Biology subjects

Brueggemann, M.

Publications and source records attributed to Brueggemann, M..

10 recordsLinked to original sources

bioRxiv2026

RNA G-quadruplexes mediate cooperativity in HNRNPH binding and splicing regulation

Alternative splicing, regulated by RNA-binding proteins (RBPs), enables the generation of diverse transcript isoforms critical for cellular function. However, how RNA secondary structure impacts RBP binding and function remains poorly understood. Here, we unravel how RNA G-quadruplexes (rG4s) facilitate cooperativity in splicing regulation by the RBP heterogeneous nuclear ribonucleoprotein H (HNRNPH). Through high-throughput in vivo and in vitro studies combined with theoretical modeling, we dissect how rG4s mediate cooperative HNRNPH binding to RNA, ultimately modulating the splicing of hundreds of exons. rG4 unfolding by HNRNPH exposes multiple G-rich binding sites, thereby establishing indirect cooperativity, which is further amplified to achieve switch-like splicing regulation. HNRNPH-mediated regulation is evident in breast cancer patients, with tumors showing rG4-disrupting variants and global HNRNPH alterations, driving distinct splicing patterns that distinguish tumor subtypes. Overall, our findings offer valuable insights into the mechanistic role of RNA secondary structures in cooperative RBP binding and splicing regulation and highlight the clinical relevance of HNRNPH-dependent splicing in cancer. HighlightsO_LIHundreds of cassette exons are cooperatively regulated by HNRNPH. C_LIO_LIUnfolding of RNA G-quadruplexes (rG4s) at HNRNPH binding sites facilitates indirect cooperativity in RNA binding. C_LIO_LIMulti-step splicing amplifies the response into highly switch-like regulation. C_LIO_LIrG4-disrupting variants and changing HNRNPH expression are associated with breast cancer phenotypes. C_LI

molecular biology↗

bioRxiv2026

A Novel Natural Killer Cell Expansion Technology for the Development of Cellular Immunotherapies

Adoptive cell therapy based on Natural Killer (NK) cells holds great promise for the treatment of cancer. For all approaches aiming at utilizing NK cells in immunotherapy, efficient ex vivo expansion technologies for the generation on of cytotoxic NK cells are a prerequisite for clinical translation. In this study, a novel multifunctional fusion protein consisting of a CD20-directed Fab-fragment, an agonistic anti-4-1BB single-chain Fragment variable (scFv), the Sushi domain of the interleukin (IL)-15 receptor and human IL-15 was generated. This molecule triggered strong NK cell expansion when bound to co-cultivated autologous B cells, due to trans-presentation of IL-15 and binding to 4-1BB/CD137. Expansion rates of up to 7,500-fold were achieved and the NK cells showed high cytotoxic capacity against a panel of tumor cell lines representing various tumor entities. Importantly, the activated NK cells did not show cytolytic activity against non-malignant B cells indicating that NK cells amplified by our novel approach were still physiologically regulated. The cytotoxic activity of the expanded NK cells was further enhanced by combination with therapeutic antibodies. Our molecule was additionally able to trigger efficient proliferation of NK cells from cord blood as well as multiple myeloma (MM) and acute myeloid leukemia (AML) patients. In conclusion, our novel platform technology provides ex vivo expansion of NK cells by using a single multifunctional fusion protein and may be well-suited for the development of NK cell-based immunotherapies. Key pointsA novel fusion protein that enables NK cell expansion from different sources including peripheral blood, bone marrow and cord blood

immunology↗

bioRxiv2025

Blinatumomab-driven T-cell activation in αβ and γδ T-cell subsets: Insights from in vitro assays

Blinatumomab (BLN) is a bispecific T-cell engager that has revolutionized the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), significantly improving outcomes in both adults and children. By simultaneously binding to CD19 on B cells and CD3 on T cells, BLN triggers target cell-dependent T-cell activation, resulting in the cytolysis of CD19+ BCP-ALL cells. Despite the remarkable clinical advancements achieved with BLN, the immunological mechanisms underlying treatment response or failure remain poorly characterized. {gamma}{delta} T cells are attractive candidates for adoptive T-cell therapy due to potent cytotoxicity, capacity to present antigens, broad lysis of different tumor entities, and low alloreactivity. Because {gamma}{delta} T cells can also be redirected by BLN, we systematically studied BLN-driven effector functions of conventional {beta} and unconventional {gamma}{delta} T cells. We evaluated cytotoxicity and cytokine/effector release in freshly isolated and in vitro-expanded {beta} and {gamma}{delta} T cells from healthy adults against CD19 BCP-ALL lines (NALM-6, HAL-01), and profiled dynamic phenotypic alterations by multiparametric flow cytometry. CD19 targets were consistently reduced in the presence of BLN. Freshly isolated {beta}, especially CD8, displayed superior BLN-mediated cytotoxicity as compared to {gamma}{delta} T cells, with donor-dependent variability in {gamma}{delta} killing. Notably, zoledronate-expanded V{gamma}9V{delta}2 {gamma}{delta} T-cell lines achieved cytotoxicity comparable to PHA-expanded {beta} cells. However, {gamma}{delta} T-cell-killing benefited from higher BLN concentration when challenged with high tumor load. BLN induced CD3 down-modulation in {beta} T cells but not in {gamma}{delta} T cells, alongside higher soluble Fas ligand in {beta} cultures, consistent with stronger early activation, preceding activation-induced cell death. {gamma}{delta} T cells showed no such changes, suggesting reduced susceptibility to activation-induced cell death. Single-cell RNA and flow analyses corroborated these findings, showing robust activation/exhaustion programs in {beta} T cells and a stable effector-memory state with low checkpoint expression in {gamma}{delta} T cells. Together, these data reveal subset-specific BLN responses and support expanded V{gamma}9V{delta}2 {gamma}{delta} T cells as a rational adoptive partner to BLN -- particularly in settings of favorable antigen density/low tumor burden -- providing complementary cytotoxicity with potentially reduced inflammatory liability. These findings provide a framework for combining {gamma}{delta} T-cell-based therapies in BLN-treated patients for improving BLN efficacy in BCP-ALL patients.

immunology↗

bioRxiv2025

A novel Fc-optimized antibody drug conjugate targeting CD7 for the therapy of T-cell acute lymphoblastic leukemia

While treatment for patients with T-cell acute lymphoblastic leukemia (T-ALL) has improved in the last decades, therapeutic options for patients refractory to standard therapy or with relapsing disease are limited. In particular, no immunotherapy option has been approved in T-ALL yet. Here, a novel dual antibody engineering approach for targeting CD7 was evaluated. The chimeric CD7 antibody chimTH69 was modified by Fc engineering to improve antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In addition, it was conjugated to monomethyl auristatin E (MMAE), a microtubule-disrupting agent. The resulting Fc-optimized antibody-drug conjugate (ADC), designated chimTH69-DE-vcMMAE, showed a unique set of effector functions in vitro. It triggered ADCC by mononuclear cells at picomolar concentrations, mediated ADCP by macrophages and directly inhibited the growth of a panel of T-ALL cell lines by delivering the cytotoxic compound to induce G2 cell cycle arrest and apoptosis. In addition, due to its specific linker design, chimTH69-DE-vcMMAE demonstrated bystander killing activity against CD7-negative leukemia cells. In mice, CD7-directed therapy with chimTH69-DE-vcMMAE inhibited the growth of subcutaneous CCRF-CEM T-ALL xenografts. Moreover, chimTH69-DE-vcMMAE exerted strong antileukemic effects in a phase II-like patient-derived xenograft preclinical trial in pediatric and adult patients when applied in an experimental overt leukemia setting. ChimTH69-DE-vcMMAE induced minimal residual disease-negativity in one PDX model. These findings indicate that targeting CD7 with the novel Fc-optimized ADC is a potent strategy to trigger anti-leukemia responses and may open a novel therapeutic avenue for T-ALL treatment. Key PointA novel antibody drug conjugate targeting CD7 showed efficient anti-leukemia activity in preclinical models of T-ALL.

cancer biology↗

bioRxiv2025

IntegrateALL: an end-to-end RNA-seq analysis pipeline for multilevel data extraction and interpretable subtype classification in B-precursor ALL

Transcriptome sequencing (RNA-seq) is emerging as a diagnostic standard for B-cell precursor acute lymphoblastic leukemia (B-ALL). Expression-based classifiers reach [~]95% accuracy, but reproducible end-to-end solutions that also integrate transcript-derived genomic drivers and quantitative virtual karyotyping are lacking. We developed IntegrateALL, a Snakemake pipeline that standardizes RNA-seq analysis from FASTQ to rule-based subtype assignment across 26 WHO-HAEM5/ICC entities by integrating expression-based subtype prediction, gene fusion- / hotspot SNV calling and virtual karyotyping. We introduce KaryALL, a machine-learning classifier that uses normalized expression and minor-allele-frequency features (RNASeqCNV) to distinguish near haploid, hypodiploid and high hyperdiploid B-ALL and chromosome-21 gains/iAMP21 (accuracy: 0.98 / F1-score: 0.96 on 615 independent test samples). SNP-array concordance supported RNA-based karyotyping. Applied to 774 unselected B-ALL cases, IntegrateALL yielded unambiguous subtype assignments in 81.5%, based on concordance of gene expression class with a defining driver (75.3% of all cases) or, in selected cases, high-confidence expression-based classification alone (6.2%); the remainder (18.5%) were flagged for manual curation. Independent validation (3 cohorts; n=436, including pediatric cases) reproduced these distributions. Across all patients (n=1,210), 2.6% harbored two subtype defining drivers, including hyperdiploidy in fusion-driven subtypes where it was not expected or subtype-defining SNVs (e.g., PAX5 P80R / IKZF1 N159Y) co-occurring with BCR::ABL1-positive/-like, KMT2A- or DUX4-fusions. In most dual-driver cases, one subtype gene expression signature predominated, indicating a hierarchy of oncogenic control and the value of systematic driver screening alongside expression-based calls. IntegrateALL provides an adaptable fully reproducible workflow for molecular B-ALL characterization by systematically integrating genomic drivers and downstream gene regulation.

cancer biology↗

bioRxiv2025

Myeloid cell-mediated killing of B-ALL by CD38 and CD20IgA antibody variants is enhanced by CD47/SIRPα interference

Enhancing myeloid effector cell recruitment may improve immunotherapy by monoclonal antibodies - including that against acute lymphoblastic leukemia (ALL). To assess expression of target antigens in B-ALL, we compared mRNA profiling of 559 patient leukemia samples across 18 molecular subtypes with that of representative cell lines. The latter served as target cells to compare human IgG1 or IgA2 variants against CD19, CD20 or CD38 in antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cell-mediated cytotoxicity (ADCC) by polymorphonuclear leukocytes (PMN). Interestingly, antibodies against broadly expressed CD19 were negligibly effective in mediating ADCP or ADCC. Antibodies against CD20 or CD38, the former variably expressed across subtypes, triggered ADCP by macrophages both as IgG1 and IgA2. However, PMN mediated ADCC against CD20 or CD38 was only observed with IgA2 variants, but not with respective IgG1 antibodies. Blocking the myeloid checkpoint molecule CD47 with a CD47 antibody or a soluble SIRP-Fc fusion protein enhanced ADCP and ADCC by IgA2 antibodies. The binding site for SIRP on CD47 contains an N-terminal pyroglutamate (pGlu), whose formation is catalyzed by glutaminyl-peptide cyclotransferase like (QPCTL). The direct involvement of pGlu in CD47/SIRP interactions was shown by using engineered CD47 variants. Both CD47 and QPCTL were broadly expressed across BCP-ALL subtypes, indicating QPCTL inhibitors as additional therapeutic option. Importantly, the combination of anti-CD38 IgA2 and CD47 blockade was effective against xenografted B-ALL cells in human FcRI (CD89) transgenic (tg) NXG mice. Together, these studies support the combination of anti-CD38 IgA2 with CD47 interference to improve myeloid effector cell recruitment for immunotherapy of B-ALL. Data sharing statementRNA-Seq data from BCP-ALL patients and B cells from healthy donors are available in the European Genome-Phenome Archive (EGA) accession numbers EGAS00001006107 and EGAS00001007305, respectively. Mass spectrometry data of analyzed proteins will be made available after manuscript acceptance on PRIDE - PRoteomics IDEntifications Database. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/669665v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@15220daorg.highwire.dtl.DTLVardef@eace04org.highwire.dtl.DTLVardef@17b87daorg.highwire.dtl.DTLVardef@1142822_HPS_FORMAT_FIGEXP M_FIG C_FIG Key pointsO_LIIgG1 and IgA2 antibodies against CD38 or CD20 were effective in recruiting macrophages for ADCP, but only IgA2 triggered ADCC by PMN C_LIO_LIMyeloid effector cell activation was enhanced by interfering with the CD47/SIRP axis, especially when IgA2 antibodies were applied C_LI

cancer biology↗

bioRxiv2025

Context-dependent effects of mutations on complex splicing decisions

Alternative pre-mRNA splicing is an essential step in human gene regulation, and mutation-induced aberrant splicing is frequently found in diseases and therapy resistance. Splicing regulation is highly dependent on sequence and cellular context, posing a challenge to predict outcomes of splicing-related disease mutations. Here, we use kinetic modeling to derive the underlying quantitative principles, describing splice site competition and downstream effects on a wide spectrum of splice isoforms. Employing statistical learning on a large-scale mutagenesis dataset for CD19 mRNA splicing, our model quantitatively describes the generation of 93 RNA isoforms. It takes into account various splicing events such as cassette exon skipping, intron retention, and extensive alternative 3 and 5 splice site usage, which are implicated in CART therapy resistance in leukemia. Beyond CD19, by analyzing genome-wide RNA sequencing data and large-scale screening of synthetic splicing decisions, we find that splice site distance is an important parameter controlling the switch from canonical to alternative/cryptic splice site usage, as mutations located in between two nearby splice sites show a pronounced directionality, regulating up- and downstream effectors in opposite direction. Taken together, our work demonstrates that a quantitative description of splice site competition provides insights into context-dependent, complex isoform changes and cryptic splice site activation in health and disease.

systems biology↗

bioRxiv2024

Long-read transcriptome sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations

BackgroundMutations in splicing factor 3B subunit 1 (SF3B1) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS). These mutations have a different effect on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options. ResultsWe applied long-read transcriptome sequencing to 44 MDS and CLL patients with and without SF3B1 mutations and found > 60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3 splice sites. Our data highlighted a constrained window at canonical 3 splice sites in which dynamic splice site switches occurred in SF3B1-mutated patients. Using transcriptome-wide RNA binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3 splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1K700E. ConclusionsOur work presents the hitherto most complete long-read transcriptome sequencing study in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosis results most likely from the different cell types expanded during cancerogenesis rather than different mechanism of action of the mutated SF3B1. These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases. HighlightsO_LILong-read transcriptome sequencing data enables the identification of > 60% of novel isoforms in the transcriptomes of CLL and MDS patients and isogenic cell lines. C_LIO_LISF3B1 mutations trigger common splicing alterations upon SF3B1 mutations across patient cohorts, most frequently decreased intron retention and increased alternative 3 splice site usage. C_LIO_LIMutation effect depends on alternative 3 splice site and branch point positioning that coincide with bimodal SF3B1 binding at these sites C_LIO_LIMolecular dynamics simulations predict reduced binding of SF3B1K700E to mRNA at the second binding pocket harboring the polypyrimidine tract. C_LI

cancer biology↗

bioRxiv2023

Mutational and transcriptional landscape of pediatric B-cell precursor lymphoblastic lymphoma

Pediatric B-cell precursor (BCP) lymphoblastic malignancies are neoplasms with manifestation either in bone marrow/blood (BCP acute lymphoblastic leukemia, BCP-ALL) or less common in extramedullary tissue (BCP lymphoblastic lymphoma, BCP-LBL). Although both presentations are similar in morphology and immunophenotype molecular studies are virtually restricted to BCP-ALL so far. The lack of molecular studies on BCP-LBL is probably due to its rarity and the restriction to tiny, mostly formalin-fixed paraffin embedded (FFPE) tissues. Here we present the first comprehensive mutational and transcriptional analysis of what we consider the largest BCP-LBL cohort described to date (n=97). Whole exome sequencing indicates a mutational spectrum of BCP-LBL strikingly similar to that found in BCP-ALL. However, epigenetic modifiers were more frequently mutated in BCP-LBL, whereas BCP-ALL was more frequently affected by mutation in genes involved in B-cell development. Integrating copy number alterations, somatic mutations and gene expression by RNA-sequencing revealed virtually all molecular subtypes originally defined in BCP-ALL to be present in BCP-LBL too, with only 7% of lymphomas that were not assigned to a subtype. Therefore, the results here described may pave the way for molecular risk adapted treatment protocols for BCP-LBL patients. KeypointsComprehensive molecular characterization of B-cell precursor lymphoblastic lymphoma allows molecular subtyping analogous to leukemias Compared to leukemias, lymphomas show more alterations in epigenetic modifiers and less in B-cell development genes

cancer biology↗

bioRxiv2023

The gene expression classifier ALLCatchR identifies B-precursor ALL subtypes and underlying developmental trajectories across age

Current classifications (WHO-HAEM5 / ICC) define up to 26 molecular B-cell precursor acute lymphoblastic leukemia (BCP-ALL) disease subtypes, which are defined by genomic driver aberrations and corresponding gene expression signatures. Identification of driver aberrations by RNA-Seq is well established, while systematic approaches for gene expression analysis are less advanced. Therefore, we developed ALLCatchR, a machine learning based classifier using RNA-Seq expression data to allocate BCP-ALL samples to 21 defined molecular subtypes. Trained on n=1,869 transcriptome profiles with established subtype definitions (4 cohorts; 55% pediatric / 45% adult), ALLCatchR allowed subtype allocation in 3 independent hold-out cohorts (n=1,018; 75% pediatric / 25% adult) with 95.7% accuracy (averaged sensitivity across subtypes: 91.1% / specificity: 99.8%). High confidence predictions were achieved in 84.6% of samples with 99.7% accuracy. Only 1.2% of samples remained unclassified. ALLCatchR outperformed existing tools and identified novel candidates in previously unassigned samples. We established a novel RNA-Seq reference of human B-lymphopoiesis. Implementation in ALLCatchR enabled projection of BCP-ALL samples to this trajectory, which identified shared patterns of proximity of BCP-ALL subtypes to normal lymphopoiesis stages. ALLCatchR sustains RNA-Seq routine application in BCP-ALL diagnostics with systematic gene expression analysis for accurate subtype allocations and novel insights into underlying developmental trajectories.

cancer biology↗