Gerrit Wolters-Eisfeld

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies, largely due to intrinsic or acquired resistance to systemic therapy. In addition to dysregulated glucose metabolism, emerging evidence implicates amino acid metabolism as a key determinant of tumor growth and treatment adaptation. To identify biomarkers associated with chemoresistance, we performed SILAC-based mass spectrometry secretome profiling of gemcitabine-resistant and chemosensitive PDAC cell models, followed by qRT-PCR and immunoblot validation. Functional relevance was assessed by stable shRNA-mediated KYNU knockdown, proliferation assays, and Transwell migration assays. Clinical significance was evaluated by immunohistochemical analysis of KYNU expression in a PDAC tissue microarray (n = 368) and correlation with overall survival. Transcriptomic and pathway enrichment analyses were used to characterize resistance-associated programs. Kynureninase (KYNU), a tryptophan-catabolic enzyme, was upregulated and preferentially secreted by gemcitabine-resistant cells and induced by gemcitabine, interferon-γ, and hypoxia. KYNU knockdown significantly reduced proliferation without affecting migration. Resistant cells displayed higher KYNU expression and a broader therapy-adapted cellular state. In patient samples, high KYNU expression was associated with significantly shorter overall survival (p = 0.0198). These findings identify KYNU as a marker of a therapy-adapted PDAC phenotype and support its potential as a prognostic and treatment-response biomarker.

Copy number alterations (CNAs) are hallmarks of cancer, yet investigation of their oncogenic role has been hindered by technical limitations and missing model systems. Here we generated a genome-wide DNA methylation and CNA atlas of 106 genetic mouse models across 31 pediatric tumor types, including 18 new models for pediatric glioma. We demonstrated their epigenetic resemblance to human disease counterparts and identified entity-specific patterns of immune infiltration. We discovered that mouse tumors harbor highly recurrent CNA signatures that occur distinctly based on the tumor subgroup and driving oncogene and showed that these CNAs share syntenic regions with the matching human tumor types, thereby revealing a conserved but previously underappreciated role in subgroup-specific tumorigenesis that can be analyzed using the presented models. Our study provides insights into globally available mouse models for pediatric solid cancers and enables access to functional CNA interrogation, with the potential to unlock new translational targets in pediatric cancers.

Supplementary Fig. S7. Uniform manifold approximation and projection (UMAP) of leukemia-infiltrating immune cells vs. healthy control bone-marrow derived immune cells.

Methylome, bulk-RNA and sc RNA-seq data

Supplementary Fig. S6. Functional categorization of ML-PBNKC

Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is a molecularly distinct tyrosine kinase–driven cancer that has a high relapse rate and poor response toward combinatorial chemotherapy. Tyrosine kinase inhibitors (TKI) in the clinic improve the survival of patients with Ph-like ALL. Engineered antibody and cell-based immunotherapies can advance treatment for this genetic subtype of ALL. Allogeneic memory-like natural killer (ML-NK) cells have been used to treat leukemia and have shown a low risk of graft-versus-host reaction, which may be combined with leukemia epitope–targeting antibodies. However, mutation or pathway-directed TKI of Ph-like ALL can interfere with memory function and antibody-dependent NK cell–mediated cytotoxicity (ADCC). In this study, we explored the potential of ML-NK cells and Fc-enhanced CD19-ADCC in combination with TKI directed against kinase-driven leukemia models, including patient-derived xenografted Ph-like ALL. We demonstrate that receptor cross-linking in coculture with K562 feeder cells generated a more robust memory-like state of NK cells than coactivation with soluble IL-12, IL-15, and IL-18, as determined by genomic and functional studies. After receptor cross-linking and subsequent ILs preactivation, the optimized ML-NK cells showed enhanced antileukemic effector functions, which could compensate for exhausted B-cell precursor leukemia-infiltrating primary NK cells. TKI differentially affected multiple features of NK cell biology including viability, expansion, metabolism, receptor repertoire, and cytotoxicity. ADCC was maintained upon exposure to specific Abelson (ABL) or Janus kinase (JAK) inhibitors, in contrast to the multitarget TKI dasatinib impeding spleen tyrosine kinase–dependent ADCC. In conclusion, optimized ML-NK cell and CD19 antibody–based immunotherapy combined with carefully selected TKI demonstrates significant in vitro treatment efficacy in kinase-driven leukemia.

Supplementary Fig. S9: NK cell receptor expression under TKI treatment.

Supplementary Fig. S2. DNA methylation array probe distribution and differential gene expression in pre-activated vs. canonical NK cells.

Supplementary Fig. S4: Cell surface expression of select IL-preactivated DMEGs in canonical vs. ML-PBNKC.