166004 20260116163027.0 doi 10.1136/jitc-2025-012706 sideral 147396 ART-2026-147396 eng Pesini, Cecilia Mutant calreticulin enables potent and selective CAR-T cell therapy in preclinical models of myeloproliferative neoplasms 2026 Background The adoptive transfer of T cells engineered to express chimeric antigen receptors (CAR-T) has shown high efficacy and safety in treating various hematologic malignancies. However, many hematologic disorders, such as BCR::ABL1-negative myeloproliferative neoplasms (MPNs), lack effective treatment options. Some of these neoplasms are marked by a recurrent mutation that results in the expression of mutant calreticulin (mCALR), a neoantigen absent in healthy tissues, making it a highly specific and appealing target for CAR-T cell therapy. Methods Five distinct CARs were designed based on available monoclonal antibody sequences that target mCALR and were subsequently used to generate CAR-T cells. The most effective construct was selected through functional in vitro assays against mCALR-positive cell lines. Its efficacy was then evaluated in cell lines, patient-derived cells, and orthotopic xenograft models, assessing tumor burden, CAR-T cell infiltration, and animal survival. Bulk and single-cell RNA sequencing were performed on patient-derived cells and residual tumor cells from CART-treated mice, respectively, to investigate potential resistance mechanisms. The impact of the most relevant pathway alteration on CAR-T efficacy was also analyzed. Pharmacological rescue assays using targeted agents were then conducted. Results Among the five constructs, one demonstrated superior and specific cytotoxicity against mCALR-expressing cells, with no activity against mCALR-negative controls. This CAR-T cell also eliminated patient-derived MPN cells and controlled disease progression in xenograft models, which correlated with the persistence of CAR-T cells and tumor infiltration. Transcriptomic profiling of patient samples and residual tumor cells in spleens of treated mice revealed upregulation of anti-apoptotic proteins. Functional assays confirmed reduced CAR-T efficacy in Bcl-2 high cells, which was restored by co-treatment with venetoclax, indicating a viable combination approach to overcome resistance. Conclusions This study demonstrates, for the first time, the successful targeting of mCALR with CAR-T cells as a therapeutic strategy for MPNs. The chosen construct shows strong preclinical efficacy against established cell lines and patient-derived cells. Additionally, transcriptomic profiling uncovered apoptosis resistance mechanisms and supports a combination strategy with BH3 mimetics, such as venetoclax. These findings provide a compelling rationale for ongoing preclinical development and future clinical application of anti-mCALR CAR-T cells for the treatment of MPNs. Access copy available to the general public Unrestricted info:eu-repo/grantAgreement/ES/AEI/PID2020-113963RB-I00 info:eu-repo/grantAgreement/ES/AEI/PID2024-157582OB-I00 info:eu-repo/grantAgreement/ES/AEI/RYC2022-036627-I info:eu-repo/grantAgreement/ES/DGA/B29-23R info:eu-repo/grantAgreement/ES/ISCIII/B61-24 info:eu-repo/grantAgreement/ES/DGA/LMP139-21 info:eu-repo/grantAgreement/EC/H2020/101018587/EU/Individual and Collective Migration of the Immune Cellular System/ICoMICS This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 101018587-ICoMICS info:eu-repo/grantAgreement/ES/ISCIII/CB21-13-00087 info:eu-repo/grantAgreement/ES/ISCIII/PI25-00236 info:eu-repo/grantAgreement/ES/ISCIII/RD24-0014-0015 info:eu-repo/grantAgreement/ES/MICINN/PID2022-136554OA-I00 info:eu-repo/semantics/openAccess by-nc https://creativecommons.org/licenses/by-nc/4.0/deed.es info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Gil-Bellido, Mario Millán, Lorena S. Oñate, Carmen Calvo-Pérez, Adanays Santiago, Llipsy (orcid)0000-0002-1861-5981 Iglesias, Eldris Bernal, Jorge Paúl Araujo-Voces, Miguel Paz Artigas, Laura Universidad de Zaragoza (orcid)0000-0001-6139-5905 García-Martínez, Laura Roig, Francisco J Movilla Meno, Nieves Universidad de Zaragoza (orcid)0000-0002-0163-8378 Garcia-Aznar, José Manuel Universidad de Zaragoza (orcid)0000-0002-9864-7683 Menéndez-Jandula, Bárbara Olave, María Teresa Universidad de Zaragoza (orcid)0000-0002-6326-5992 Azaceta Reinares, Gemma Universidad de Zaragoza (orcid)0000-0001-5068-7355 Garrote, Marta Alvarez-Larrán, Alberto Gálvez, Eva M Sánchez Martínez, Diego Arias, Maykel A Universidad de Zaragoza (orcid)0000-0002-9730-2210 Pardo, Julian Universidad de Zaragoza (orcid)0000-0003-0154-0730 Ramirez-Labrada, Ariel Universidad de Zaragoza (orcid)0000-0002-3888-7036 1003 443 Universidad de Zaragoza Dpto. Anatom.Histolog.Humanas Area Histología 1007 610 Universidad de Zaragoza Dpto. Medicina, Psiqu. y Derm. Area Medicina 1011 566 Universidad de Zaragoza Dpto. Microb.Ped.Radio.Sal.Pú. Área Inmunología 5004 605 Universidad de Zaragoza Dpto. Ingeniería Mecánica Área Mec.Med.Cont. y Teor.Est. 1002 050 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Biología Celular 14, 1 (2026), e012706 [17 pp.] J. immunotherap. cancer Journal for immunotherapy of cancer 2051-1426 7895756 http://zaguan.unizar.es/record/166004/files/texto_completo.pdf Versión publicada 3285982 http://zaguan.unizar.es/record/166004/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:166004 articulos driver 2026-01-16-14:54:43 ARTICLE