Abstract Peritoneal metastatic colorectal cancer (pmCRC) has the worst outcome compared to metastatic CRC patients with metastases in other organs, such as liver or lung. More importantly, despite of the improvement of both systemic and peritoneal specific treatment, 50% to 90% of patients experience relapse and progression of the disease, leading to premature death. Precision oncology has successfully improved the overall survival of several solid and non-solid malignancies. For CRC, large volumes of information have been acquired regarding the molecular aberrations, characterizing both lung and liver metastases from CRC, thus leading to a more personalized treatment approach. On the contrary, this has not yet been done for pmCRC. In a comprehensive effort to close this gap and to identify new predictive signatures to drug responses in pmCRC, we previously established a novel platform of matched preclinical pmCRC models, including 14 patient-derived xenografts (PDX) of peritoneal metastases from a total of 10 pmCRC patients and showed that tumors intrinsically resistant to 5-Fluorouracil (5-FU) were enriched in alterations of the DNA damage response and repair (DDR) machinery. We therefore hypothesize, that those tumors are responsive to DDR inhibitors such as olaparib. In order to prove our hypothesis, we generated a new cohort of 48 pmCRC PDX, characterized by RNA sequencing. Based on both transcriptomic and mutational profiles, we classified 14 PDX as homologous recombination deficient (HRD) and 34 PDX as proficient. The most common alteration identified in the HRD positive group were loss-of-function frameshift insertions/deletions in BRCA1/2, in addition to other homologous recombination repair genes (HRRmut). Clinical data, in particular treatment response data, were combined with the genomic profiles of the respective PDX models, supporting the prediction of 5-FU resistance. Subsequently, a total of 12 pmCRC models predicted to be resistant to 5-FU-based treatment regimens, but sensitive to PARP due to HRD, were treated with 5-FU or olaparib alone, as well as in combination. Response data will be presented. Our study highlights the importance of molecular profiling for better personalized treatment. Citation Format: Mathias Dahlmann, Beate Rau, Safak Gül-Klein, Bernadette Brzezicha, Marlen Keil, Antje Wengner, Jens Hoffmann, Sebastian Stintzing, Ulrike S. Stein, Wolfgang Walther, Loredana Vecchione. Repurposing PARP inhibitors in molecularly defined subgroups of peritoneal metastatic colorectal cancer (pmCRC): Preclinical analysis of patient-derived xenograft (PDX) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4476.

Background/Objectives: Amplitude-modulated radiofrequency (AMRF) fields have emerged as promising non-temperature-induced strategies in oncology. While conventional hyperthermia (HT) relies on thermal stress, the biological impact of AMRF, particularly in combination with radiotherapy (RT), remains insufficiently characterized. Methods: We assessed RF and AMRF, alone or with RT, using phenotypic analyses of proliferation, apoptosis, and necrosis across four cancer cell lines (HT29, SW620, U343, U138). Transcriptomic profiling with Kyoto Encyclopedia of Genes and Genomes (KEGG), GO:BP, and Reactome enrichment was performed in SW620 and U138 cells, selected for their strong phenotypic responses. Results: Across the panel, AMRF was associated with broader cytotoxic responses than RF or HT in most but not all cell lines. AMRF+RT produced the strongest necrotic responses, with cell-line-specific exceptions identified explicitly in the Results (the absence of a significant AMRF+RT apoptotic effect in SW620 and the absence of a significant AMRF+RT necrotic response in U343). In SW620 cells, AMRF was associated with extensive transcriptional reprogramming involving immune modulation, extracellular matrix remodeling, and cell cycle regulation, whereas RF alone showed narrower and delayed effects. In contrast, U138 cells showed elevated apoptosis and necrosis but limited transcriptional changes-a phenotype-transcriptome divergence that points to mechanisms operating downstream of transcription and warrants functional investigation in dedicated follow-up studies. Conclusions: AMRF and AMRF+RT emerge as promising non-temperature-induced anticancer modalities in the cell-line models profiled here, with the pattern of response varying between cell lines. These findings expand the biological impact of RF-based treatments and set the grounds for further investigation in mechanistic and translational studies.

We have previously identified MACC1 and IER2 as functional biomarkers in the context of colorectal cancer. In silico correlation analysis suggested a possible functional connection between the expressions of these biomarkers, given that a significant positive correlation between IER2 and MACC1 RNA was observed. In loss- and gain-of-function experiments, we found that MACC1 positively regulates the expression of IER2. Furthermore, pulldown experiments provided evidence for MACC1-IER2 protein-protein interactions. Functionally, MACC1 enhanced proliferation of HCT116 cells overexpressing IER2 but not of HCT116 cells with knockdown of IER2 expression. Patients with high expressions of both biomarkers lived significantly shorter, whereas those with low concentrations of both markers showed the longest survival. Taken together, these findings show a functional interplay between the colorectal biomarkers MACC1 and IER2, which, in turn, has an impact on the survival of colorectal cancer patients.

Understanding the mechanisms of metastasis is one of the most pressing issues in cancer therapy. Metastasis-associated in colon cancer 1 (MACC1) is an important biomarker and functional driver of tumor progression and metastasis. However, the molecular mechanisms underlying its activity remain incompletely understood. Here, we demonstrate that MACC1 acts as an important adaptor protein that promotes hyperactivation of receptor tyrosine kinase (RTK) signaling pathways in colorectal cancer (CRC) cells. Based on mass spectrometry-based interactomics, we identified key MACC1 interactors, including GRB2, SHP2, SHC1, and STAT5B, that preferentially associate with tyrosine-phosphorylated residues Y365, Y379, and Y789. Site-directed mutagenesis of Y379 and Y789 reduced MACC1-induced migration, proliferation, and ERK phosphorylation. Using digital Western blotting (DigiWest), we observed a broad MACC1-dependent hyperactivation of downstream signaling effectors, including MEK, ERK, β-catenin, SRC, FAK, CREB, and VASP. Targeting MACC1-induced signaling with clinically relevant inhibitors effectively reversed MACC1-driven clonogenicity. Our findings support a role for MACC1 in promoting hyperactivation of RTK-associated signaling and reveal pharmacological vulnerabilities of potential relevance to metastasis-prone cancers characterized by elevated MACC1 expression.

Cancer metastasis remains the most lethal characteristic of tumors mediating the majority of cancer-related deaths and continues to be a leading cause of mortality. Exemplified for colorectal cancer (CRC) metastasis results in around 0.9 million deaths annually worldwide. Identifying key molecules responsible for metastasis, understanding their biological functions and therapeutically targeting these molecules is therefore of tremendous value. Metastasis Associated in Colon Cancer 1 (MACC1), a gene first described in 2009, is such a key driver of metastatic processes, initiating cellular proliferation, migration, invasion, and metastasis in vivo. Since its discovery, the value of MACC1 as a prognostic biomarker has been confirmed and linked to poor overall outcome in more than 20 cancer types. Despite numerous studies identifying MACC1 as a promising target for solid cancer, little is known about the structural features of MACC1 protein and its oligomeric states. Therefore, we asked whether MACC1 can form protein complexes and if so, which region is critical for complexation/oligomerization. Using AlphaFold-Multimer, the MACC1 homodimer structure was predicted and the residues involved in dimerization were evaluated. The notion of MACC1 dimerization was validated further using bioluminescence resonance energy transfer (BRET) in HEK293 and HCT116 cell system. To our surprise, BRET revealed that MACC1 exists as homodimer in living cells. Further, we report that mutation of the predicted dimer interface residues Val212, Ile214 and Cys216 close to the N-terminus of MACC1 in the ZU5 domain hinders homodimerization and affects MACC1-mediated cell migration in vitro and metastasis formation in vivo. Taken together, these findings identify an important role of MACC1 dimers for the MACC1-mediated metastasis phenotype. We provide with this study a framework to better understand the MACC1 signaling landscape and to facilitate the rational development of novel therapies aiming at MACC1 oligomerization to intervene in MACC1-mediated metastasis. Malti Dumbani, Benedikt Kortüm, Simona Kostova, Fabian Zincke, Alexandra Forrai, Dennis Kobelt, Wolfgang Walther, Erich Wanker, Oliver Daumke, Christoph Buhlheller, Ulrike S. Stein. Dimerization of MACC1: A newly identified feature and its role in cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 84.

Breast cancer (BC) is a heterogeneous and complex disease, with varying prognosis. Patient-derived xenograft models (PDX) are able to reflect this heterogeneity and can therefore be used to support the development of novel therapeutic strategies against BC. Preclinical experiments with larger cohorts of BC PDX can be employed to experimentally model a clinical phase II study with new drugs or drug combinations. The predictive value of these preclinical trials was shown earlier in co-clinical trials, where treatment efficacies were compared between patients and their corresponding PDX. Here, we present experimental preclinical data of a cohort of triple negative BC PDX models (TNBC; ER-/PR-/Her2-). Within the scope of different research projects, we established PDX models by collecting breast cancer tissue samples from surgery and engrafting them subcutaneously on immunodeficient mice. In total, 39 PDXs have been successfully engrafted. They were phenotypically characterized and screened for their dug sensitivity to standard of care and targeted drugs (e.g. docetaxel, paclitaxel, bevacizumab, everolimus). The immunohistochemical stainings for estrogen/progesterone/androgen/Her2 receptors, Ki-67, and CK5/6 of the original tumor and the PDX were comparable. The breast cancer PDX models were further analyzed for their mutational and HLA status and were tested for orthotopic growth as well as their potential to form metastases. In our hands, 16 out of 39 PDX models were classified as TNBC subtype (41%). This cohort showed heterogeneous in vivo tumor growth and various chemosensitivity (60% taxane responders, 20% anti VEGF/Her2 responders). The RNAseq analyses detected different genetic alterations (TP53/HDR/BRAF/BRCA mutations, PTEN loss). 7 out of 16 TNBC PDX were implanted orthotopically into the mammary fad pad of NOG mice and metastasized into liver, lung and spleen. Macroscopic metastases were found in the same organs after intravenous injection of tumor cell suspensions from the PDX. To evaluate sensitivity to immune therapy, selected TNBC PDX were used for efficacy studies in HLA matched humanized mice. These models were treated with nivolumab and pembrolizumab, to evaluate response to these check point inhibitors. In summary, our extensively characterized cohort of TNBC PDX reflects the clinical disease situation and can been successfully applied as translational tool for the assessment of determinants for metastasis, tumor progression and drug responsiveness or resistance. Furthermore, TNBC PDX models are applicable for immune-therapeutic testing including combination settings to evaluate novel therapies for TNBC. Diana Behrens, Verena Kiver, Theresia Scheller, Mathias Dahlmann, Bernadette Brzezicha, Britta Buettner, Wolfgang Walther, Jens Hoffmann. Triple negative breast cancer (TNBC) PDX models for preclinical investigation of novel therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3823.

Colorectal cancer (CRC) metastasis continues to account for a substantial proportion of cancer-related deaths worldwide. Calcium-binding protein S100A4 is a known executor of CRC metastasis. S100A4 has been correlated to metastasis formation in the past, and therefore pharmaceutical intervention reduces the metastatic phenotype. Herein, a high-throughput screen (HTS) of 105,600 compounds from the EMBL screening library using an S100A4 promoter-driven luciferase construct transfected into HCT116 cells identified novel compounds for S100A4 transcriptional inhibition. The most promising inhibitors identified were tested for S100A4 transcriptional inhibition, their impact on wound healing, migration, proliferation and viability of cancer cells. Subsequently, the leading candidate E12 was tested in vivo in a xenograft mouse model (HCT116/CMVp- Luc). After several testing rounds, E12 a 2-(4-fluorobenzenesulfonamido)benzamide-based compound showed the strongest inhibition of S100A4 expression at mRNA (EC50 < 1 µM; 48 h) and protein level and concomitant restriction of metastatic abilities in two CRC cell lines with a tolerable viability reduction. In vivo, a reduction in metastasis formation was demonstrated, displayed by reduced overall bioluminescence of tumors and human satellite DNA in the liver of treated mice. This study exhibited E12's promising potential for S100A4 targeted metastasis inhibition therapy to improve the outcome of metastasized CRC patients.

Abstract Background: The preclinical evaluation of novel cancer treatments demands comprehensive model systems in vitro that provide meaningful data before entering in vivo studies. Here we evaluate the capabilities of live cell imaging systems to evaluate novel immune therapies. Using integrated immune and tumor cell models in vitro we demonstrate, that these model systems can generate reliable data of pharmacodynamic activity of biologicals, small molecules or combinatorial approaches for further preclinical in vivo characterization. Methods: Target tumor cell killing was assessed in vitro with immune cells (T- and NK-cells) and engagers. Tumor cells were transduced with a fluorescent marker to discriminate tumor cells from immune cells. The technology was used to determine inhibition of cell motility (re-invasion) after scratching of tumor cell monolayers. Cells were monitored using the IncuCyte. Dose-response-curves of single treatments and all combinations were generated in parallel. Active therapies were selected for further in vivo validation of immune cell killing. Humanized mice were generated by injection of CD34+ HSC or human immune cell subsets. Immune cell engraftment was monitored by FACS. To analyze the effect biologicals or small molecules, tumor cells were transplanted into these humanized mice. Tumor development and therapeutic effects were monitored by BLI measurements. Results and conclusion: Tumor cell killing by immune cells and monolayer scratch assay in 96 well format were successfully monitored in the IncuCyte. Here, data can be generated over time without the need of new samples at every time point compared with conventional end point measurements. Using antibodies directing immune cells to attack target cells extensive cell killing was observed over time. These data predicted in vivo treatment outcome in mice co-engrafted with human immune cells. After successful humanization of mice, immune cells can be directed to kill target tumor cells. Small molecule combinations were tested in vitro utilizing the metastasis/2D scratch assay. After setup of dose-response curves for two molecules combinatorial treatments were tested. Here we found a synergistic increase in efficacy. These combinations were tested in vivo to evaluate their abilities to inhibit cell motility and distant metastasis. Here we show that the in vitro assays predicted correctly the highest efficacy of combined treatments compared to mono treatments.The IncuCyte System provides data that translate our integrated model systems into in vivo studies. We have shown that activated immune cells can kill target tumor cells in vitro. These data have been validated in vivo using immune cell humanized mice. Further, immune cells, biologicals and small molecule based treatments can be tested either alone or in combination, allowing the preselection of active combinations for further development. Citation Format: Dennis Kobelt, Maria Stecklum, Simone Rhein, Wolfgang Walther, Jens Hoffmann. Integrated tumor models for immune oncology: Using live cell imaging for prediction of treatment efficacy in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2048.

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1566.

Abstract Introduction: The development of new drugs in cancer therapy comprises toxicity and efficacy tests with increasing complexity. First and foremost, in vitro experiments are performed with well-established cancer cell lines, which are subsequently validated in animal experiments as prerequisite for clinical trials. Until a few years ago, there were gaps in the complexity chain between in vitro and in vivo experiments. Ethical considerations have led to use of systems like organ on a chip or mini-organ 3D in vitro models. In cancer research, cell cultures or organoids are generated from patient tumor material. However, these cultures only reflect the tumor of one patient, which is why panels of patient tumors should be used to evaluate the effectiveness of drugs on different patients. In vivo panel screens of patient derived xenografts (PDX) mouse models need high numbers of animals and takes several months. A pre-screen with in vitro models generated from in vivo PDX tissues allows large scale and faster pre-screens complementing in vivo systems for more focused in vivo analyses. Methods: Currently we have a pool of more than 600 established PDX models of 21 tumor entities. From this pool, cancer tissues of glioblastoma, mesothelioma, gastric, head/neck, lung and breast cancer were processed in single cell suspensions and cultured under defined conditions to obtain adherent cells or spheroids. The generated PDX in vitro cultures were analyzed for cellular impurities, cancer stem cell content and perpetuation of in vivo PDX characteristics. FACS analyses for tumor specific markers, chemo sensitivity assays and growth characteristics of the PDX derived cell lines (especially for glioblastoma) were analyzed. Results: From PDX tissues used, 90% grew as adherent and/or spheroid PDX derived in vitro cultures, in which mouse cells were entirely depleted. A high percentage of these cultures showed enriched cancer stem cell features and stem cell marker expression. Tumor marker expression and standard drug sensitivity data correlate to the in vivo PDX and derived in vitro cell culture models. RNAseq data were used to predict drug sensitivities in silico for untested drugs and drug combinations on our newly established PDX derived glioblastoma cell lines. Initial screens with predicted candidates were performed. Promising conditions were successfully repeated in corresponding animal PDX models. Conclusion: The newly developed technology for establishment if in vitro cell cultures from PDX efficiently generates stably growing cell lines possessing all key features of the original PDX. These cell lines can be used for initial pre-screens to optimize and improve selection of pharmacologically active drugs or drug combinations before initiating in vivo PDX studies. Citation Format: Lars Winkler, Joshua Alcaniz, Maria Stecklum, Wolfgang Walther, Jens Hoffmann. Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4250.

Background The preclinical evaluation of novel immune therapies demands humanized mouse models with functional human immune cells. In previous studies we have demonstrated, that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system with functional T-, B-, and NK cells, as well as monocytes and dendritic cells in immunodeficient mice. By transplantation of cell-line-derived (CDX) or patient-derived (PDX) tumor xenografts on humanized mice, we successfully generated a full human tumor-immune-cell model for different tumor entities. Finally, we validated the functionality of these models using checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo), Pembrolizumab (Pembro), cell therapies and immune cell engagers. Methods HSC-humanized mice were generated by i.v. transplantation of CD34+stem cells to immunodeficient NOG mice. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections. CDX and PDX from different entities (i.e. lymphoma, neuroblastoma, and breast cancer) were transplanted on those humanized mice. These models were used to evaluate novel immune therapies. Blood and tumor samples were analysed by FACS for immune cell infiltration and activation. Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. 14 weeks after HSC inoculation up to 20% of the human immune cells in the blood were functional T-cells, characterized by a high PD-1 expression. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Checkpoint inhibitor treatments induced tumor growth delay in selected models. FACS analysis of tumors revealed an increased percentage of tumor infiltrating T-cells. We identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other cell therapies. Conclusions We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSC on immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been employed for preclinical evaluation of novel checkpoint inhibitors, cell therapies and immune cell engagers. Our models allow preclinical, translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Wolfgang Walther, Jens Hoffmann. Humanized mouse models for preclinical evaluation of novel immune cell therapies, checkpoint inhibitors, and immune cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1650.

Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.

The IL-6–gp130–STAT3 signaling axis is a major regulator of inflammation. Activating mutations in the gene encoding gp130 and germline gain-of-function mutations in STAT3 (STAT3 GOF ) are associated with multi-organ autoimmunity, severe morbidity, and adverse prognosis. To dissect crucial cellular subsets and disease biology involved in activated gp130 signaling, the gp130-JAK-STAT3 axis was constitutively activated using a transgene, L-gp130 , specifically targeted to T cells. Activating gp130 signaling in T cells in vivo resulted in fatal, early onset, multi-organ autoimmunity in mice that resembled human STAT3 GOF disease. Female mice had more rapid disease progression than male mice. On a cellular level, gp130 signaling induced the activation and effector cell differentiation of T cells, promoted the expansion of T helper type 17 (T H 17) cells, and impaired the activity of regulatory T cells. Transcriptomic profiling of CD4 + and CD8 + T cells from these mice revealed commonly dysregulated genes and a gene signature that, when applied to human transcriptomic data, improved the segregation of patients with transcriptionally diverse STAT3 GOF mutations from healthy controls. The findings demonstrate that increased gp130-STAT3 signaling leads to T H 17-driven autoimmunity that phenotypically resembles human STAT3 GOF disease.