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Boettcher, M.

Publications and source records attributed to Boettcher, M..

18 recordsLinked to original sources

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↗

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↗

Ryanodine receptor 1 is dispensable for CD4+ T-cell differentiation and effector function in intestinal inflammation models

T-cell receptor signaling is necessary for the activation and differentiation of CD4 T cells. Calcium (Ca2+) signaling is essential for this process, and the complexity of Ca2+ channels presents a potential therapeutic target for modulating the strength of T-cell receptor signaling and further differentiation of CD4 T cells. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing second messenger that triggers Ca2+ release through ryanodine receptor 1 (RYR1) in T cells. While the molecular and biophysical properties of NAADP-induced Ca2+ microdomains in T cells have been thoroughly investigated, and the function of the NAADP-HN1L/JPT2-RYR1 axis has been proven in T-cell activation and proliferation, its role in intestinal inflammation in vivo remains to be elucidated. In this study, we generated a conditional knockout mouse with Ryr1 deleted in {beta} T cells to investigate the functional relevance of RYR1 signaling in CD4 T cells. Ryr1 deletion in CD4+ T cells decreased TCR-induced Ca2+ microdomain formation, reduced peak Ca2+ amplitude and delayed initial velocity of global Ca2+ signaling in vitro. However, Ryr1 expression in CD4 T cells was dispensable for their pathogenicity in murine models of intestinal inflammation. Thus, Ryr1 expression in CD4+ T cells plays a redundant role in intestinal inflammation.

immunology↗

Perturb-seq identifies TCF7 as a central nexus linking MAPK- and Wnt-driven gene expression

The MAPK pathway is a central signaling cascade whose dysregulation contributes to numerous diseases. While its upstream regulation is well studied, the mechanisms by which MAPK activation leads to diverse transcriptional outcomes remain incompletely understood. To address this shortcoming, we mapped the target gene sets controlled by 22 RAF-inducible transcription factors using targeted Perturb-seq and integrated these data with time-resolved transcriptional profiling. Network reconstruction revealed a topology dominated by two central hubs, EGR1 and FOS, which co-regulate partially overlapping target gene sets. In addition, we uncovered a positive feedback loop between EGR1, a canonical RAF-MAPK effector, and TCF7, a transcription factor typically linked to Wnt signaling. Through this interaction, TCF7 emerges as a nexus that integrates MAPK and Wnt pathway inputs. Together, these findings define the architecture of RAF-MAPK-driven transcriptional regulation and demonstrate how cross-talk between oncogenic signaling pathways can be encoded in transcriptional networks. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/694273v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@53d6d5org.highwire.dtl.DTLVardef@3d6b5borg.highwire.dtl.DTLVardef@5333e6org.highwire.dtl.DTLVardef@74519e_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

Dysregulated circRNA expression profile and associated miRNA sponging in abnormal lung development in congenital diaphragmatic hernia

RationaleCircular RNAs (circRNAs) can function as disease biomarkers. Their profile in abnormal lung development in congenital diaphragmatic hernia (CDH) is unknown. ObjectiveTo evaluate circRNA expression profile in CDH-associated abnormal lung development. MethodsWe profiled circRNAs in rat CDH and control lungs at embryonic day (E)15 and E21 by microarray. We validated identified circRNAs using back-splice junction amplicon sequencing, RT-qPCR, and in situ hybridization. We modified a CircRNA Function prediction Tool to predict CircRNA::micro(mi)RNA::messenger(m)RNA interactions and compared these with Oxford Nanopore RNA sequencing and existing human CDH datasets. Measurements and Main ResultsMicroarrays revealed a unique circRNA biosignature during CDH lung development. CircAnp32e was expressed in a sex-specific and spatiotemporal expression pattern in the epithelium at E15. The predicted mature sequence of circAnp32e overlapped >90% with its human orthologue. CircRNA::miRNA::mRNA interaction networks in E15 and E21 revealed enrichment in inflammation/infection, smooth muscle cell function, cell proliferation/cell cycle regulation, and response to hypoxia pathways. Parental genes of differential expressed circRNAs at E15 enriched pathways linked to cell proliferation/cell cycle/cancer, while at end-gestation, inflammation and cardiovascular processes were also overrepresented. Rat and human CDH lungs showed overlapping pathways with additional enrichment for RNA processing and protein binding/modification in humans. ConclusionA unique circRNA signature during abnormal lung development in CDH may mediate inflammatory responses, smooth-muscle-cell function, and cell proliferation regulation via miRNA sponging. Overlap of downstream pathways in rat and human CDH suggest conserved functions across species. CircRNAs may serve as biomarkers to guide prenatal management and mitigate aberrant lung development in CDH.

developmental biology↗

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↗

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↗

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↗

Regulatory logic of human cortex evolution by combinatorial perturbations

Comparative genomic studies between contemporary and extinct hominins revealed key evolutionary modifications, but their number has hampered a system level investigation of their combined roles in scaffolding modern traits. Through multi-layered integration we selected 15 genes carrying nearly fixed sapiens-specific protein-coding mutations and developed a scalable design of combinatorial CRISPR-Cas9 bidirectional perturbations to uncover their regulatory hierarchy in cortical brain organoids. Interrogating the effects of overexpression and downregulation for all gene pairs in all possible combinations, we defined their impact on transcription and differentiation and reconstructed their regulatory architecture. We uncovered marked cell type-specific effects, including the promotion of alternative fates and the emergence of interneuron populations, alongside a core subnetwork comprising KIF15, NOVA1, RB1CC1 and SPAG5 acting as central regulator across cortical cell types.

systems biology↗

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↗

Targeted Perturb-seq Reveals EGR1 and FOS as Key Regulators of the Transcriptional RAF-MAPK Response

The MAPK pathway is an important cellular signaling cascade whose dysregulation causes a variety of diseases. While the upstream regulators of this cascade have been extensively characterized, the understanding of how its activation translates into different transcriptional responses remains poorly understood. This study attempts to fill this knowledge gap by using targeted Perturb-seq against 22 transcription factors in an inducible model system for RAF-MAPK signaling. A topology-based modeling approach is applied to the obtained data to construct a directional interaction network. By removing coherent feed-forward loops and integrating the expression kinetics of transcription factors, a parsimonious network structure is derived that distinguishes direct from indirect interactions between the investigated transcription factors and their targets. In particular, EGR1 and FOS are found to act as orthogonal upstream regulators of the RAF-MAPK response. The results presented here provide valuable insights into the organization of the transcriptional network downstream of RAF-MAPK signaling and thus provide a basis for a better understanding of this complex process.

systems biology↗

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↗

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↗

Identification of X-chromosomal genes that drive global X-dosage effects in mammals

X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Here, double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signalling pathway and delaying differentiation. To identify the genetic basis of these sex differences, we have performed a series of CRISPR knockout screens in murine embryonic stem cells to comprehensively identify X-linked genes that cause the female pluripotency phenotype. We found multiple genes that act in concert, among which Klhl13 plays a central role. We show that this E3 ubiquitin ligase substrate adaptor protein promotes pluripotency factor expression, delays differentiation and represses MAPK target genes, and we identify putative substrates. We thus elucidate the mechanisms that drive sex-induced differences in pluripotent cells with implications for gender medicine in the context of induced pluripotent stem cell based therapies.

genetics↗

High throughput CRISPR screening identifies genes involved in macrophage viability and inflammatory pathways

AbstractMacrophages are critical cells of the innate immune system involved in the recognition and destruction of invading microbes in addition to the resolution of inflammation and maintenance of homeostasis. Understanding the genes involved in all aspects of macrophage biology is essential to gaining new insights into immune system dysregulation during diseases that range from autoinflammatory to cancer. Here we utilize high throughput clustered regularly interspaced short palindromic repeats (CRISPR) screening to generate a resource that identifies genes required for macrophage viability and function. First, we employ a pooled based CRISPR/Cas nuclease active screening approach to identify essential genes required for macrophage viability by targeting genes within coding exons. In addition, we also target 3UTRs to gain insights into new cis-regulatory regions that control expression of these essential genes. Second, using our recently generated NF-{kappa}B reporter macrophage line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen to identify regulators of inflammation. We identify a number of novel positive and negative regulators of the NF-{kappa}B pathway as well as unraveling complexities of the TNF signaling cascade showing it can function in an autocrine manner to negatively regulate the pathway. Utilizing a single complex library design we are capable of interrogating various aspects of macrophage biology, generating a resource for future studies.\n\nAuthor SummaryExcess inflammation is associated with a variety of autoimmune diseases and cancers. Macrophages are important mediators of this inflammatory response. Defining the genes involved in their viability and effector function is needed to completely understand these two important aspects of macrophage biology. Here we screened over 21,000 genes and generated a resource guide of genes required for macrophage viability as well as novel positive and negative regulators of NF-{kappa}B signaling.

immunology↗

Leukocyte-derived High-mobility group box 1 controls innate immune responses against Listeria monocytogenes.

High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern with key proinflammatory functions following tissue injury. Moreover, HMGB1 neutralization was shown to alleviate LPS-induced shock, suggesting a role for the protein as a master therapeutic target for inflammatory and infectious diseases. Here, we report that HMGB1 neutralization impedes immune responses to Listeria monocytogenes, a wide-spread bacterium with pathogenic relevance for humans and rodents. Using genetic deletion strategies and neutralizing antibodies, we demonstrate that hepatocyte HMGB1, a major driver of post-necrotic inflammation in the liver, is dispensable for pathogen defense during moderately severe infection with listeria. In contrast, antibody-mediated HMGB1 neutralization and HMGB1 deficiency in myeloid cells effectuate rapid and uncontrolled bacterial dissemination in mice despite preserved basic leukocyte functionality and autophagy induction. During overwhelming infection, hepatocyte injury may contribute to increased HMGB1 serum levels and excessive inflammation in the liver, supporting context-dependent roles for HMGB1 from different cellular compartments during infection. We provide mechanistic evidence that HMGB1 from circulating immune cells contributes to the timely induction of hepatic immune regulatory gene networks, early inflammatory monocyte recruitment to the liver and promotion of neutrophil survival, which are mandatory for pathogen control. In summary, our data establish HMGB1 as a critical co-factor in the immunological clearance of listeria, and argue against HMGB1 neutralization as a universal therapeutic strategy for sepsis.\n\nAuthor summaryHigh-mobility group box 1 (HMGB1) is an abundantly expressed nucleoprotein with signaling properties following secretion or release into the extracellular space. Given its central immune-regulatory roles during tissue injury and LPS-induced septic shock, interventions aimed at HMGB1 signaling have been advocated as therapeutic options for various disease conditions. Here, we show that antibody-mediated HMGB1 neutralization interferes with immunological defense against Listeria monocytogenes, a gram-positive bacterium with high pathogenic relevance for rodents and humans, effectuating uncontrolled bacterial growth and inflammation. Using conditional knockout animals, we demonstrate that while leukocyte functionality is preserved in HMGB1-deficient myeloid cells, HMGB1 released in response to Listeria triggers hepatic inflammatory monocyte recruitment and activation of transcriptional immune networks required for the early control of bacterial dissemination. Hepatocyte HMGB1, a key driver of post-necrotic inflammation in the liver, is dispensable for the immune response during moderately severe infection, but likely contributes to excessive hepatitis when infection is uncontrolled and cellular injury is high. We demonstrate a critical and non-redundant role for HMGB1 in the immune-mediated clearance of listeriosis and argue against HMGB1 neutralization as a universal therapeutic option in the context of infection.

immunology↗

Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding

While pooled loss- and gain-of-function screening approaches have become increasingly popular to systematically investigate mammalian gene function, they have thus far ignored the fact that cell populations are heterogeneous. Here we introduce multi-level barcoded sgRNA libraries to (i) monitor differences in the behavior of multiplexed clonal cell lines, (ii) trace sub-clonal lineages of cells expressing the same sgRNA, (iii) derive in-sample screen replicates and (iv) reduce the number of cells and sequencing read counts required to reach statistical significance. Using our approach, we illustrate how clonal heterogeneity impairs the results of pooled genetic screens and demonstrate the ability of multi-level barcoding to resolve cellular heterogeneity related issues.

genomics↗

Decoding directional genetic dependencies through orthogonal CRISPR/Cas screens

Genetic interaction studies are a powerful approach to identify functional interactions between genes. This approach can reveal networks of regulatory hubs and connect uncharacterised genes to well-studied pathways. However, this approach has previously been limited to simple gene inactivation studies. Here, we present an orthogonal CRISPR/Cas-mediated genetic interaction approach that allows the systematic activation of one gene while simultaneously knocking out a second gene in the same cell. We have developed this concept into a quantitative and scalable combinatorial screening platform that allows the parallel interrogation of hundreds of thousands of genetic interactions. We demonstrate that the established platform works robustly to uncover genetic interactions in human cancer cells and to interpret the direction of the flow of genetic information.

molecular biology↗