Biology subjects

Mougiakakos, D.

Publications and source records attributed to Mougiakakos, D..

11 recordsLinked to original sources

bioRxiv2026

A pro-inflammatory effector-memory T cell program links peripheral EBV priming to CNS autoimmunity

T and B cell-mediated immunosurveillance of the central nervous system (CNS) can become maladaptive in multiple sclerosis (MS). Epstein-Barr virus (EBV) is a likely driver of MS, yet how exactly EBV-specific T cells contribute remains unclear. We generated an antigen-resolved atlas of blood and cerebrospinal fluid (CSF) T cells from untreated MS patients and controls. T cell clones carrying a GZMK effector-memory program in blood were CSF-biased, particularly in MS, and associated with EBV reactivity. In CSF, EBV-reactive GZMK CD8 T cells engaged B lineage cells. Activated CD8 T cells promoted atypical B cell and plasmablast formation, whereas GZMK-enriched effector-memory cells induced IL-6 and IL-8 release from myeloid cells. Together, these findings identify an infection-imprinted, CNS-biased state and support a model in which EBV-specific CD8 T cells contribute to inflammation through a non-canonical mode of B cell and myeloid cell activation.

immunology↗

bioRxiv2026

Organelle proteomics reveals novel metabolic vulnerabilities in FLT3-ITD cells

In acute myeloid leukemia (AML), the insertion site of internal tandem duplications (ITDs) within the FLT3 gene critically determines the sensitivity to tyrosine kinase inhibitors (TKIs). Despite recent advances, patients harboring ITDs in the tyrosine kinase domain (TKD) still lack effective therapeutic options. To elucidate the molecular basis underlying the differential TKI sensitivity of FLT3-ITD cells, we integrated high-resolution mass spectrometry-based (phospho)proteomics with subcellular fractionation. Our analysis revealed that midostaurin induces the subcellular redistribution of approximately 2500 proteins involved in crucial biological processes, including cell cycle control, autophagy, and metabolism. Functional analyses further demonstrated that the ITD insertion site determines the autophagy response to midostaurin and modulates mitochondrial metabolism, influencing organelle architecture and ATP production, even at steady state. Importantly, by integrating subcellular proteomic dataset with functional metabolic assays, we uncovered a lipid-dependent vulnerability of FLT3-ITD cells: lipid restriction enhances FLT3 trafficking to the plasma membrane, and markedly reduces cell viability, restoring midostaurin sensitivity of resistant FLT3-ITD cells. Together, our findings reveal that the FLT3-ITD insertion site orchestrates a coordinated remodeling of subcellular protein organization, autophagy, and metabolism, and identify lipid-mediated control of FLT3 compartmentalization as a therapeutically actionable mechanism to overcome TKI resistance in FLT3-ITD AML.

cancer biology↗

bioRxiv2025

TCR activation impairs CAR-T cytotoxicity against separate target cells

Chimeric antigen receptor T cells (CAR-T) are effective therapeutics against cancer and autoimmunity, but whether the endogenous T-cell receptor (TCR) is beneficial, detrimental or irrelevant for CAR-T function and patient outcome remains unclear. We here traced anti-CD19 CAR-T clonotypes in patients with B-cell malignancies pre- and post-infusion using single-cell RNA-, TCR-, and CITE-sequencing. A cytotoxic phenotype, but not CAR-mediated in vitro reactivity to tumor cells, predicted CAR-T persistence. To test the functional impact of endogenous TCR activity on CAR-T behavior, we combined CAR transduction with orthotopic TCR replacement. This revealed that TCR signaling adds to activation of CAR-T cells, but gradually compromises CAR-mediated cytotoxicity, when TCR and CAR antigens are presented by different target cells. Therefore, spatial antigen separation alters TCR/CAR interplay with implications for therapeutic CAR-T design.

immunology↗

bioRxiv2025

Targeting Kinases to Reshape the Immunopeptidome landscape in Chronic Myeloid Leukemia

Antileukemia immunity is essential for disease control and maintaining treatment-free remission in chronic myeloid leukemia (CML). Immunotherapeutic approaches hold significant promise for enhancing immune-mediated disease control. However, successful immunotherapy relies on identifying CML-associated antigens and achieving robust antigen presentation. Protein kinases are central regulators of signaling, and protein turnover, influencing which peptides are processed and presented on HLA molecules. Indeed, previous works with CDK4/6 or MEK inhibitors showed that kinases influence immune recognition of cancer cells. However, the molecular mechanisms underlying the modulation of the immunopeptidome by kinases inhibitors treatment remained unclear. Here, we investigated whether pharmacological inhibition of key kinases, including BCR-ABL, JNK and LCK, could be leveraged to reshape the immunopeptidome of CML cells, enhancing the exposure of specific tumor-associated antigens. By employing high-sensitive mass spectrometry (MS)-based immunopeptidomics, we quantified more than 20,000 HLA-I peptides. Pharmacological suppression of LCK, JNK and BCR-ABL drastically remodels the antigen landscape, impacting the exposure of about 4,000 HLA-I ligands. By integrating immunopeptidomics, phospho-immunopeptidomics, and proteomic, our study revealed three complementary molecular mechanisms of kinase-dependent antigen control: direct phosphorylation of HLA-I peptides (i), modulation of source protein stability (ii), and control of transcription factors governing antigen expression (iii). Moreover, comparative HLA ligandome analysis of benign hematological specimens and CML primary samples identified a panel of about 90 frequently presented CML-exclusive peptides, whose presentation can be significantly increased by LCK and JNK inhibition. Our study establishes a framework to rationally combine kinase inhibitors with immunotherapies to enhance antigen visibility and improve antileukemic immunity.

cancer biology↗

bioRxiv2025

CD36 is a metabolic checkpoint for Th2 cell tissue residency during allergic airway inflammation

The prevalence of allergic diseases, including asthma, continues to rise in industrialized societies, yet the mechanisms sustaining pathogenic T helper 2 (Th2) responses remain incompletely understood. Here, we show that patients with allergic asthma exhibit elevated lipophilic volatile organic compounds in exhaled air and altered fatty acid-metabolism gene expression in sputum-derived Th2 cells. Using a mouse model of house dust mite-induced allergic airway inflammation, we find that the lipid transporter CD36 is dispensable for T follicular helper and germinal center B cell responses but is critical for maintaining lung-resident memory Th2 cells. CD36 regulates GATA3 and PPAR{gamma} expression in lung-resident memory Th2 cells and their interaction with type-2 conventional dendritic cells during airway inflammation. In human T cells, pharmacological inhibition of CD36 does not impair initial activation but blocks terminal Th2 differentiation. These findings identify CD36 as a metabolic checkpoint that sustains Th2 effector function and tissue residency, and establish lipid metabolism as a yet unrecognized therapeutic target in allergic asthma. SummaryAllergic asthma is marked by rising prevalence yet the drivers of persistent T helper 2 (Th2) immunity remain unclear. We show that asthma patients exhibit altered fatty acid-metabolism signatures in sputum Th2 cells and elevated lipophilic volatile organic compounds in exhaled air. In a mouse model of house dust mite-induced airway inflammation, the lipid transporter CD36 was dispensable for germinal center responses but essential for lung-resident memory Th2 cells, controlling GATA3 and PPAR{gamma} expression and promoting cDC2 interactions. Pharmacological inhibition of CD36 in human T cells preserved activation but blocked terminal Th2 differentiation. These findings identify CD36 as a metabolic checkpoint that sustains Th2 effector function and tissue residency, and nominate it as a therapeutic target in allergic asthma. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/676489v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@117da6eorg.highwire.dtl.DTLVardef@105934corg.highwire.dtl.DTLVardef@109903corg.highwire.dtl.DTLVardef@dc9345_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Patient-Specific Pharmacogenomics unveils xCT Regulation Pathways in Colon Cancer

Colorectal cancer (CRC) represents the third-leading cause of cancer-related deaths. Knowledge covering diverse cellular and molecular data from individual patients has become valuable for diagnosis, prognosis, and treatment selection. Here, we present an in-depth comparative mRNA-seq and microRNA-seq analysis of tissue samples from 32 CRC, pairing tumors with adjacent healthy tissues. The differential expression gene (DEG) analysis revealed an interconnection between nutrients, metabolic programs, and cell cycle pathways. We focused on the impact of overexpressed SLC7A11 (xCT) and SLC3A2 genes which compose the cystine/glutamate transporter (Xc-) system. To assess the oncogenic potency of the Xc-system in a cellular setting, we applied a knowledge-based approach for analyzing gene perturbations from CRISPR screens across various cell types as well as using a variety of functional assays in five primary patient-derived organoid cell models to functionally verify our hypothesis. We identified a previously undescribed cell surface protein signature predicting chemotherapy resistance and further highlighted the causality and potential of pharmacological blockage of ferroptosis as promising avenue for cancer therapy. Biological processes such as redox homeostasis, ion/amino acid transporters and de novo nucleotide synthesis were associated with these co-dependent genes in patient specimens. This study highlighted a number of overlooked genes as potential clinical targets for CRC and promotes stem cell-based, patient-individual in vitro model systems as a versatile partner platform to functionally validate in silico predictions, with focus on SLC7A11 and its associated genes in tumorigenesis.

cancer biology↗

bioRxiv2025

Glycolytic flux sustains human Th1 identity and effector function via STAT1 glycosylation

Fate decisions of T helper (Th) cells are tightly linked to their metabolic states, but precise mechanistic links remain unknown, especially in humans. Using in vitro stimulation in combination with gene editing we studied how metabolic regulation shapes human Th1 cell identity and effector function. Differentiated Th1 cells displayed elevated STAT1 phosphorylation at Tyr701 and Ser727 as well as heightened T-bet and IFN{gamma} expression, which were dampened by CRISPR/Cas9-mediated STAT1 deletion. Metabolic profiling revealed enhanced glycolytic activity in Th1 in comparison to Act.T cells, evidenced by increased extracellular acidification rate, ATP production via glycolysis, glucose uptake, lactate secretion and NADH abundance. SCENITH analysis demonstrated elevated glycolysis-dependent anabolic activity of Th1 cells. Inhibition of glycolysis reduced IFN{gamma} production and STAT1 phosphorylation independent of JAK1/2 activity, STAT1 abundance or SHP-2 activity, implicating glycolysis directly in sustaining STAT1-mediated Th1 functionality. Mechanistically, O-Glycosylation, facilitated by O-Glycosyltransferase, emerged as pivotal in modulating STAT1 activity, as evident through immunoprecipitation and Western blot analysis. Pharmaceutical O-Glycosyltransferase inhibition prevented Th1 differentiation as well as STAT1 O-glycosylation. CRISPR/Cas9 mediated mutation of the O-glycosylation sites Ser499 and Thr510 sites diminished STAT1 Ser727 phosphorylation and IFN{gamma} synthesis. Together, this study highlights glycolysis as key regulator of human Th1 cell identity and effector function, with STAT1 O-Glycosylation selectively maintaining Th1 effector capacity. This mechanism could be explored to safeguard Th1 cells in antiviral immunity and autoimmunity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/636644v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1ee58aeorg.highwire.dtl.DTLVardef@1001af6org.highwire.dtl.DTLVardef@1ea2ed7org.highwire.dtl.DTLVardef@1a943fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Phosphoproteomics reveals novel BCR::ABL1-independent mechanisms of resistance in chronic myeloid leukemia

BCR::ABL1 drives chronic myeloid leukemia (CML) disease and treatment, as revealed by the success of tyrosine kinase inhibitor (TKI) therapy. However, additional poorly characterized molecular pathways, acting as BCR::ABL1 independent mechanisms, play crucial roles in CML, contributing to leukemic stem cells (LSCs) persistence, TKI resistance and disease progression. Here, by combining high sensitive mass spectrometry (MS)-based phosphoproteomics with the SignalingProfiler pipeline, we obtained two signaling maps offering a comprehensive description of the BCR::ABL1 dependent and independent pro-survival signalling mechanisms. We leveraged these maps to unbiasedly and systematically discover therapeutic vulnerabilities, by implementing the Druggability Score computational algorithm. By this strategy, and in combination with in vitro and in ex vivo functional assays, we show a crucial role of acquired FLT3-dependency in resistant CML models. In conclusion, we reposition FLT3, one of the most frequently mutated drivers of acute leukemia, as a potential therapeutic target for TKI resistant CML patients.

systems biology↗

bioRxiv2023

Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior-knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

cancer biology↗

bioRxiv2022

A key role of the WEE1-CDK1 axis in mediating TKI-therapy resistance in FLT3-ITD positive acute myeloid leukemia patients

Internal tandem duplications (ITDs) in the FLT3 gene are frequently identified and confer a poor prognosis in patient affected by acute myeloid leukemia (AML). The insertion site of the ITDs in FLT3 significantly impacts the sensitivity to tyrosine kinase inhibitors (TKIs) therapy, affecting patients clinical outcome. To decipher the molecular mechanisms driving the different sensitivity to TKIs therapy of FLT3-ITD mutation, we used high-sensitive mass spectrometry-based (phospho)proteomics and deep sequencing. Here, we present a novel generally-applicable strategy that supports the integration of unbiased large-scale datasets with literature-derived signaling networks. The approach produced FLT3-ITDs specific predictive models and revealed a crucial and conserved role of the WEE1-CDK1 axis in TKIs resistance. Remarkably, we found that pharmacological inhibition of the WEE1 kinase synergizes and strengthens the pro-apoptotic effect of TKIs therapy in cell lines and patient-derived primary blasts. In conclusion, this work proposes a new molecular mechanism of TKIs resistance in AML and suggests a combination therapy as option to improve therapeutic efficacy.

cancer biology↗

bioRxiv2021

Mitochondrial function is essential for humoral immunity by controlling flux of the TCA cycle, phosphatidic acid and mTOR activity in B cells

The function of mitochondrial respiration during B cell fate decisions and differentiation remains equivocal. This study reveals that selection for mitochondrial fitness occurs during B cell activation and is essential for subsequent plasma cell differentiation. By expressing a mutated mitochondrial helicase in transitional B cells, we depleted mitochondrial DNA during B cell maturation, resulting in reduced oxidative phosphorylation. Although no changes in follicular B cell development were evident, germinal centers, class switch recombination to IgG, plasma cell generation and humoral immunity were diminished. Defective oxidative phosphorylation led to aberrant flux of the tricarboxylic acid cycle and lowered the amount of saturated phosphatidic acid. Consequently, MTOR activity and BLIMP-1 induction were curtailed whereas HIF1, glycolysis and AMPK activity were amplified. Exogenous phosphatidic acid increased mTOR activity in activated B cells. Hence, mitochondrial function is required and selected for in activated B cells for the successful generation of functional plasma cells.

immunology↗