Immunotherapies against HER2-Positive Breast Cancer

Immunotherapy; Resistance; VaccinesImmunoteràpia; Resistència; VacunesInmunoterapia; Resistencia; VacunasBreast cancer is the leading cause of cancer-related deaths among women worldwide. HER2-positive breast cancer, which represents 15–20% of all cases, is characterized by the overexpression of the HER2 receptor. Despite the variety of treatments available for HER2-positive breast cancer, both targeted and untargeted, many patients do not respond to therapy and relapse and eventually metastasize, with a poor prognosis. Immunotherapeutic approaches aim to enhance the antitumor immune response to prevent tumor relapse and metastasis. Several immunotherapies have been approved for solid tumors, but their utility for HER2-positive breast cancer has yet to be confirmed. In this review, we examine the different immunotherapeutic strategies being tested in HER2-positive breast cancer, from long-studied cancer vaccines to immune checkpoint blockade, which targets immune checkpoints in both T cells and tumor cells, as well as the promising adoptive cell therapy in various forms. We discuss how some of these new approaches may contribute to the prevention of tumor progression and be used after standard-of-care therapies for resistant HER2-positive breast tumors, highlighting the benefits and drawbacks of each. We conclude that immunotherapy holds great promise for the treatment of HER2-positive tumors, with the potential to completely eradicate tumor cells and prevent the progression of the disease.This research was funded by Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Salud Carlos III

Protocol to generate a patient derived xenograft model of acquired resistance to immunotherapy in humanized mice

Cancer; Cell isolation; Stem cellsCàncer; Aïllament cel·lular; Cèl·lules mareCáncer; Aislamiento celular; Células madreImmunotherapy has revolutionized cancer treatment, but preclinical models are required to understand immunotherapy resistance mechanisms underlying patient relapse. This protocol describes how to generate an acquired resistance humanized in vivo model to immunotherapies in patient-derived xenografts (PDX). We detail steps to inject human CD34+ cells into NSG mice, followed by generation of immunoresistant PDX in humanized mice. This approach recapitulates the human immune system, allowing investigators to generate preclinical resistance models to different immunotherapies for identifying the resistant phenotype. For complete details on the use and execution of this protocol, please refer to Martínez-Sabadell et al., 2022 and Arenas et al. (2021).This work was supported by Asociación Española Contra el Cancer (GCAEC19017ARRI), Breast Cancer Research Foundation (BCRF-21-008), and Instituto de Salud Carlos III (PI19/01181). A.M.S. was funded by the Spanish Government (PFIS FI20/00188). P.O.R. was funded by the BBVA. E.J.A. was funded by the AECC (POSTD211413AREN). VHIO would like to acknowledge the Cellex Foundation for providing research facilities and equipment and the Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) from the Institute of Health Carlos III (ISCIII) for their support on this research. Authors acknowledge financial support for the Cancer Immunology and Immunotherapy (CAIMI-2) program funded by BBVA Foundation. The Graphical abstract was created with BioRender.com

HER2 and p95HER2 differentially regulate miRNA expression in MCF-7 breast cancer cells and downregulate MYB proteins through miR-221/222 and miR-503

Mecanismes de la malaltia; Càncer de mamaMecanismos de la enfermedad; Cáncer de mamaDisease Mechanisms; Breast CancerThe HER2 oncogene and its truncated form p95HER2 play central roles in breast cancer. Here, we show that although HER2 and p95HER2 generally elicit qualitatively similar changes in miRNA profile in MCF-7 breast cancer cells, a subset of changes are distinct and p95HER2 shifts the miRNA profile towards the basal breast cancer subtype. High-throughput miRNA profiling was carried out 15, 36 and 60 h after HER2 or p95HER2 expression and central hits validated by RT-qPCR. miRNAs strongly regulated by p95HER2 yet not by HER2, included miR-221, miR-222, miR-503, miR-29a, miR-149, miR-196 and miR-361. Estrogen receptor-α (ESR1) expression was essentially ablated by p95HER2 expression, in a manner recapitulated by miR-221/-222 mimics. c-Myb family transcription factors MYB and MYBL1, but not MYBL2, were downregulated by p95HER2 and by miR-503 or miR-221/-222 mimics. MYBL1 3′UTR inhibition by miR-221/222 was lost by deletion of a single putative miR-221/222 binding sites. p95HER2 expression, or knockdown of either MYB protein, elicited upregulation of tissue inhibitor of matrix metalloprotease-2 (TIMP2). miR-221/222 and -503 mimics increased, and TIMP2 knockdown decreased, cell migration and invasion. A similar pathway was operational in T47D- and SKBr-3 cells. This work reveals important differences between HER2- and p95HER2- mediated miRNA changes in breast cancer cells, provides novel mechanistic insight into regulation of MYB family transcription factors by p95HER2, and points to a role for a miR-221/222– MYB family–TIMP2 axis in regulation of motility in breast cancer cells.This work was supported by the Danish Council for Independent Research (grants no. 12-126942 and 12-127290 to SFP), by the Hartmann foundation (SFP), Fondation Juchum (SFP), Kirsten og Freddy Johansens Fond (SFP), the Breast Cancer Research Foundation (BCRF-17-008) (JA), Instituto de Salud Carlos III (PI16/00253) (JA) and the Harboe foundation (SFP). Katrine Franklin Mark is gratefully acknowledged for excellent technical assistance. We are grateful to Pascal Pineau from Institut Pasteur, France for the MYBL1 3′UTR/psiCHECK2 construct

Epstein–Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report

B cells; Epstein–Barr virus; Breast cancerCélulas B; Virus de Epstein-Barr; Cáncer de mamaLimfòcits B; Virus d'Epstein-Barr; Càncer de mamaEBV-specific T cells have been recently described to be involved in fatal encephalitis and myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) high-throughput sequencing was performed to monitor the T-cell clonotypes present in the different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after three passes in mice along 6 months. Eighteen of these (42%) were also found in the TNBC biopsy. TCR infiltrating the XABCL tissue showed a very restricted T-cell repertoire as compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL-LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain clonotypes were selected by the B cells. A number of these TCRs had previously been reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides that might have been expanding these T cells. The HLA class I and class II-associated peptides from XABCL-LCL were then compared with published repertoires from LCL of different HLA typing. Proteins from the antigen processing and presentation pathway remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II-presented peptides were derived from cancer-related proteins. These results suggest that bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor-infiltrating T cells and influence the TCR repertoire in the tumor site.This project was funded by Roche Farma, S.A. grant SP181123001 and the Spanish Ministry of Science, Innovation and Universities grant RTI2018-097414-B-I00. Partial financial support was received from the “El Paseíco de la Mama” 2015. This study received partial funding from Roche Farma, S.A. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication

Targeting HER2-AXL heterodimerization to overcome resistance to HER2 blockade in breast cancer

Breast cancer; HeterodimerizationCáncer de mama; HeterodimerizaciónCàncer de mama; HeterodimeritzacióAnti-HER2 therapies have markedly improved prognosis of HER2-positive breast cancer. However, different mechanisms play a role in treatment resistance. Here, we identified AXL overexpression as an essential mechanism of trastuzumab resistance. AXL orchestrates epithelial-to-mesenchymal transition and heterodimerizes with HER2, leading to activation of PI3K/AKT and MAPK pathways in a ligand-independent manner. Genetic depletion and pharmacological inhibition of AXL restored trastuzumab response in vitro and in vivo. AXL inhibitor plus trastuzumab achieved complete regression in trastuzumab-resistant patient-derived xenograft models. Moreover, AXL expression in HER2-positive primary tumors was able to predict prognosis. Data from the PAMELA trial showed a change in AXL expression during neoadjuvant dual HER2 blockade, supporting its role in resistance. Therefore, our study highlights the importance of targeting AXL in combination with anti-HER2 drugs across HER2-amplified breast cancer patients with high AXL expression. Furthermore, it unveils the potential value of AXL as a druggable prognostic biomarker in HER2-positive breast cancer.A.A.-A., E.J.A., and F.B.-M. were supported by Asociación Española contra el Cáncer AECC (PRDVA18013LLUC to A.A.-A., POSTD211413AREN to E.J.A., and AECC_Postdoctoral17-1062 to F.B.-M.). A.M.-S. was funded by the Spanish Government (PFIS FI20/00188). J.Ar. is supported by Breast Cancer Research Foundation (BCRF-20-08), Instituto de Salud Carlos III Project reference number AC15/00062, and the EC under the framework of the ERA-NET TRANSCAN-2 initiative cofinanced by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociación Española Contra el Cáncer (AECC). A.P. was supported by Instituto de Salud Carlos III—PI19/01846, Breast Cancer Now—2018NOVPCC1294. P.E. and A.L. were funded by Instituto de Salud Carlos III and cofinanced by FEDER (PI18/01219 to P.E. and CB16/12/00481 to A.L.). J.M.C. was funded by Sociedad Española de Oncología Médica (Rio Hortega-SEOM) and Compromiso ADAMED

Gasdermin B over-expression modulates HER2-targeted therapy resistance by inducing protective autophagy through Rab7 activation

Gasdermin B; HER2 breast cancer; Protective autophagyGasdermin B; Càncer de mama HER2; Autofàgia protectoraGasdermin B; Cáncer de mama HER2; Autofagia protectoraBackground Gasdermin B (GSDMB) over-expression promotes poor prognosis and aggressive behavior in HER2 breast cancer by increasing resistance to therapy. Decoding the molecular mechanism of GSDMB-mediated drug resistance is crucial to identify novel effective targeted treatments for HER2/GSDMB aggressive tumors. Methods Different in vitro approaches (immunoblot, qRT-PCR, flow cytometry, proteomic analysis, immunoprecipitation, and confocal/electron microscopy) were performed in HER2 breast and gastroesophageal carcinoma cell models. Results were then validated using in vivo preclinical animal models and analyzing human breast and gastric cancer samples. Results GSDMB up-regulation renders HER2 cancer cells more resistant to anti-HER2 agents by promoting protective autophagy. Accordingly, the combination of lapatinib with the autophagy inhibitor chloroquine increases the therapeutic response of GSDMB-positive cancers in vitro and in zebrafish and mice tumor xenograft in vivo models. Mechanistically, GSDMB N-terminal domain interacts with the key components of the autophagy machinery LC3B and Rab7, facilitating the Rab7 activation during pro-survival autophagy in response to anti-HER2 therapies. Finally, we validated these results in clinical samples where GSDMB/Rab7/LC3B co-expression associates significantly with relapse in HER2 breast and gastric cancers. Conclusion Our findings uncover for the first time a functional link between GSDMB over-expression and protective autophagy in response to HER2-targeted therapies. GSDMB behaves like an autophagy adaptor and plays a pivotal role in modulating autophagosome maturation through Rab7 activation. Finally, our results provide a new and accessible therapeutic approach for HER2/GSDMB + cancers with adverse clinical outcome.This study has been supported by the Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación (PID2019-104644RB-I00) -GMB-, the Instituto de Salud Carlos III (CIBERONC, CB16/12/00449 -JA-, CB16/12/00231 -DLN- and CB16/12/00295 -GMB-, PI19/01181 -JA-, PI18/00795, CP17/00063 and RTI2018-095611-A-I00 -DLN- and ERA-NET TRANSCAN-2 -JA- [all partly supported by FEDER funds]) and by the AECC Scientific Foundation (FC_AECC PROYE19036MOR -GMB- and LABAE19004LLOB -DLN-). Furthermore, this work was supported by Breast Cancer Research Foundation (BCRF-19–08) -JA-. We are also grateful to the CERCA Programme (Generalitat de Catalunya) for institutional support. MGC and DS contracts are funded by CIBERONC, KG is a recipient of a PFIS fellowship (FI19/00188), RRB is recipient of a Ramón y Cajal grant (RyC-2016–19671) and DLN is recipient of a Miguel Servet grant (MS17/00063) (all partly supported by FEDER funds). We are also grateful to MD Anderson BIOBANK for providing tumor samples. The bank (reference # B.0000745) belongs to the National Registry of Biobanks coordinated by the Carlos III Health Institute

The target antigen determines the mechanism of acquired resistance to T cell-based therapies

Cancer; Antigen; ResistanceCáncer; Antígeno; ResistenciaCàncer; Antigen; ResistènciaDespite the revolution of immunotherapy in cancer treatment, patients eventually progress due to the emergence of resistance. In this scenario, the selection of the tumor antigen can be decisive in the success of the clinical response. T cell bispecific antibodies (TCBs) are engineered molecules that include binding sites to the T cell receptor and to a tumor antigen. Using gastric CEA+/HER2+ MKN45 cells and TCBs directed against CEA or HER2, we show that the mechanism of resistance to a TCB is dependent on the tumor antigen. Acquired resistant models to a high-affinity-CEA-targeted TCB exhibit a reduction of CEA levels due to transcriptional silencing, which is reversible upon 5-AZA treatment. In contrast, a HER2-TCB resistant model maintains HER2 levels and exhibit a disruption of the interferon-gamma signaling. These results will help in the design of combinatorial strategies to increase the efficacy of cancer immunotherapies and to anticipate and overcome resistances.This work was supported by Asociación Española Contra el Cancer (AECC), Breast Cancer Research Foundation (BCRF-21-008), and Instituto de Salud Carlos III (PI19/01181). A.M.S. was funded by the Spanish Government (PFIS FI20/00188). B.M. was funded by a fellowship from PERIS (Departament de Salut, Generalitat de Catalunya). M.R.A. was funded by Agency for Management of University and Research Grants (AGAUR, 2022 FI_B2 00080). P.O.R. was funded by the BBVA. E.J.A. was funded by the AECC (POSTD211413AREN). VHIO acknowledges the Cellex Foundation for providing research facilities and equipment, the Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) from the Institute of Health Carlos III (ISCIII), and the Department of Health (Generalitat de Catalunya, SLT008/18/00198 SLT008/18/00205) for their support on this research. The authors acknowledge financial support from the State Agency for Research (Agencia Estatal de Investigación) (CEX2020-001024-S/AEI/10.13039/501100011033) and for the Cancer Immunology and Immunotherapy (CAIMI-2) program funded by BBVA Foundation. We would like to remark the funding from B.M PERIS (Spain). The authors thank Dr. Anne Freimoser-Grundschober and Roche for helping provide the TCBs. The graphical abstract was created with BioRender.com

Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance

Cáncer de mama y de ovario; Inhibición WEE1Càncer de mama i d'ovari; Inhibició WEE1Breast and ovarian cancer; WEE1 inhibitionPurpose: PARP inhibitors (PARPi) induce synthetic lethality in homologous recombination repair (HRR)-deficient tumors and are used to treat breast, ovarian, pancreatic, and prostate cancers. Multiple PARPi resistance mechanisms exist, most resulting in restoration of HRR and protection of stalled replication forks. ATR inhibition was highlighted as a unique approach to reverse both aspects of resistance. Recently, however, a PARPi/WEE1 inhibitor (WEE1i) combination demonstrated enhanced antitumor activity associated with the induction of replication stress, suggesting another approach to tackling PARPi resistance. Experimental Design: We analyzed breast and ovarian patient-derived xenoimplant models resistant to PARPi to quantify WEE1i and ATR inhibitor (ATRi) responses as single agents and in combination with PARPi. Biomarker analysis was conducted at the genetic and protein level. Metabolite analysis by mass spectrometry and nucleoside rescue experiments ex vivo were also conducted in patient-derived models. Results: Although WEE1i response was linked to markers of replication stress, including STK11/RB1 and phospho-RPA, ATRi response associated with ATM mutation. When combined with olaparib, WEE1i could be differentiated from the ATRi/olaparib combination, providing distinct therapeutic strategies to overcome PARPi resistance by targeting the replication stress response. Mechanistically, WEE1i sensitivity was associated with shortage of the dNTP pool and a concomitant increase in replication stress. Conclusions: Targeting the replication stress response is a valid therapeutic option to overcome PARPi resistance including tumors without an underlying HRR deficiency. These preclinical insights are now being tested in several clinical trials where the PARPi is administered with either the WEE1i or the ATRi.This work was supported by the Spanish Instituto de Salud Carlos III (ISCIII), an initiative of the Spanish Ministry of Economy and Innovation partially supported by European Regional Development FEDER Funds (FIS PI17/01080 to V. Serra, PI12/02606 to J. Balmaña); European Research Area-NET, Transcan-2 (AC15/00063), Asociación Española contra el Cáncer (AECC; LABAE16020PORTT), the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR; 2017 SGR 540), La Marató TV3 (654/C/2019), and ERAPERMED2019–215 to V. Serra. We also acknowledge the GHD-Pink program, the FERO Foundation, and the Orozco Family for supporting this study (to V. Serra). V. Serra was supported by the Miguel Servet Program (ISCIII; CPII19/00033); M. Castroviejo-Bermejo and C. Cruz (AIOC15152806CRUZ) by AECC; A. Herencia-Ropero by Generalitat de Catalunya-PERIS (SLT017/20/000081); M. Palafox by Juan de la Cierva (FJCI-2015–25412); A. Lau by AECC and Generalitat de Catalunya-PERIS (INVES20095LLOP, SLT002/16/00477); A. Gris-Oliver by FI-AGAUR (2015 FI_B 01075). This work was supported by Breast Cancer Research Foundation (BCRF-19–08), Instituto de Salud Carlos III Project Reference number AC15/00062, and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociación Española Contra el Cáncer (to J. Arribas). The xenograft program in the Caldas laboratory was supported by Cancer Research UK and also received funding from an EU H2020 Network of Excellence (EuroCAN). The RPPA facility is funded by NCI #CA16672

Therapy-Induced Senescence Enhances the Efficacy of HER2-Targeted Antibody–Drug Conjugates in Breast Cancer

Eficàcia; Conjugats de fàrmacs; Càncer de mamaEficacia; Conjugados de medicamentos; Cáncer de mamaEfficacy; Drug conjugates; Breast cancerAntibody–drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as [vic-]trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression. Significance: Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.This work was supported by Breast Cancer Research Foundation (BCRF-20-008), Instituto de Salud Carlos III (project reference numbers AC15/00062, CB16/12/00449 and PI19/01181), the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Fundación Mutua Madrileña and Asociación Española Contra el Cáncer. S. Duro-Sánchez is supported by the Spanish Ministerio de Universidades by the grant Formación de Profesorado Universitario (FPU20/05388). A. Esteve-Codina is funded by ISCIII /MINECO (PT17/0009/0019) and co-funded by FEDER. The authors acknowledge Alyson MacInnes for reviewing and editing the article

High p16 expression and heterozygous RB1 loss are biomarkers for CDK4/6 inhibitor resistance in ER+ breast cancer

Breast cancer; Cancer models; Predictive markersCáncer de mama; Modelos de cáncer; Marcadores predictivosCàncer de pulmó; Models de càncer; Marcadors predictiusCDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies