Genomic characterization of metastatic breast cancers.

Metastasis is the main cause of death for patients with breast cancer. Many studies have characterized the genomic landscape of breast cancer during its early stages. However, there is evidence that genomic alterations are acquired during the evolution of cancers from their early to late stages, and that the genomic landscape of early cancers is not representative of that of lethal cancers1-7. Here we investigated the landscape of somatic alterations in 617 metastatic breast cancers. Nine driver genes (TP53, ESR1, GATA3, KMT2C, NCOR1, AKT1, NF1, RIC8A and RB1) were more frequently mutated in metastatic breast cancers that expressed hormone receptors (oestrogen and/or progesterone receptors; HR+) but did not have high levels of HER2 (HER2-; n = 381), when compared to early breast cancers from The Cancer Genome Atlas. In addition, 18 amplicons were more frequently observed in HR+/HER2- metastatic breast cancers. These cancers showed an increase in mutational signatures S2, S3, S10, S13 and S17. Among the gene alterations that were enriched in HR+/HER2- metastatic breast cancers, mutations in TP53, RB1 and NF1, together with S10, S13 and S17, were associated with poor outcome. Metastatic triple-negative breast cancers showed an increase in the frequency of somatic biallelic loss-of-function mutations in genes related to homologous recombination DNA repair, compared to early triple-negative breast cancers (7% versus 2%). Finally, metastatic breast cancers showed an increase in mutational burden and clonal diversity compared to early breast cancers. Thus, the genomic landscape of metastatic breast cancer is enriched in clinically relevant genomic alterations and is more complex than that of early breast cancer. The identification of genomic alterations associated with poor outcome will allow earlier and better selection of patients who require the use of treatments that are still in clinical trials. The genetic complexity observed in advanced breast cancer suggests that such treatments should be introduced as early as possible in the disease course.

Author Correction: Genomic characterization of metastatic breast cancers.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

In skeletal muscle and neural crest cells, SMCHD1 regulates biological pathways relevant for Bosma syndrome and facioscapulohumeral dystrophy phenotype.

Many genetic syndromes are linked to mutations in genes encoding factors that guide chromatin organization. Among them, several distinct rare genetic diseases are linked to mutations in SMCHD1 that encodes the structural maintenance of chromosomes flexible hinge domain containing 1 chromatin-associated factor. In humans, its function as well as the impact of its mutations remains poorly defined. To fill this gap, we determined the episignature associated with heterozygous SMCHD1 variants in primary cells and cell lineages derived from induced pluripotent stem cells for Bosma arhinia and microphthalmia syndrome (BAMS) and type 2 facioscapulohumeral dystrophy (FSHD2). In human tissues, SMCHD1 regulates the distribution of methylated CpGs, H3K27 trimethylation and CTCF at repressed chromatin but also at euchromatin. Based on the exploration of tissues affected either in FSHD or in BAMS, i.e. skeletal muscle fibers and neural crest stem cells, respectively, our results emphasize multiple functions for SMCHD1, in chromatin compaction, chromatin insulation and gene regulation with variable targets or phenotypical outcomes. We concluded that in rare genetic diseases, SMCHD1 variants impact gene expression in two ways: (i) by changing the chromatin context at a number of euchromatin loci or (ii) by directly regulating some loci encoding master transcription factors required for cell fate determination and tissue differentiation.

Targeted molecular characterization shows differences between primary and secondary myelofibrosis.

INTRODUCTION: In BCR-ABL1-negative myeloproliferative neoplasms, myelofibrosis (MF) is either primary (PMF) or secondary (SMF) to polycythemia vera or essential thrombocythemia. MF is characterized by an increased risk of transformation to acute myeloid leukemia (AML) and a shortened life expectancy. METHODS: Because natural histories of PMF and SMF are different, we studied by targeted next generation sequencing the differences in the molecular landscape of 86 PMF and 59 SMF and compared their prognosis impact. RESULTS: PMF had more ASXL1 (47.7%) and SRSF2 (14%) gene mutations than SMF (respectively 27.1% and 3.4%, P = .04). Poorer survival was associated with RNA splicing mutations (especially SRSF2) and TP53 in PMF (P = .0003), and with ASXL1 and TP53 mutations in SMF (P < .0001). These mutations of poor prognosis were associated with biological features of scoring systems (DIPSS and MYSEC-PM score). Mutations in TP53/SRSF2 in PMF or TP53/ASXL1 in SMF were more frequent as the risk of these scores increased. This allowed for a better stratification of MF patients, especially within the DIPSS intermediate-1 risk group (DIPSS) or the MYSEC-PM high risk group. AML transformation occurred faster in SMF than in PMF and patients who transformed to AML were more SRSF2-mutated and less CALR-mutated at MF sampling. CONCLUSIONS: PMF and SMF have different but not specific molecular profiles and different prognosis depending on the molecular profile. This may be due to differences in disease history. Combining mutations and existing scores should improve prognosis assessment.

Common origin of sequential cutaneous CD30+ lymphoproliferations with nodal involvement evidenced by genome-wide clonal evolution.

AIMS: This study sought to clarify the molecular pathways underlying the putative evolution from lymphomatoid papulosis (LyP) to cutaneous anaplastic large-cell lymphoma (c-ALCL) and lymph node invasion (LNI). METHODS AND RESULTS: We analysed nine sequential tumours from the same patient presenting with parallel evolution of LyP (n = 3) and c-ALCL (n = 1) with LNI (n = 1), combined with systemic diffuse large B-cell lymphoma (DLBCL) (n = 4). Clonality analysis showed a common clonal T-cell origin in the five CD30+ lesions, and a common clonal B-cell origin in the four DLBCL relapses. Array-comparative genomic hybridisation and targeted next-generation sequencing analysis demonstrated relative genomic stability of LyP lesions as compared with clonally related anaplastic large-cell lymphoma (ALCL) tumours, which showed 4q and 22q13 deletions involving the PRDM8 and TIMP3 tumour suppressor genes, respectively. The three analysed CD30+ lesions showed mostly private (specific to each sample) genetic alterations, suggesting early divergence from a common precursor. In contrast, DLBCL tumours showed progressive accumulation of private alterations, indicating late divergence. CONCLUSIONS: Sequential cutaneous and nodal CD30+ tumours were clonally related. This suggests that LyP, c-ALCL and LNI represent a continuous spectrum of clonal evolution emerging from a common precursor of cutaneous CD30+ lymphoproliferations. Therefore, nodal ALCL tumours in the context of LyP should be considered as a form of transformation rather than composite lymphoma.

Stromal Expression of MARCKS Protein in Ovarian Carcinomas Has Unfavorable Prognostic Value.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Identification of new therapeutic targets is crucial. MARCKS, myristoylated alanine-rich C-kinase substrate, has been implicated in aggressiveness of several cancers and MARCKS inhibitors are in development. Using immunohistochemistry (IHC), we retrospectively assessed MARCKS expression in epithelial and stromal cells of 118 pre-chemotherapy EOC samples and 40 normal ovarian samples from patients treated at Salah Azaiez Institute. We compared MARCKS expression in normal versus cancer samples, and searched for correlations with clinicopathological features, including overall survival (OS). Seventy-five percent of normal samples showed positive epithelial MARCKS staining versus 50% of tumor samples (p = 6.02 × 10-3). By contrast, stromal MARCKS expression was more frequent in tumor samples (77%) than in normal samples (22%; p = 1.41 × 10-9). There was no correlation between epithelial and stromal IHC MARCKS statutes and prognostic clinicopathological features. Stromal MARCKS expression was correlated with shorter poor OS in uni- and multivariate analyses. Stromal MARCKS overexpression in tumors might contribute to cancer-associated fibroblasts activation and to the poor prognosis of EOC, suggesting a potential therapeutic interest of MARCKS inhibition for targeting the cooperative tumor stroma.

Brief reports: A distinct DNA methylation signature defines breast cancer stem cells and predicts cancer outcome.

Self-renewal and differentiation are two epigenetic programs that regulate stem cells fate. Dysregulation of these two programs leads to the development of cancer stem cells (CSCs). Recent evidence suggests that CSCs are relatively resistant to conventional therapies and responsible for metastasis formation. Deciphering these processes will help understand oncogenesis and allow the development of new targeted therapies. Here, we have used a whole genome promoter microarray to establish the DNA methylation portraits of breast cancer stem cells (bCSCs) and non-bCSCs. A total of 68 differentially methylated regions (DMRs) were more hypomethylated in bCSCs than in non-bCSCs. Using a differentiation assay we demonstrated that DMRs are rapidly hypermethylated within the first 6 hours following induction of CSC differentiation whereas the cells reached the steady-state within 6 days, suggesting that these DMRs are linked to early CSC epigenetic regulation. These DMRs were significantly enriched in genes coding for TGF-ß signaling-related proteins. Interestingly, DMRs hypomethylation was correlated to an overexpression of TGF-ß signaling genes in a series of 109 breast tumors. Moreover, patients with tumors harboring the bCSC DMRs signature had a worse prognosis than those with non-bCSC DMRs signature. Our results show that bCSCs have a distinct DNA methylation landscape with TGF-ß signaling as a key epigenetic regulator of bCSCs differentiation.

Claudin-low breast cancers: clinical, pathological, molecular and prognostic characterization.

BACKGROUND: The lastly identified claudin-low (CL) subtype of breast cancer (BC) remains poorly described as compared to the other molecular subtypes. We provide a comprehensive characterization of the largest series of CL samples reported so far. METHODS: From a data set of 5447 invasive BC profiled using DNA microarrays, we identified 673 CL samples (12,4%) that we describe comparatively to the other molecular subtypes at several levels: clinicopathological, genomic, transcriptional, survival, and response to chemotherapy. RESULTS: CL samples display profiles different from other subtypes. For example, they differ from basal tumors regarding the hormone receptor status, with a lower frequency of triple negative (TN) tumors (52% vs 76% for basal cases). Like basal tumors, they show high genomic instability with many gains and losses. At the transcriptional level, CL tumors are the most undifferentiated tumors along the mammary epithelial hierarchy. Compared to basal tumors, they show enrichment for epithelial-to-mesenchymal transition markers, immune response genes, and cancer stem cell-like features, and higher activity of estrogen receptor (ER), progesterone receptor (PR), EGFR, SRC and TGFß pathways, but lower activity of MYC and PI3K pathways. The 5-year disease-free survival of CL cases (67%) and the rate of pathological complete response (pCR) to primary chemotherapy (32%) are close to those of poor-prognosis and good responder subtypes (basal and ERBB2-enriched). However, the prognostic features of CL tumors are closer to those observed in the whole BC series and in the luminal A subtype, including proliferation-related gene expression signatures (GES). Immunity-related GES valuable in basal breast cancers are not significant in CL tumors. By contrast, the GES predictive for pCR in CL tumors resemble more to those of basal and HER2-enriched tumors than to those of luminal A tumors. CONCLUSIONS: Many differences exist between CL and the other subtypes, notably basal. An unexpected finding concerns the relatively high numbers of ER-positive and non-TN tumors within CL subtype, suggesting a larger heterogeneity than in basal and luminal A subtypes.

ESPL1 is a candidate oncogene of luminal B breast cancers.

ESPL1/separase is a putative oncogene of luminal B breast cancers. Histoclinical correlations of its expression have never been explored in large series of breast tumors, and specifically in the luminal subtype. In a pooled series of invasive breast carcinomas profiled using DNA microarrays, we identified 3,074 luminal cases, including 1,307 luminal B tumors, in which we searched for correlations between ESPL1 mRNA expression and molecular and histoclinical features. Compared to normal breast samples, ESPL1 was overexpressed in 52 % of luminal tumors, and much more frequently in luminal B (83 %) than luminal A tumors (29 %). In luminal breast cancers, higher ESPL1 expression was associated with poor-prognosis criteria (age ≤ 50 years, ductal type, advanced stage, large tumor size, lymph node-positive status, high grade, PR-negative status, luminal B subtype) and with poor metastasis-free survival in both uni- and multivariate analyses. This independent prognostic value was also observed in luminal B tumors only, and persisted when compared with gene expression signatures (PAM50, Recurrence Score, Mammaprint, EndoPredict) currently proposed to refine the indications of adjuvant chemotherapy in hormone receptor-positive/HER2-negative breast cancer. We also confirmed the observations made with experimental mouse models: ESPL1-overexpressing luminal tumors showed complex genomic profiles and molecular features of chromosomal instability and loss of tumor suppressor genes (P53 and Rb). Our results reinforce the idea that ESPL1 is a candidate oncogene in luminal B cancers. Its expression may help improve the prognostication. Inhibiting ESPL1 may represent a promising therapeutic approach for these poor-prognosis tumors.

Early lesions of follicular lymphoma: a genetic perspective.

The pathogenesis of follicular lymphoma is a multi-hit process progressing over many years through the accumulation of numerous genetic alterations. Besides the hallmark t(14;18), it is still unclear which other oncogenic hits contribute to the early steps of transformation and in which precursor stages these occur. To address this issue, we performed high-resolution comparative genomic hybridization microarrays on laser-capture micro-dissected cases of follicular lymphoma in situ (n=4), partial involvement by follicular lymphoma (n=4), and duodenal follicular lymphoma (n=4), assumed to represent, potentially, the earliest stages in the evolution of follicular lymphoma. Cases of reactive follicular hyperplasia (n=2), uninvolved areas from follicular lymphoma in situ lymph nodes, follicular lymphoma grade 1-2 (n=5) and follicular lymphoma grade 3A (n=5) were used as controls. Surprisingly, alterations involving several relevant (onco)genes were found in all entities, but at significantly lower proportions than in overt follicular lymphoma. While the number of alterations clearly assigns all these entities as precursors, the pattern of partial involvement by follicular lymphoma alterations was quantitatively and qualitatively closer to that of follicular lymphoma, indicating significant selective pressure in line with its faster rate of progression. Among the most notable alterations, we observed and validated deletions of 1p36 and gains of the 7p and 12q chromosomes and related oncogenes, which include some of the most recurrent oncogenic alterations in overt follicular lymphoma (TNFRSF14, EZH2, MLL2). By further delineating distinctive and hierarchical molecular and genetic features of early follicular lymphoma entities, our analysis underlines the importance of applying appropriate criteria for the differential diagnosis. It also provides a first set of candidates likely to be involved in the cascade of hits that pave the path of the various progression phases to follicular lymphoma development.

Array comparative genomic hybridization and sequencing of 23 genes in 80 patients with myelofibrosis at chronic or acute phase.

Myelofibrosis is a myeloproliferative neoplasm that occurs de novo (primary myelofibrosis) or results from the progression of polycythemia vera or essential thrombocytemia (hereafter designated as secondary myelofibrosis or post-polycythemia vera/ essential thrombocythemia myelofibrosis). To progress in the understanding of myelofibrosis and to find molecular prognostic markers we studied 104 samples of primary and secondary myelofibrosis at chronic (n=68) and acute phases (n=12) from 80 patients, by using array-comparative genomic hybridization and sequencing of 23 genes (ASXL1, BMI1, CBL, DNMT3A, EZH2, IDH1/2, JAK2, K/NRAS, LNK, MPL, NF1, PPP1R16B, PTPN11, RCOR1, SF3B1, SOCS2, SRSF2, SUZ12, TET2, TP53, TRPS1). We found copy number aberrations in 54% of samples, often involving genes with a known or potential role in leukemogenesis. We show that cases carrying a del(20q), del(17) or del(12p) evolve in acute myeloid leukemia (P=0.03). We found that 88% of the cases were mutated, mainly in signaling pathway (JAK2 69%, NF1 6%) and epigenetic genes (ASXL1 26%, TET2 14%, EZH2 8%). Overall survival was poor in patients with more than one mutation (P=0.001) and in patients with JAK2/ASXL1 mutations (P=0.02). Our study highlights the heterogeneity of myelofibrosis, and points to several interesting copy number aberrations and genes with diagnostic and prognostic impact.

Comprehensive genome characterization of solitary fibrous tumors using high-resolution array-based comparative genomic hybridization.

Solitary fibrous tumors (SFTs) are rare spindle cell tumors with limited therapeutic options. Their molecular basis is poorly known. No consistent cytogenetic abnormality has been reported. We used high-resolution whole-genome array-based comparative genomic hybridization (Agilent 244K oligonucleotide chips) to profile 47 samples, meningeal in >75% of cases. Few copy number aberrations (CNAs) were observed. Sixty-eight percent of samples did not show any gene CNA after exclusion of probes located in regions with referenced copy number variation (CNV). Only low-level CNAs were observed. The genomic profiles were very homogeneous among samples. No molecular class was revealed by clustering of DNA copy numbers. All cases displayed a "simplex" profile. No recurrent CNA was identified. Imbalances occurring in >20%, such as the gain of 8p11.23-11.22 region, contained known CNVs. The 13q14.11-13q31.1 region (lost in 4% of cases) was the largest altered region and contained the lowest percentage of genes with referenced CNVs. A total of 425 genes without CNV showed copy number transition in at least one sample, but only but only 1 in at least 10% of samples. The genomic profiles of meningeal and extra-meningeal cases did not show any differences.

Signaling pathway switch in breast cancer.

Next generation sequencing studies have drawn the general landscape of breast cancers and identified hundreds of new, actual therapeutic targets. Two major signaling pathways seem to be altered in a vast proportion of breast cancers. The PI3 kinase/AKT pathway is activated and the JUN/MAPK pathway is repressed. Via the regulation of the cell cycle this metabolic switch impacts on the balance between self-renewal, proliferation and differentiation of the tumor-initiating cells.

ZNF703 mRNA-Targeting Antisense Oligonucleotide Blocks Cell Proliferation and Induces Apoptosis in Breast Cancer Cell Lines.

The luminal B molecular subtype of breast cancers (BC) accounts for more than a third of BCs and is associated with aggressive clinical behavior and poor prognosis. The use of endocrine therapy in BC treatment has significantly contributed to the decrease in the number of deaths in recent years. However, most BC patients with prolonged exposure to estrogen receptor (ER) selective modulators such as tamoxifen develop resistance and become non-responsive over time. Recent studies have implicated overexpression of the ZNF703 gene in BC resistance to endocrine drugs, thereby highlighting ZNF703 inhibition as an attractive modality in BC treatment, especially luminal B BCs. However, there is no known inhibitor of ZNF703 due to its nuclear association and non-enzymatic activity. Here, we have developed an antisense oligonucleotide (ASO) against ZNF703 mRNA and shown that it downregulates ZNF703 protein expression. ZNF703 inhibition decreased cell proliferation and induced apoptosis. Combined with cisplatin, the anti-cancer effects of ZNF703-ASO9 were improved. Moreover, our work shows that ASO technology may be used to increase the number of targetable cancer genes.

BRCA1-methylated triple negative breast cancers previously exposed to neoadjuvant chemotherapy form RAD51 foci and respond poorly to olaparib.

Background: About 15% of Triple-Negative-Breast-Cancer (TNBC) present silencing of the BRCA1 promoter methylation and are assumed to be Homologous Recombination Deficient (HRD). BRCA1-methylated (BRCA1-Me) TNBC could, thus, be eligible to treatment based on PARP-inhibitors or Platinum salts. However, their actual HRD status is discussed, as these tumors are suspected to develop resistance after chemotherapy exposure. Methods: We interrogated the sensitivity to olaparib vs. carboplatin of 8 TNBC Patient-Derived Xenografts (PDX) models. Four PDX corresponded to BRCA1-Me, of which 3 were previously exposed to NeoAdjuvant-Chemotherapy (NACT). The remaining PDX models corresponded to two BRCA1-mutated (BRCA1-Mut) and two BRCA1-wild type PDX that were respectively included as positive and negative controls. The HRD status of our PDX models was assessed using both genomic signatures and the functional BRCA1 and RAD51 nuclear foci formation assay. To assess HR restoration associated with olaparib resistance, we studied pairs of BRCA1 deficient cell lines and their resistant subclones. Results: The 3 BRCA1-Me PDX that had been exposed to NACT responded poorly to olaparib, likewise BRCA1-WT PDX. Contrastingly, 3 treatment-naïve BRCA1-deficient PDX (1 BRCA1-Me and 2 BRCA1-mutated) responded to olaparib. Noticeably, the three olaparib-responsive PDX scored negative for BRCA1- and RAD51-foci, whereas all non-responsive PDX models, including the 3 NACT-exposed BRCA1-Me PDX, scored positive for RAD51-foci. This suggested HRD in olaparib responsive PDX, while non-responsive models were HR proficient. These results were consistent with observations in cell lines showing a significant increase of RAD51-foci in olaparib-resistant subclones compared with sensitive parental cells, suggesting HR restoration in these models. Conclusion: Our results thus support the notion that the actual HRD status of BRCA1-Me TNBC, especially if previously exposed to chemotherapy, may be questioned and should be verified using the BRCA1- and RAD51-foci assay.

Genome profiling of pancreatic adenocarcinoma.

Pancreatic adenocarcinoma is one of the most aggressive human cancers. It displays many different chromosomal abnormalities and mutations. By using 244 K high-resolution array-comparative genomic hybridization (aCGH) we studied the genome alterations of 39 fine-needle aspirations from pancreatic adenocarcinoma and eight human adenocarcinoma pancreatic cell lines. Using both visual inspection and GISTIC analysis, recurrent losses were observed on 1p, 3p, 4p, 6, 8p, 9, 10, 11q, 15q, 17, 18, 19p, 20p, 21, and 22 and comprised several known or suspected tumor suppressor genes such as ARHGEF10, ARID1A, CDKN2A/B, FHIT, PTEN, RB1, RUNX1-3, SMAD4, STK11/LKB1, TP53, and TUSC3. Heterozygous deletion of the 1p35-p36 chromosomal region was identified in one-third of the tumors and three of the cell lines. This region, commonly deleted in human cancers, contains several tumor suppressor genes including ARID1A and RUNX3. We identified frequent genetic gains on chromosome arms 1q, 3q, 5p, 6p, 7q, 8q, 12q, 15q, 18q, 19q, and 20q. Amplifications were observed in 16 tumors. AKT2, CCND3, CDK4, FOXA2, GATA6, MDM2, MYC, and SMURF1 genes were gained or amplified. The most obvious amplification was located at 18q11.2 and targeted the GATA6 gene, which plays a predominant role in the initial specification of the pancreas and in pancreatic cell type differentiation. In conclusion, we have identified novel biomarkers and potential therapeutic targets in pancreatic adenocarcinoma.

Investigation of Molecular Features Involved in Clinical Responses and Survival in Advanced Endometrial Carcinoma Treated by Hormone Therapy.

Hormone therapy (HT) is an effective treatment for metastatic endometrial carcinoma (mEC), with limited toxicity and low cost. We focused on molecular analysis of mECs treated by HT and, for the first time to date, we compared the genomic profiles of paired metastasis and primary ECs. The main objective was to identify predictive factors of the response to HT as well as specific altered signaling pathways driving mEC biology. From 1052 patients with EC treated by HT in two French cancer centers, 32 with endometrioid EC and 6 with high grade serous EC were included. We evaluated hormone receptors (HR) and mismatch repair proteins expression by immunohistochemistry and gene alterations by targeted next-generation sequencing and array-based comparative genomic hybridization. Several variables were tested in univariate and multivariate analyses to identify potential associations with (i) the clinical benefit of HT (CBHT) and (ii) a longer response (>18 months) (LRHT) and overall survival (OS). We compared the biological and genomic profiles of 11 primary/metastatic EC pairs. Thirty tumors (78.9%) were HR-positive and 6 (15.8%) showed microsatellite instability (MSI). The genomic profiles of 34 tumors showed an average altered genome of 3.26%, DNA repair homologous recombination deficiency in five tumors (14.7%), and 17 regions significantly targeted by amplification/deletion. Thirty-three tumors had 273 variants (158 genes, median of 7 mutations/sample), including 112 driver mutations. TP53, PTEN, PPP2R1A, ARID1A, FGFR2, and PIK3CA were the most frequently mutated. Based on the genomic status, nine oncogenic pathways were altered in more than 25% of primary EC. Clinically, 22 (57.9%) and 6 (15.8%) patients presented CBHT and LRHT, respectively. Neither oncogenic pathways alterations nor the variables tested were associated with CBHT and LRHT. Only patient's age, mitotic index and the presence of at least one HR were associated with OS. Paired analysis of the primary/metastatic samples showed that among the 22 mutations acquired in the metastatic counterparts, the most frequently targeted genes were involved in pathways that might confer a selective advantage to cancer metastasis including hormone resistance. In conclusion, only patient's age, mitotic index and the presence of at least one HR were associated with OS. The identification of gene mutations newly acquired in metastasis might help to better understand the formation of EC metastasis and select the best actionable candidates for HT-treated patients at the metastatic stage.

Overcoming Resistance to Anti-Nectin-4 Antibody-Drug Conjugate.

Antibody-drug conjugates (ADC) represent a fast-growing drug class in oncology. However, ADCs are associated with resistance, and therapies able to overcome it are of utmost importance. Recently, enfortumab vedotin-ejfv (EV) was approved in nectin-4+ metastatic urothelial cancer. We previously described PVRL4/nectin-4 as a new therapeutic target in breast cancer and produced an efficient EV-like ADC comprising a human anti-nectin-4 mAb conjugated to monomethyl auristatin-E (MMAE) named N41mab-vcMMAE. To study the consequence of the long-term treatment with this ADC, we developed a preclinical breast cancer model in mice, and report a mechanism of resistance to N41mab-vcMMAE after 9-month treatment and a way to reverse it. RNA-sequencing pointed to an upregulation in resistant tumors of ABCB1 expression, encoding the multidrug resistance protein MDR-1/P-glycoprotein (P-gp), associated with focal gene amplification and high protein expression. Sensitivity to N41mab-vcMMAE of the resistant model was restored in vitro by P-gp pharmacologic inhibitors, like tariquidar. P-gp is expressed in a variety of normal tissues. By delivering the drug to the tumor more specifically than classical chemotherapy, we hypothesized that the combined use of ADC with P-gp inhibitors might reverse resistance in vivo without toxicity. Indeed, we showed that the tariquidar/N41mab-vcMMAE combination was well tolerated and induced a rapid regression of ADC-resistant tumors in mice. In contrast, the tariquidar/docetaxel combination was toxic and poorly efficient. These results show that ABC transporter inhibitors can be safely used with ADC to reverse ADC-induced resistance and open new opportunities in the fight against multidrug resistance.