Markus Templin

Advances in cell therapy engineering have driven chimeric antigen receptor T cell manufacturing from manual procedures to highly automated and technically sophisticated production systems. To meet the increasing demand for high-throughput manufacturing, current efforts focus on accelerating production while maintaining/increasing product quality. Yet, to date, the precise daily progression of the T cell states during manufacturing have not been examined in detail. Here we characterize anti-CD19 CAR-T cell manufacturing on a CliniMACS Prodigy platform under current good manufacturing practice (cGMP) conditions, integrating cytotoxic activity profiling with comprehensive phenotyping across a complete production cycle. Using over 130 extra- and intracellular markers related to T cell activation, CAR signaling, metabolism, apoptosis, and hypoxia, we identify a pivotal transition point between process days 5 and 7 marking the shift from early activation to a stable/durable, functionally more specific T cell phenotype. These data shed light on cellular remodeling throughout semi-automated CAR-T manufacturing and provide a mechanistic framework for modulating T cell phenotypes across distinct production phases.

Aberrant cellular signaling underlies cancer development and progression. Identifying alterations in these pathways yields critical insights for personalized oncology. Clinically, assessing the activation status of signaling proteins complements genetic and histopathological analyses, improving therapeutic evaluation and accuracy. In this study, we employed the high-throughput Western blot system DigiWest for the characterization of gastrointestinal tumors, both retrospectively and in a proof-of-concept direct clinical application. Retrospective analyses of pancreatic and colorectal carcinomas ( n = 20) compared with matched normal tissues revealed distinct protein expression and activation patterns differentiating tumor subtypes and defining clinically relevant subgroups. By resolving individualized, treatment-relevant signaling signatures we demonstrate the feasibility of molecular-level personalization in samples with high clinical heterogeneity. In the clinical proof-of-concept, single core needle biopsies from 14 patients with gastrointestinal tumors who underwent Molecular Tumor Board presentation were analyzed. The resulting proteomic profiles uncovered patient-specific, targetable pathway activation patterns and showed concordance with mutational data and therapy recommendations. Collectively, these findings establish DigiWest as a valuable, robust complementary tool to sequencing-based approaches for personalized diagnostics and treatment evaluation in precision oncology.

We have identified a new inherited bone marrow failure syndrome with severe congenital neutropenia (CN) caused by autosomal recessive mutations in the coatomer protein complex I (COPI) subunit zeta 1 (COPZ1) gene. A stop-codon COPZ1 mutation and a missense (MS) mutation were found in 3 patients from 2 unrelated families. Although 2 affected siblings with a stop-codon COPZ1 mutation suffered from CN that involves other hematologic lineages and nonhematologic tissues, the patient with a MS COPZ1 mutation had isolated neutropenia. Both COPZ1 mutations were localized to a highly evolutionarily conserved region. The resulting truncated (TR) COPZ1 protein was predicted to display diminished interaction with its COPI complex partner, COPG1. These findings were consistent with the observed block in retrograde protein transport from the Golgi apparatus to the endoplasmic reticulum (ER) in human fibroblasts carrying TR COPZ1. Human CD34+ cells with TR or MS COPZ1 had significantly impaired granulocytic differentiation, and in zebrafish embryos, TR Copz1 also resulted in defective myelopoiesis. Intracellularly, TR COPZ1 downregulated JAK/STAT/CEBPE/G-CSFR signaling and hypoxia-responsive pathways, while inducing STING, interferon-stimulated genes, stimulating oxidative phosphorylation activity, and increasing reactive oxygen species levels in hematopoietic cells. MS COPZ1 deregulated interferon and JAK/STAT signaling but less than the TR protein. Finally, treatment with the small molecule HIF1α stabilizer IOX2 or transduction of cells with COPZ2 restored defective granulopoiesis in COPZ1-mutated human CD34+ cells, offering potential therapeutic options.

Acute pulmonary inflammation is a common disease on intensive care. Due to the lack of specific treatments, lethality is still very high. The ionotropic GABA_A-receptors are known from the central nervous system (CNS) and have recently been detected in the lung. These receptors have been shown to influence inflammatory processes. Opposing data has been reported, where both, GABA site agonists and antagonists achieved anti-inflammatory effects. The distribution of the 19 known GABA_A-receptor subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ and ρ1-3) and their role in inflammation remain unclear. In murine models of LPS- and bacteria-induced inflammation, Muscimol (GABA_A-receptor agonist) and Bicuculline (antagonist) were administered before the onset of inflammation. Transcription of GABA_A-receptor subunits was evaluated by real-time polymerase chain reaction. Neutrophil counts and adhesion molecule expression in wild type and GABAα1 knock-in mice were determined by flow-cytometry. Myeloperoxidase, neutrophil extracellular traps and cytokines were determined. In a model of ventilator-induced lung injury, blood gas analysis was performed using arterial blood. A multiplex western blot was done to assess signaling proteins. Muscimol and Bicuculline inhibited neutrophil influx in the bronchoalveolar lavage but did not change neutrophil activation. Both altered surface expression of adhesion molecules on neutrophils and reduced release of interleukin-6 (IL-6). The increased α1 subunit expression on lung epithelium and endothelium after inflammation was abolished by Muscimol and Bicuculline. In GABAα1-knock-in mice the protective effects of both agents were no longer observed. Only Muscimol lowered protein extravasation, improved blood gas analysis and lung function. A multiplex assay ascribed these anti-inflammatory effects to the influence of the IL-6 and phosphoinositide 3-kinase signaling pathways. In conclusion, Muscimol and Bicuculline exert various protective effects in two murine models of acute pulmonary inflammation. The multiple effects of Muscimol were linked to the inhibition of the proinflammatory signaling pathways IL-6 and PI3K.

Background: Atypical teratoid rhabdoid tumors (ATRT) are incurable high-grade pediatric brain tumors. Despite intensive research efforts, the prognosis for ATRT patients under currently established treatment protocols is poor. While novel therapeutic strategies are urgently needed, the generation of molecular-driven treatment concepts is a challenge mainly due to the absence of actionable genetic alterations. Results: We here use a functional genomics approach to identify genetic dependencies in ATRT, validate selected hits using a functionally instructed small molecule drug library, and observe preferential activity in ATRT cells without subgroup-specific selectivity. CDK4/6 inhibitors are among the most potent drugs and display anti-tumor efficacy due to mutual exclusive dependency on CDK4 or CDK6. Chemogenetic interactor screens reveal a broad spectrum of G1 phase cell cycle regulators that differentially enable cell cycle progression and modulate response to CDK4/6 inhibition in ATRT cells. In this regard, we find that the ubiquitin ligase substrate receptor AMBRA1 acts as a context-specific inhibitor of cell cycle progression by regulating key components of mitosis including aurora kinases. Conclusions: Our data provide a comprehensive resource of genetic and chemical dependencies in ATRTs, which will inform further preclinical evaluation of novel targeted therapies for this tumor entity. Furthermore, this study reveals a unique mechanism of cell cycle inhibition as the basis for tumor suppressive functions of AMBRA1.

Background: Schizophrenia (SCZ) is a severe psychiatric disorder associated with alterations in early brain development. Details of underlying pathomechanisms remain unclear, despite genome and transcriptome studies providing evidence for aberrant cellular phenotypes and pathway deregulation in developing neuronal cells. However, mechanistic insight at the protein level is limited. Methods: Here, we investigate SCZ-specific protein expression signatures of neuronal progenitor cells (NPC) derived from patient iPSC in comparison to healthy controls using high-throughput Western Blotting (DigiWest) in a targeted proteomics approach. Results: SCZ neural progenitors displayed altered expression and phosphorylation patterns related to Wnt and MAPK signaling, protein synthesis, cell cycle regulation and DNA damage response. Consistent with impaired cell cycle control, SCZ NPCs also showed accumulation in the G2/M cell phase and reduced differentiation capacity. Furthermore, we correlated these findings with elevated p53 expression and phosphorylation levels in SCZ patient-derived cells, indicating a potential implication of p53 in hampering cell cycle progression and efficient neurodevelopment in SCZ. Conclusions: Through targeted proteomics we demonstrate that SCZ NPC display coherent mechanistic alterations in regulation of DNA damage response, cell cycle control and p53 expression. These findings highlight the suitability of iPSC-based approaches for modeling psychiatric disorders and contribute to a better understanding of the disease mechanisms underlying SCZ, particularly during early development.

Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream and the liver, the hepatic effects of OA are not well understood, highlighting a significant research gap. This study aims to comprehensively elucidate the impact of OA on the liver by examining the transcriptome, proteome, and phosphoproteome alterations in human HepaRG liver cells exposed to non-cytotoxic OA concentrations. We employed an integrative multi-omics approach, encompassing RNA sequencing, shotgun proteomics, phosphoproteomics, and targeted DigiWest analysis. This enabled a detailed exploration of gene and protein expression changes, alongside phosphorylation patterns under OA treatment. The study reveals concentration- and time-dependent deregulation in gene and protein expression, with a significant down-regulation of xenobiotic and lipid metabolism pathways. Up-regulated pathways include actin crosslink formation and a deregulation of apoptotic pathways. Notably, our results revealed that OA, as a potent phosphatase inhibitor, induces alterations in actin filament organization. Phosphoproteomics data highlighted the importance of phosphorylation in enzyme activity regulation, particularly affecting proteins involved in the regulation of the cytoskeleton. OA's inhibition of PP2A further leads to various downstream effects, including alterations in protein translation and energy metabolism. This research expands the understanding of OA's systemic impact, emphasizing its role in modulating the phosphorylation landscape, which influences crucial cellular processes. The results underscore OA's multifaceted effects on the liver, particularly through PP2A inhibition, impacting xenobiotic metabolism, cytoskeletal dynamics, and energy homeostasis. These insights enhance our comprehension of OA's biological significance and potential health risks.

Okadaic acid (OA) is a marine biotoxin that is produced by algae and accumulates in filter-feeding shellfish, through which it enters the human food chain, leading to diarrheic shellfish poisoning (DSP) after ingestion. Furthermore, additional effects of OA have been observed, such as cytotoxicity. Additionally, a strong downregulation of the expression of xenobiotic-metabolizing enzymes in the liver can be observed. The underlying mechanisms of this, however, remain to be examined. In this study, we investigated a possible underlying mechanism of the downregulation of cytochrome P450 (CYP) enzymes and the nuclear receptors pregnane X receptor (PXR) and retinoid-X-receptor alpha (RXRα) by OA through NF-κB and subsequent JAK/STAT activation in human HepaRG hepatocarcinoma cells. Our data suggest an activation of NF-κB signaling and subsequent expression and release of interleukins, which then activate JAK-dependent signaling and thus STAT3. Moreover, using the NF-κB inhibitors JSH-23 and Methysticin and the JAK inhibitors Decernotinib and Tofacitinib, we were also able to demonstrate a connection between OA-induced NF-κB and JAK signaling and the downregulation of CYP enzymes. Overall, we provide clear evidence that the effect of OA on the expression of CYP enzymes in HepaRG cells is regulated through NF-κB and subsequent JAK signaling.

Human platelet lysate (HPL) is an efficient alternative for animal serum supplements, significantly enhancing stromal cell proliferation. However, the molecular mechanism behind this growth-promoting effect remains elusive. The aim of this study was to investigate the effect of HPL on cell cycle gene expression in different human stromal cells and to identify the main key players that mediate HPL’s growth-enhancing effect. RT-qPCR and an antibody array revealed significant upregulation of cell cycle genes in stromal cells cultured in HPL. As HPL is rich in growth factors that are ligands of tyrosine kinase receptor (TKR) pathways, we used TKR inhibitors and could significantly reduce cell proliferation. Genome profiling, RT-qPCR and Western blotting revealed an enhanced expression of the transcription factors signal transducer and activator of transcription 3 (STAT3) and MYC, both known TKR downstream effectors and stimulators of cell proliferation, in response to HPL. In addition, specifically blocking STAT3 resulted in reduced cell proliferation and expression of cell cycle genes. Our data indicate that HPL-enhanced cell proliferation can, at least in part, be explained by the TKR-enhanced expression of STAT3 and MYC, which in turn induce the expression of genes being involved in the promotion and control of the cell cycle.

In cancer, the complex interplay between tumor cells and the tumor microenvironment results in the modulation of signaling processes. By assessing the expression of a multitude of proteins and protein variants in cancer tissue, wide-ranging information on signaling pathway activation and the status of the immunological landscape is obtainable and may provide viable information on the treatment response. Archived breast cancer tissues from a cohort of 84 patients (no adjuvant therapy) were analyzed by high-throughput Western blotting, and the expression of 150 proteins covering central cancer pathways and immune cell markers was examined. By assessing CD8α, CD11c, CD16 and CD68 expression, immune cell infiltration was determined and revealed a strong correlation between event-free patient survival and the infiltration of immune cells. The presence of tumor-infiltrating lymphocytes was linked to the pronounced activation of the Jak/Stat signaling pathway and apoptotic processes. The elevated phosphorylation of PPARγ (pS112) in non-immune-infiltrated tumors suggests a novel immune evasion mechanism in breast cancer characterized by increased PPARγ phosphorylation. Multiplexed immune cell marker assessment and the protein profiling of tumor tissue provide functional signaling data facilitating breast cancer patient stratification.