doi:10.1136/jitc-2025-012706engPesini, CeciliaGil-Bellido, MarioMillán, Lorena S.Oñate, CarmenCalvo-Pérez, AdanaysSantiago, LlipsyIglesias, EldrisBernal, Jorge PaúlAraujo-Voces, MiguelPaz Artigas, LauraGarcía-Martínez, LauraRoig, Francisco JMovilla Meno, NievesGarcia-Aznar, José ManuelMenéndez-Jandula, BárbaraOlave, María TeresaAzaceta Reinares, GemmaGarrote, MartaAlvarez-Larrán, AlbertoGálvez, Eva MSánchez Martínez, DiegoArias, Maykel APardo, JulianRamirez-Labrada, ArielMutant calreticulin enables potent and selective CAR-T cell therapy in preclinical models of myeloproliferative neoplasmsART-2026-147396Background 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.2026http://zaguan.unizar.es/record/16600410.1136/jitc-2025-012706http://zaguan.unizar.es/record/166004oai:zaguan.unizar.es:166004info:eu-repo/grantAgreement/ES/AEI/PID2020-113963RB-I00info:eu-repo/grantAgreement/ES/AEI/PID2024-157582OB-I00info:eu-repo/grantAgreement/ES/AEI/RYC2022-036627-Iinfo:eu-repo/grantAgreement/ES/DGA/B29-23Rinfo:eu-repo/grantAgreement/ES/ISCIII/B61-24info:eu-repo/grantAgreement/ES/DGA/LMP139-21info:eu-repo/grantAgreement/EC/H2020/101018587/EU/Individual and Collective Migration of the Immune Cellular System/ICoMICSThis project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 101018587-ICoMICSinfo:eu-repo/grantAgreement/ES/ISCIII/CB21-13-00087info:eu-repo/grantAgreement/ES/ISCIII/PI25-00236info:eu-repo/grantAgreement/ES/ISCIII/RD24-0014-0015info:eu-repo/grantAgreement/ES/MICINN/PID2022-136554OA-I00Journal for immunotherapy of cancer 14, 1 (2026), e012706 [17 pp.]by-nchttps://creativecommons.org/licenses/by-nc/4.0/deed.esinfo:eu-repo/semantics/openAccess