phenotype. Senescence is triggered by a number positive for SA-b-gal were used. of signals, including oncogene expression, replicative stress, or expo- Where indicated, doxorubicin-treated cells were induced to express sure to a variety of chemicals (10). The observation that many common p95HER2 by removing doxycycline from the media and adding fresh antitumor therapies such as DNA-damaging agents (e.g., doxorubicin) media to the plates for 5 additional days. and Cdk4/6 inhibitors (Cdk4/6i, e.g., palbociclib) induce senescence has led to increasing interest in this distinct cellular phenotype and its SA-b-gal activity assay effect on antitumor efficacy (11). SA-b-gal activity was detected by colorimetric Senescence b-Galac- One of the hallmarks of cellular senescence is the enlargement of the tosidase Staining Kit (Cell Signaling Technology) following the man- lysosomal compartment (12). In fact, the most commonly used marker ufacturer’s indications. Chemiluminescent detection was performed of cellular senescence, beta-galactosidase (SA-b-gal) activity (13), is a using Galacto-Light Plus beta-Galactosidase Reporter Gene Assay surrogate marker of lysosomal enlargement (14). It is not clear whether System Kit (Applied Biosystems) but with a citric acid-sodium phos- this enlargement reflects increased lysosomal activity (15) or a hyper- phate buffer at pH 5.5. trophy of defective lysosomes caused by increasing reactive oxygen species levels (16). Stranded mRNA sequencing library preparation and Given the importance of lysosomal function in the mechanism of sequencing action of ADCs, in this article we present data aimed to clarify whether RNA was isolated with RNeasy Mini Kit (Qiagen). RNA sequencing therapies inducing senescence and, hence, potentially increasing lyso- (RNA-seq) libraries were prepared following the TruSeq Stranded somal function, affect the efficacy of ADCs targeting HER2. mRNA LT Sample Prep Kit protocol (Illumina) and validated on Agilent 2100 Bioanalyzer with DNA 7500 assay (Agilent). The RNA- seq libraries were sequenced on HiSeq 2000 (Illumina). Materials and Methods Patient-derived tumor xenografts RNA-seq data processing and analysis PDTX118 and PDTX251M were collected at Vall d’Hebron Uni- RNA-seq reads were mapped against the human genome (GRCh38) versity Hospital (Barcelona) following institutional guidelines. Our using STAR (29) with ENCODE parameters. Genes were quantified study was approved by Vall d’Hebron Institute of Oncology (VHIO) using RSEM. The gene annotation file was downloaded from gencode Animal Care and Use Committee in accordance with the Declaration release 28 and differential gene expression was performed with of Helsinki. Written informed consent was obtained from patients. DESeq2 (RRID:SCR_015687). Gene set enrichment analysis (GSEA) was performed using GSEA Cell lines v4.2.1 provided by the Broad Institute (RRID:SCR_003199) using MCF7 (RRID:CVCL_0031), MCF10A (RRID:CVCL_0598), BT474 predefined gene sets. Senescence (FRIDMAN_SENESCENCE_UP; (RRID:CVCL_0179), SKBR3 (RRID:CVCL_0033), HEK293T (RRID: broadinstitute.org/gsea/) and senescence-associated secretory pheno- CVCL_0063), and MDA-MB-231 (RRID:CVCL_0062) cell lines were type (SASP; ref. 20) signatures were obtained from published gene sets. purchased from ATCC and maintained in DMEM:F12 medium The lysosomal signature was obtained from Kyoto Encyclopedia of (Gibco). MDA-MB-231/luc cells, MCF7-TetOff/p95HER2 cells (17), Genes and Genomes database. Genes corresponding to these signa- MCF10A-HER2 (18), and BT474-R3 (19) were maintained as tures are shown in Supplementary Table SI. AACRJournals.org Cancer Res; 82(24) December 15, 2022 4671 Duro-Sánchez et al. Senescence reversibility assay Abcam. Quantification of CTSB IHC was performed using the When cells were senescent, we withdrew treatments and cultured image software QuPath (University of Edinburgh, Edinburgh, Scot- cells in fresh media, or cultured in doxycycline supplemented media. land) considering five image fields of representative tumors from Pictures were taken at indicated days. each group. Cell viability assays Flow cytometry Cells were seeded in 96-well plates and after 24 hours treated with Cells were harvested with accutase (Gibco), resuspended in blocking T-DM1 or SYD985 at described concentrations. Seven days later, cells buffer, incubated with trastuzumab and with anti-human AlexaFluor- viability was determined by crystal violet staining. 488 (Invitrogen). In the In vivo experiment, cells were incubated with EpCAM-AlexaFluor-647 (BioLegend). Cells were resuspended in Bystander effect assays Zombie Aqua viability marker and acquired on BD FACSCelesta (BD Cells were treated as described, collected, and plated in coculture Biosciences). Flow cytometry data were analyzed with FlowJo software with MDA-MB-231/luc cells at ratios of 5 or 10:1 in 96-well plates. The Downloaded from http://aacrjournals.org/cancerres/article-pdf/82/24/4670/3230524/4670.pdf by Autonomous University of Barcelona user on 06 February 2023 (BD BioSciences, RRID:SCR_008520). day after, cells were treated with T-DM1 (0.01, 0.03, or 0.82 nmol/L) or SYD985 (0.03 or 0.82 nmol/L) for 5 days or T-DXd (3.25 nmol/L) for Statistical analysis 7 days. Bystander cell death was measured using Luciferase Assay For in vitro experiments, data are presented as average  SD and System (Promega). were analyzed by two-sided Student t test. Results were considered Bystander assays employing increasing concentrations of T-DM1 statistically significant at P values <0.05. For in vivo experiments, mice and SYD985 were also performed with MCF7-TetOff/p95HER2 cells were randomly assigned to treatment groups and we used two-way overexpressing p95HER2. ANOVA with subsequent Bonferroni correction for analysis. We did not use statistical methods to predetermine sample size in animal qRT-PCR studies, but we made efforts to use the minimum number of animals. qRT-PCR was performed using TaqMan Gene Expression Probes Sample sizes of 3 to 4 mice per group were chosen and two flanks were and TaqMan Universal Master Mix II with UNG (Applied Biosystems, implanted to reduce the number of animals. All experiments were RRID:SCR_005039) following the manufacturer’s recommendations. repeated at least three times, except the in vivo experiments, and a Western blotting single representative experiment is shown. All measurements were Cells were lysed and sonicated for protein extraction. Protein performed at least in triplicate. extracts were quantified, resolved in SDS-PAGE gels, transferred onto nitrocellulose membranes (GE Healthcare Biosciences), and subjected Data availability statement to immunoblot analysis. For detailed explanation and list of antibodies, Data generated in this study are available within the article and see extended Materials and Methods. Supplementary Data or can be facilitated upon request to the corre- sponding author. Cathepsin B activity assay To measure the proteolytic activity of cathepsin B (CTSB), we used the Cathepsin B Activity Fluorometric Assay Kit (Biovision) according Results to the manufacturer’s instructions. Diverse inducers of senescence cause an increased lysosomal phenotype CTSB chemical inhibition We have previously shown that expression of the oncoprotein MCF7-TetOff/p95HER2 cells induced to express p95HER2 were p95HER2 induces a distinct senescence phenotype in MCF7 cells (17). seeded in 96-well plates and pretreated with the CTSB inhibitor CA- We compared this phenotype with those induced by certain antitumor 074-Me or vehicle. Next, MDA-MB-231/luc cells were plated onto therapies, namely the DNA-damaging compound doxorubicin and the MCF7-TetOff/p95HER2 cultures and cocultures were treated with Cdk4/6i palbociclib. As expected, both p95HER2 and the antitumor different concentrations of SYD985 for 5 days. Bystander cell death treatments markedly inhibited cell proliferation (Fig. 1A), and was measured as described previously. increased the number of cells positive for SA-b-gal activity (Fig. 1B and C). Transcriptomic analyses confirmed an enrichment in a defined In vivo analysis senescence signature in cells treated with doxorubicin or Cdk4/6i, Tumor fragments were implanted into the fourth fat pad of 6–8 weeks comparable with that observed in cells expressing p95HER2 (Fig. 1D, old NOD/SCID mice (Janvier, RRID:IMSR_CRL:394). b-estradiol was left; Supplementary Table S1). added to drinking water. For PDTXSR1 and PDTX251M, mice were Despite these similarities, the senescence phenotypes induced by treated intravenously with SYD985 (10 mg/kg), via oral gavage with p95HER2 and those induced by the antitumor treatments showed palbociclib (50 mg/kg, daily for 5 days), both or PBS. For the parental marked differences. For example, while the expression of p95HER2 or PDTX118 experiment, mice were treated intravenously with T-DM1 or treatment with doxorubicin induced a fully penetrant irreversible cell- SYD985 (both 10 mg/kg). Mice were sacrificed when tumor volume cycle arrest (see also Supplementary Fig. S1A; ref. 17), the cell-cycle approached 1,200–2,000 mm3 or due to unacceptable toxicity. Tumor arrest induced by Cdk4/6i is transient, as cells resumed proliferation samples were then collected for analyses. Mice were treated in accor- 1 week after the treatment was halted (Supplementary Fig. S1A). dance with the Ethical Committee for the Use of Experimental Animals Furthermore, the induction of a secretory phenotype was clearly more at the Vall d’Hebron Institute of Oncology. pronounced in p95HER2-induced senescent cells than in therapy- induced senescent cells (Supplementary Fig. S1B). In fact, some of the IHC analysis components of the secretory phenotype induced during p95HER2- IHC for HER2 was performed as described previously (19). For induced senescence, such as IL8, EPHA2, MMP1, or IL11, were not CTSB, slides were incubated overnight with primary antibody from secreted by therapy-induced senescent cells (Supplementary Fig. S1C). 4672 Cancer Res; 82(24) December 15, 2022 CANCER RESEARCH Effect of Therapy-Induced Senescence on ADCs Targeting HER2 A 20 B 4 D (normalized to day 0) Senescence signature Lysosomal signature *** SA-β-gal activity Cell number 15 3 0.6_ 0.6_ NES = 2.59 NES = 2.54 10 2 *** *** 0.3_ 0.3_ ** ** ** 5 1 0.0_ 0.0_ 0 0 Doxorubicin Vehicle Doxorubicin Vehicle 0.6_ 0.6_ NES = 2.21 NES = 2.62 Downloaded from http://aacrjournals.org/cancerres/article-pdf/82/24/4670/3230524/4670.pdf by Autonomous University of Barcelona user on 06 February 2023 C 0.3_ 0.3_ Vehicle Doxorubicin 0.0_ 0.0_ Cdk4/6i Vehicle Cdk4/6i Vehicle 0.4_ Enrichment score 0.4_ NES = 1.85 NES = 1.71 Cdk4/6i p95HER2 0.2_ 0.2_ 0.0_ 0.0_ −0.2_ p95HER2 Vehicle p95HER2 Vehicle Figure 1. Diverse senescence inducers increase lysosomal function. A, MCF7 cells were treated with vehicle, doxorubicin (50 nmol/L), the Cdk4/6i palbociclib (700 nmol/L) or induced to express p95HER2 for 4 or 5 days. Then, cells were counted; n ¼ 3. B, SA-b-gal activity was quantified using Galacto-Light Plus b-Galactosidase Reporter Gene Assay System Kit (Applied Biosystems) from cells treated as in A. Results were normalized to those of cells treated with vehicle; n ¼ 4. C, Representative pictures of SA-b-gal staining of MCF7 cells treated as in A. D, Transcriptomic profiles of cells treated as in A were determined by RNA-seq. Senescence and lysosomal GSEA signatures in treated cells were compared with that in cells treated with vehicle. P value corresponds to the NOM P value obtained by GSEA in the HALLMARK database.   , P < 0.01;    , P < 0.001. These differences are likely due to upregulation of secreted compo- (1þ by IHC analysis), now considered HER2-low. HER2-low tumors nents by p95HER2 signaling (see ref. 17). These p95HER2-induced include 50% of all breast cancers (21), and can be potentially targeted secreted factors are added to the components of the senescence- with trastuzumab (22, 23). In all cases, treatment with doxorubicin or associated secretory phenotype induced by other triggers. In agree- Cdk4/6i resulted in the inhibition of cellular proliferation and an ment with this conclusion, while MMP1 is not secreted upon doxo- increase in the number of SA-b-gal–positive cells, comparable to what rubicin treatment, expression of p95HER2 in doxorubicin-induced was observed in Fig. 1 (Supplementary Fig. S2D). senescent cells promoted its expression. In contrast, Lumican is The treatments with the inducers of senescence had little effect or expressed by senescent cells independently of p95HER2 expression decreased the sensitivity to the ADCs, irrespectively of the levels of (Supplementary Fig. S1D). HER2. In the majority of cases, treatments inducing senescence did not Notwithstanding these differences, transcriptomics revealed similar affect sensitivity to T-DM1 (Fig. 2). Only MCF10A cells overexpres- enrichments in lysosomal signatures across the various treatments sing HER2 and treated with the Cdk4/6i showed a higher sensitivity to (Fig. 1D), suggesting that the lysosomal enlargement is a common T-DM1 (Fig. 2). Similarly, the induction of cellular senescence did not consequence of senescence induction. Given the relevance of lyso- increase sensitivity to SYD985 (Fig. 2). somal function for the efficacy of ADCs, we analyzed the effect of To further assess the relevance of the lysosomal phenotype on the combining these with therapies that induce senescence. efficacy of ADCs, we used cells that are resistant to T-DM1 due to lysosomal dysfunction (24). Again, no improved response to SYD985 Effect of therapy-induced senescence on sensitivity to was observed in these cells upon treatment with Cdk4/6i (Supple- anti-HER2 ADCs mentary Fig S2C). We concluded that in the majority of cases For subsequent analyses, we induced cellular senescence in a panel senescence induction does not affect cellular sensitivity to ADCs, of breast cancer cell lines. Different concentrations of doxorubicin or despite the aforementioned lysosomal phenotype. Cdk4/6i were required to maximize the induction of cellular senes- Some degree of lysosomal enlargement may precede the full cell- cence (Supplementary Fig. S2A; Supplementary Table S2). The cell cycle arrest characteristic of cellular senescence. Because chemother- lines chosen expressed different levels of HER2 (Supplementary apeutics linked to ADCs are particularly active on proliferating cells, Fig. S2B), including cells with ERBB2 amplification and HER2 over- concentrations of doxorubicin or Cdk4/6i that induce a lysosomal expression (defined as 3þ according to IHC semiquantitative analy- enlargement but only a partial proliferation arrest may increase the sis). In addition, we used cell lines that have historically been con- activity of ADCs. Thus, we treated cells with different concentrations sidered HER2-negative but express readily detectable level of HER2 of doxorubicin or Cdk4/6i and measured lysosomal function using as a AACRJournals.org Cancer Res; 82(24) December 15, 2022 4673 Duro-Sánchez et al. T-DM1 SYD985 T-DM1 SYD985 IC50 IC50 IC50 IC50 Vehicle 0.70 Vehicle 0.01 Vehicle 0.06 Vehicle 0.08 Doxorubicin >0.5 Doxorubicin 0.02 Doxorubicin 0.07 Doxorubicin 0.07 % MCF10A-HER2 100 Cdk4/6 inh 100 0.012 Cdk4/6 inh 0.01 100 Cdk4/6 inh 0.08 100 Cdk4/6 inh 0.13 80 80 80 80 % BT474 60 60 60 60 40 40 40 40 20 20 20 20 0 0 0 0 0.0 0.1 0.5 0.0 0.5 0.0 0.1 0.5 0.0 0.1 1.0 10.0 Vehicle 0.02 Vehicle 0.27 Downloaded from http://aacrjournals.org/cancerres/article-pdf/82/24/4670/3230524/4670.pdf by Autonomous University of Barcelona user on 06 February 2023 100 100 120 Doxorubicin 0.04 Doxorubicin 0.08 120 Cdk4/6 inh 0.14 100 % HCC1954 80 Cdk4/6 inh 0.0780 100 % T47D 80 60 60 80 60 60 40 40 40 Vehicle 33.9 40 Vehicle 32.71 20 20 20 Doxorubicin >100 20 Doxorubicin >50 0 Cdk4/6 inh 178.6 Cdk4/6 inh >50 0 0 0 0.0 0.1 0.5 0.0 0.1 1.0 10.0 0.1 1.0 10.0 100.0 0.1 1.0 10.0 140 Vehicle 0.04140 Vehicle 0.11 100 120 Doxorubicin 1.85120 Doxorubicin 4.83 100 Cdk4/6 inh 0.09 Cdk4/6 inh 0.11 80 % SKBR3 100 100 % MCF7 80 80 80 60 60 60 60 40 40 40 40 Vehicle 13.91 Vehicle >50 20 Doxorubicin >50 20 Doxorubicin >50 20 20 Cdk4/6 inh 27.45 Cdk4/6 inh >50 0 0 0 0 0.0 0.1 0.5 0.0 0.0 0.1 1.0 10.0 0.0 0.1 0.5 5.0 50.0 0.0 0.1 0.5 5.0 50.0 [T-DM1] nmol/L [SYD985] nmol/L [T-DM1] nmol/L [SYD985] nmol/L Figure 2. Effect of doxorubicin or Cdk4/6i on the sensitivity to T-DM1 or SYD985. The indicated cell lines were treated with a concentration of doxorubicin (ranging from 12.5 to 50 nmol/L, depending on cell line, for 4 days) or Cdk4/6i (700 to 3,000 nmol/L for 5 days), replated and treated with different concentrations of T-DM1 or SYD985 for 7 additional days without doxorubicin or Cdk4/6i. Then, cell numbers were estimated with the Crystal Violet staining assay. surrogate marker glucosamine (N-Acetyl)-6-sulfatase (GNS; ref. 25). levels of the target antigen, the lysosomal compartment does not limit Certain concentrations of doxorubicin or Cdk4/6i led to an increased the bystander effect of ADCs with a cleavable linker, as amplifying it by expression of GNS, evidencing some degree of increased lysosomal inducing senescence does not have an effect. As expected, T-DM1 had activity, in cells still dividing, as judged by cell counting (Supplemen- no bystander effect in similar assays (Fig. 3B, bottom graphs). tary Fig. S3A and S3B). These concentrations of doxorubicin (10 and In contrast, cellular senescence amplified the bystander effect of 25 nmol/L) or Cdk4/6i (50 and 100 nmol/L) did not lower the IC50 of SYD985 on HER2-negative cells cocultured with cells expressing SYD985 or T-DM1 (Supplementary Fig. S3C and S3D). We concluded low levels of HER2 (MCF7 and T47D cells; Fig. 3C, top graphs). We that the lysosomal phenotype promoted by senescence-inducing confirmed that this effect was independent from the trigger that therapies, even in cells that remain proliferative, does not lead to induced senescence by analyzing the effect of oncogene-induced sensitization to HER2-directed ADCs. senescence. As shown in Supplementary Fig. S4A, p95HER2-induced senescent cells also amplified the SYD985 bystander effect. Again, Effect of cellular senescence on the bystander effect of ADCs T-DM1 did not have bystander effect on these cultures (Supplemen- Next, we asked whether the induction of senescence could increase tary Fig. S4). A similar effect was observed when analyzing the the bystander effect of SYD985. To address this possibility, we first bystander effect of T-DXd (Supplementary Fig S4B), arguing that the induced senescence in cells expressing different levels of HER2 by induction of cellular senescence can increase the antitumor activity of treating them with doxorubicin or Cdk4/6i. Then, we cocultured these different ADCs with cleavable linkers. senescent cells with the HER2-negative cell line MDA-MB-231 expres- We concluded that the bystander effect of ADCs with cleavable sing a luciferase reporter (Fig. 3A). linkers and membrane permeable drugs can be increased by inducing Likely due to the elevated expression of the antigen, we observed a cellular senescence in target cells with low levels of HER2 expression. similar bystander effect of senescent cells expressing high levels of HER2 (HCC1954 and SKBR3 cells) compared with vehicle-treated Role of cathepsins in the bystander effect controls on the killing of reporter MDA-MB-231/luc cells (Fig. 3B, Cathepsins, particularly CTSB, have been previously shown to upper graphs), as expected much higher than in HER2-low cells. Thus, cleave the peptide linker of certain ADCs, including SYD985 (8, 26). the lysosomal phenotype induced by the treatments did not alter the Analysis of the transcriptome of senescent cells induced by doxoru- bystander effect of senescent cells expressing high levels of HER2 bicin or Cdk4/6i showed an upregulation of cathepsins B and L (CTSL) treated with SYD985. This result indicated that, in cells expressing high expression (Fig. 4A). 4674 Cancer Res; 82(24) December 15, 2022 CANCER RESEARCH Effect of Therapy-Induced Senescence on ADCs Targeting HER2 A Doxorubicin or Cdk4/6i ADC MDA-MB-231 HCC1954 or SKBR3 MCF7 or T47D B HCC1954 C MCF7 T47D SKBR3 125 Downloaded from http://aacrjournals.org/cancerres/article-pdf/82/24/4670/3230524/4670.pdf by Autonomous University of Barcelona user on 06 February 2023 100 ns % MDA-MB-231 ns ns ns ** * 75 ** 50 ** 25 0 SYD985 (nmol/L) 0 0.03 0 0.03 0 0.03 0 0.03 0 0.03 0 0.03 0 0.82 0 0.82 0 0.82 0 0.82 0 0.82 0 0.82 125 100 % MDA-MB-231 75 50 25 0 T-DM1 (nmol/L) 0 0.01 0 0.01 0 0.01 0 0.03 0 0.03 0 0.03 0 0.82 0 0.82 0 0.82 0 0.82 0 0.82 0 0.82 Vehicle Doxor. Cdk4/6i Vehicle Doxor. Cdk4/6i Vehicle Doxor. Cdk4/6i Vehicle Doxor. Cdk4/6i Figure 3. Effect of doxorubicin or Cdk4/6i on the bystander effect of SYD985. A, Schematic showing the experimental procedure. The indicated cells were treated with doxorubicin or Cdk4/6i for 4 or 5 days, respectively. Then, cells were harvested and cococultured with MDA-MB-231 cells expressing luciferase and cocultures were treated with SYD985 or T-DM1 for 5 additional days. At the end of the experiment, MDA-MB-231/luc cells were quantified by luminescence. B and C, Luminescence, quantified as described in A, was determined at the end of the experiments and normalized to cells treated with vehicle. n ≥ 3. ns, nonsignificant;  , P < 0.05;   , P < 0.01. To determine the functional relevance of these cathepsins, we HER2 (Fig. 5A). This HER2 status resembles those in the coculture knocked down their expression with shRNAs and functionally experiments shown in Fig. 3. We therefore selected PDTX SR1 as our characterized the resulting cells. While the knockdown of CTSB model to test the efficacy of the combination in vivo. impaired the bystander effect of SYD985 (Fig. 4B and C), the To efficiently monitor cellular senescence in in vivo experiments, knockdown of CTSL had no effect (Fig. 4D and E). Analysis of we recently generated PDTXs expressing GFP and Cherry reporters protein levels confirmed that the induction of senescence by the under the control of human p16 and IL6 promoters, respectively antitumor therapies upregulated CTSB (Fig. 4F and G) as well as (Fig. 5B). Previous characterization of similar reporters showed increased its activity (Fig. 4H). Note that, based on previous that their simultaneous expression is a bone fide marker of cellular reports (27), we identified the 34 and 24 kDa bands detected by senescence (28). Western blot as lysosomal active forms of CTSB, the mature While treatment with SYD985 or the Cdk4/6i had no effect on precursor and the final form, respectively. tumor growth, the combination had a significant antitumor effect The p95HER2-induced senescence model further confirmed the (Fig. 5C). Analysis of the percentage of cells double positive for the relevance of CTSB, as treatment with the irreversible CTSB-specific GFP and Cherry reporters showed that, as expected, the number of inhibitor CA-074Me impaired the observed bystander effect (Sup- double positive cells increased with Cdk4/6i treatment (Fig. 5D). plementary Fig. S5A). The results of these experiments in MCF7 Interestingly, the percentage of senescent cells in tumors treated with cells were successfully recapitulated in T47D cells, confirming the the combination was augmented to approximately 40%, further association between CTSB and bystander killing (Supplementary highlighting the preferential effect of DUBA on proliferating cells. Fig. S5B–S5F). Increased CTSB expression was also observed in vivo upon treatment with Cdk4/6i (Fig. 5E and F). Combination of therapy-induced senescence with ADCs To further support the efficacy of the combination, we analyzed its Using a patient-derived tumor xenograft (PDTX) sensitive to effect on a PDTX primarily resistant to SYD985 (PDTX251M) con- T-DM1 and SYD985 (Supplementary Fig. S6A), we previously gen- sidered HER2-high (Supplementary Fig. S6B and S6C). Although the erated several models of resistance to T-DM1 (19). While some of these mechanism behind this resistance is unknown, treatment with the models are sensitive to SYD985, one of them, SR1, shows resistance to Cdk4/6i also sensitized this model to SYD985 (Supplementary both ADCs and displays a lower and more heterogenous expression of Fig. S6C). AACRJournals.org Cancer Res; 82(24) December 15, 2022 4675 Duro-Sánchez et al. Discussion A 4 * The term senescence is used to define cells with diverse phenotypes Relative mRNA ** Vehicle 3 that have in common a stable cell-cycle arrest and the ability to secrete ** Doxorubicin * a collection of molecules, which include several inflammatory fac- 2 Cdk4/6i tors (29). The only undisputed marker for cellular senescence, SA- 1 b-gal activity, provides qualitative rather than exact results. While cells 0 with the characteristic strong blue staining are undoubtedly consid- ered senescent, there is no threshold of activity to clearly identify CTSB CTSK CTSL CTSS senescent cells with lower intensity of staining. Because of this shortcoming, the search for specific markers of senescent cells is B 1.3 C Vector shCTSB-1shCTSB-2 actively ongoing (30). 125 ns ns ns ns CTSB (mRNA) 1.0 100 The positivity of SA-b-gal activity likely does not identify a homo- Downloaded from http://aacrjournals.org/cancerres/article-pdf/82/24/4670/3230524/4670.pdf by Autonomous University of Barcelona user on 06 February 2023 ** * geneous phenotype but rather a variety of them. As described previ- % Cells 0.8 75 ously (31, 32), and as shown here, cell-cycle arrest induced by Cdk4/6 0.5 50 inhibition is largely reversible, in contrast with that induced by the 0.3 25 oncogene p95HER2 or the severe DNA damage resulting from doxo- 0.0 0 rubicin. Furthermore, as shown here, the SASP, which is considered a hallmark of cellular senescence, also varies markedly with different triggers of senescence. In contrast with the SASP transcriptomic signature, the signature of increased lysosomal function is similarly enriched in senescent cells D E Vector shCTSL induced by the different triggers. This result likely reflects increased 1.2 125 lysosomal activity, just as SA-b-gal does. In fact, the beta-galactosidase CTSL (mRNA) activity detectable at acidic pH, defined as SA-b-gal, is due to the action 0.9 100 * ** of a lysosomal beta-galactosidase encoded by GLB1 (14). While the 75 ** * % Cells 0.6 hypertrophic lysosomes of senescent cells have been considered 50 0.3 defective by some authors (16), the increased SA-b-gal activity, along 25 with our transcriptomic analysis and the analysis of CTSB activity, 0.0 0 indicates that it corresponds to increased lysosomal function. In summary, increased lysosomal function seems to constitute one of the most defining phenotypes of senescent cells. Following the success of next-generation HER2-directed ADCs on HER2-amplified breast cancers, these therapies are now being tested in F G H 250 patients with HER2-low breast cancer with promising effects. While 6 * kDa * ** the first-generation ADC T-DM1 showed a poorer response in HER2- CTSB (activity) 5 CTSB (protein) CTSB 200 - 35 ** low tumors, ADCs with a cleavable linker such as T-DXd have Mature 4 150 demonstrated effectivity in these low HER2-expressing tumors in 3 - 25 100 clinical trials (33), similar to the results obtained with SYD985 (8). Final 2 50 Despite these positive results, all responses were transient, under- 1 scoring the need to improve the activity of these ADCs. Given the 0 0 GAPDH relevance of the lysosomal function in the mechanism of action of ADCs, the analysis of the efficacy of combinations of ADCs and therapies inducing senescence continues to be warranted. The results presented here clearly show that lysosomal function is not a limiting factor of the efficacy of ADCs on target cells. This Figure 4. conclusion is firmly supported by the fact that the increase in the Role of cathepsins on the bystander effect of SYD985. A, Levels of the indicated cathepsins were determined by qRT-PCR in MCF7 cells treated as in Fig. 1A. n ≥ 3. lysosomal function induced by different triggers of senescence does not B, Levels of mRNA encoding CTSB in parental MCF7 cells or the same cells stably affect the sensitivity of a variety of cells expressing different levels of transfected with the empty vector or two different shRNA (sh) targeting CTSB. HER2 to T-DM1 or SYD985. Furthermore, the lysosomal activity is Results were normalized to parental cells. n ¼ 3. C, As described in Fig. 3A, also not limiting for the bystander effect of SYD985 on cells expressing cocultures were performed to analyze the bystander effect of SYD985 upon high levels for HER2. However, the lysosomal activity of senescent knockdown of CTSB. n ¼ 3. D and E, The role of cathepsin L on the bystander cells, and the subsequent upregulation of the SYD985 valine-citrulline effect of SYD985 was analyzed as in B and C. F–H, MCF7 cells treated as indicated were lysed and lysates analyzed by Western blot analysis with anti- linker-cleaving enzyme CTSB (26), does enhance the bystander effect CTSB antibodies (F and G) or processed to determine CTSB activity with a of SYD985 on cells expressing low levels of HER2. This conclusion, specific assay (H). n ¼ 3.  , P < 0.05;   , P < 0.01. based on in vitro assays, was confirmed by using a SYD985-resistance PDTX model with low and heterogenous expression of HER2. This Collectively, the results presented here show that the combina- result opens the possibility for efficacious combinations of ADCs tion of therapies inducing senescence with ADCs bearing cleavable targeting HER2 together with therapies inducing senescence, partic- linkers is effective against heterogeneous tumors expressing low ularly in breast and gastric tumors that express lower levels of HER2 levels of the targeted antigen HER2 as well as against some defined by IHC (21, 34, 35). A future challenge to be tested in the clinic primarily resistant tumors. will be not only to confirm the potential efficacy of these combinations 4676 Cancer Res; 82(24) December 15, 2022 CANCER RESEARCH Effect of Therapy-Induced Senescence on ADCs Targeting HER2 A PDTX118 (parental) SR1 B HER2 p16 promoter-GFP IL6 promoter-Cherry 25 µm 25 µm C Vehicle Cdk4/6i D 80 * Tumor volume (mm3) 2,000 2000 Downloaded from http://aacrjournals.org/cancerres/article-pdf/82/24/4670/3230524/4670.pdf by Autonomous University of Barcelona user on 06 February 2023 % p16+/IL6+ cells Vehicle Vehicle 60 1,600 SYD985 1600 SYD985 1,200 1200 40 ** 800 800 P = 0.009 20 400 400 0 0 0 12 16 20 24 28 12 16 20 24 28 SYD985 − + − + Time (days) Time (days) Cdk4/6i − − + + E Cdk4/6i F 100 *** Vehicle % CTSB+ cells Vehicle SYD985 Vehicle SYD985 80 60 CTSB 40 20 25 µm 0 SYD985 -− ++ − - + + Cdk4/6i − − + + Figure 5. Effect of the combination Cdk4/6i-SYD985 on a PDTX resistant to SYD985. A, IHC analysis of HER2 in the parental PDTX or SR1, the corresponding resistant PDTX derived from it. B, Schematic showing the generation of SR1 tumors expressing reporters under the control of human p16 or IL6 promoters. Briefly, cells from SR1 were transiently cultured, infected with virus encoding the reporters as described in ref. 25, and reimplanted into immunodeficient mice. C, PDTXs expressing the reporters described in B were implanted into NOD.SCID mice and volumes were monitored periodically. As indicated by the arrow, control group (treated with vehicle) and Cdk4/6i group (treated with palbociclib at 50 mg/kg/day) were treated with 10 mg/kg of SYD985 in single dose fashion. Narrow lines represent the growth of individual tumors, wide lines represent averages. D, At the end of the experiment, the number of double-positive cells for IL6 and p16 was quantified from independent tumors by flow cytometry. n ¼ 5. E and F, IHC analysis of CTSB in tumors treated as in C. Results from five different fields were quantified and the results are shown as averages  SD.  , P < 0.05;   , P < 0.01;   , P < 0.001. but also the toxicity profile and, based on that, define whether the and F Hoffmann-La Roche Ltd; personal fees from Pfizer, Pierre-Fabre, Seagen, strategy represents a positive balance to improve the treatment of Novartis, and Kern; and grants from Byondis, Zymeworks, Synthon, MedSir, and Solti outside the submitted work. A. Pandiella reports personal fees from Daiichi patients. In addition, we showed that the combination of SYD985 and Sankyo and Seagen outside the submitted work. J. Cortes reports personal fees from therapies inducing senescence is effective on some resistant models Roche, Celgene, Cellestia, AstraZeneca, Seattle Genetics, Daiichi Sankyo, Erytech, (Supplementary Fig. S6C), but not others (Supplementary Fig. S2C) Athenex, Polyphor, Lilly, Merck Sharp & Dohme, GSK, Leuko, Bioasis, Clovis In conclusion, our data strongly support a strategy combining next- Oncology, Boehringer Ingelheim, Elipses, Hibercell, BioInvent, Gemoab, Gilead, generation ADCs, carrying cleavable linkers and membrane permeable Menarini, Zymeworks, Reveal Genomics, Roche, Novartis, Eisai, Pfizer, Daiichi payloads, with therapies that induce cellular senescence specifically Sankyo, AstraZeneca, Gilead, Novartis, and Samsung Bioepis and grants from Roche, Ariad Pharmaceuticals, AstraZeneca, Baxalta GMBH/Servier Affaires, Bayer coupled to augmented lysosomal activity. This latter function is likely Healthcare, Eisai, F. Hoffman-La Roche, Guardanth Health, Merck Sharp & Dohme, to drive an enhanced bystander effect in low target-expressing cells that Pfizer, Piqur Therapeutics, Puma C, and Queen Mary University of London outside may result in an overall increased efficacy of antitumor treatment. the submitted work; in addition, J. Cortes has a patent for Pharmaceutical Combinations of A Pi3k Inhibitor And A Microtubule Destabilizing Agent (Javier Cortes Castan, Alejandro Piris Gimenez, Violeta Serra Elizalde. WO 2014/199294 Authors’ Disclosures A) issued and a patent for Her2 as a predictor of response to dual HER2 blockade in S. Duro-Sanchez reports grants from Byondis B.V. during the conduct of the the absence of cytotoxic therapy (Aleix Prat, Antonio Llombart, Javier Cortes. US study. M. Lalinde-Gutierrez reports grants from Byondis B.V during the conduct of 2019/ 0338368 A1) licensed. C. Saura reports personal fees from AstraZeneca, the study. S. Perez-Ramos reports grants from Byondis B.V. during the conduct of Gilead, Daiichi Sankyo, Seagen Int., Eisai Europe Ltd., Phillips Health Works, Pfizer the study. S. Escriva-de-Romaní reports grants from Byondis B.V. during the Inc., Solti, Puma Biotechnology Inc., Pierre Fabre, Roche Farma, SA, and Novartis; conduct of the study; grants and personal fees from AstraZeneca Daiichi Sankio, and nonfinancial support from Lilly SAU, Pierre Fabre, AstraZeneca, Exeter Pharma AACRJournals.org Cancer Res; 82(24) December 15, 2022 4677 Duro-Sánchez et al. LLC, AX’s Consulting SARL, AX’s Consulting SARL, and MediTech outside the Acknowledgments submitted work. J. Arribas reports grants from Breast Cancer Research Foundation, This work was supported by Breast Cancer Research Foundation (BCRF-20-008), Instituto de Salud Carlos III, ERA-NET TRANSCAN2, Asociacion Espa~ nola Contra el Cancer, Byondis, and Fundacion Mutua Madrile~ na during the conduct of the Instituto de Salud Carlos III (project reference numbers AC15/00062, CB16/12/00449 study and grants from Roche and Molecular Partners outside the submitted work; in and PI19/01181), the EC under the framework of the ERA-NET TRANSCAN-2 addition, J. Arribas has a patent for EP22382294.1 issued, a patent for EP20382457.8 initiative co-financed by FEDER, Fundacion Mutua Madrile~ na and Asociación Española Contra el Cáncer. S. Duro-Sanchez is supported by the Spanish Ministerio licensed to Mnemo Therapeutics, and a patent for EP2360187B1 licensed to de Universidades by the grant Formacion de Profesorado Universitario (FPU20/ Innovent. No disclosures were reported by the other authors. 05388). A. Esteve-Codina is funded by ISCIII /MINECO (PT17/0009/0019) and co-funded by FEDER. The authors acknowledge Alyson MacInnes for reviewing Authors’ Contributions and editing the article. S. Duro-Sanchez: Conceptualization, investigation, methodology, writing–original draft, writing–review and editing. M. Nadal-Serrano: Conceptualization, investigation, The publication costs of this article were defrayed in part by the methodology. M. Lalinde-Gutierrez: Investigation. E.J. Arenas: Project administra- payment of publication fees. Therefore, and solely to indicate this fact, Downloaded from http://aacrjournals.org/cancerres/article-pdf/82/24/4670/3230524/4670.pdf by Autonomous University of Barcelona user on 06 February 2023 tion. C. Bernad o Morales: Project administration. B. Morancho: Investigation, project this article is hereby marked “advertisement” in accordance with 18 USC administration. M. Escorihuela: Resources, investigation. S. Perez-Ramos: Investiga- section 1734. tion. S. Escriva-de-Romaní: Resources, investigation. L. Gandullo-Sanchez: Inves- tigation. A. Pandiella: Resources, investigation, writing–review and editing. A. Esteve-Codina: Resources, investigation, writing–review and editing. V. Rodilla: Note Resources. F.A. Dijcks: Resources, writing–review and editing. W.H.A. Dokter: Supplementary data for this article are available at Cancer Research Online (http:// Conceptualization, resources, supervision, funding acquisition, visualization, cancerres.aacrjournals.org/). writing–original draft, project administration, writing–review and editing. J. Cortes: Funding acquisition, investigation. C. Saura: Resources, investigation. J. Arribas: Conceptualization, resources, supervision, funding acquisition, visualization, Received March 18, 2022; revised August 19, 2022; accepted October 10, 2022; writing–original draft, project administration, writing–review and editing. published first October 12, 2022. References 1. Pegram MD, Miles D, Tsui CK, Zong Y. 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