The extracellular matrix (ECM) controls tumour dissemination. We characterise ECM organization in human and mouse tumours, identifying three regions: tumour body, proximal invasive front and distal invasive front. Invasive areas show increased matrix density, fibre thickness, length, and alignment, with unique radial fibre orientation at the distal invasive front correlating with amoeboid invasive features. Using patient samples and murine models, we find that metastases recapitulate ECM features of the primary tumour. Ex vivo culture of murine cancer cells isolated from the different tumour regions reveals a spatial cytoskeletal and transcriptional memory. Several in vitro models recapitulate the in vivo ECM organisation showing that increased matrix induces 3D confinement supporting Rho-ROCK-Myosin II activity, while radial orientation enhances directional invasion. Spatial transcriptomics identifies a mechano-inflammatory program associated with worse prognosis across multiple tumour types. These findings provide mechanistic insights into how ECM organization shapes local invasion and distant metastasis.

Knowledge of the genomic landscape of chronic lymphocytic leukemia (CLL) grows increasingly detailed, providing challenges in contextualizing the accumulated information. To define the underlying networks, we here perform a multi-platform molecular characterization. We identify major subgroups characterized by genomic instability (GI) or activation of epithelial-mesenchymal-transition (EMT)-like programs, which subdivide into non-inflammatory and inflammatory subtypes. GI CLL exhibit disruption of genome integrity, DNA-damage response and are associated with mutagenesis mediated through activation-induced cytidine deaminase or defective mismatch repair. TP53 wild-type and mutated/deleted cases constitute a transcriptionally uniform entity in GI CLL and show similarly poor progression-free survival at relapse. EMT-like CLL exhibit high genomic stability, reduced benefit from the addition of rituximab and EMT-like differentiation is inhibited by induction of DNA damage. This work extends the perspective on CLL biology and risk categories in TP53 wild-type CLL. Furthermore, molecular targets identified within each subgroup provide opportunities for new treatment approaches.

The anti-CD20 monoclonal antibodies rituximab and obinutuzumab differ in their mechanisms of action, with obinutuzumab evoking greater direct B cell death. To characterize the signaling processes responsible for improved B cell killing by obinutuzumab, we undertook a phosphoproteomics approach and demonstrate that rituximab and obinutuzumab differentially activate pathways downstream of the B cell receptor. Although both antibodies induce strong ERK and MYC activation sufficient to promote cell-cycle arrest and B cell death, obinutuzumab exceeds rituximab in supporting apoptosis induction by means of aberrant SYK phosphorylation. In contrast, rituximab elicits stronger anti-apoptotic signals by activating AKT, by impairing pro-apoptotic BAD, and by releasing membrane-bound NOTCH1 to up-regulate pro-survival target genes. As a consequence, rituximab appears to reinforce BCL2-mediated apoptosis resistance. The unexpected complexity and differences by which rituximab and obinutuzumab interfere with signaling pathways essential for lymphoma pathogenesis and treatment provide important impetus to optimize and personalize the application of different anti-CD20 treatments.

We expanded the fBALM (DNA structure fluctuation-assisted binding activated localization microscopy) method by developing a stable methodological sequence that enables dual-color imaging of high-resolution genomic DNA together with LaminB1.

Germinal center (GC) B cells are pivotal in establishing a robust humoral immune response and long-term serological immunity while maintaining antibody self-tolerance. GC B cells rely on autophagy for antigen presentation and homeostatic maintenance. However, these functions, primarily associated with the light zone, cannot explain the spatiotemporal autophagy upregulation in the dark zone of GCs. Here, combining imaging, molecular, and genomic approaches, we defined a functional mechanism controlling chromatin accessibility in GC B cells during their dark zone transition. This mechanism links autophagy and nuclear lamin B1 dynamics with their downstream effects, including somatic hypermutation and antibody affinity maturation. Moreover, the autophagy–lamin B1 axis is highly active in the aberrant ectopic GCs in the salivary glands of Sjögren’s disease, defining its role in autoimmunity.

The anti-CD20 monoclonal antibody (mAb) rituximab in combination with chemotherapy has improved outcomes for patients with CD20⁺ B-cell lymphoma. Obinutuzumab was developed as an anti-CD20 mAb with enhanced induction of direct B-cell death and antibody-dependent cellular cytotoxicity. In direct comparison to rituximab, improved outcomes with obinutuzumab have been observed for chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), but not for diffuse large B-cell lymphoma (DLBCL). The molecular basis behind these discrepancies remained unknown. Our aim was to define intracellular signaling events in the induction of direct B-cell death upon rituximab and obinutuzumab treatment. We performed LC-MS/MS phosphoproteomics on SU-DHL4 lymphoma cells treated with rituximab or obinutuzumab (0, 1 and 24 h time-points). Kinase activities were inferred by kinase-substrate enrichment analysis linking kinases to their phosphosite substrates. Immunoblotting was done where necessary to understand the nature of specific signaling events. The activity of 41 and 40 protein kinases was altered upon rituximab or obinutuzumab treatment, respectively, 32 of which were affected by both mAbs. Pathway enrichment analyses revealed up-regulated B-cell receptor (BCR) signaling and down-regulated cell cycle progression with both treatments. To delineate differences between the two mAbs we investigated signaling events in the BCR cascade in more detail. The proximal BCR kinase SYK was strongly phosphorylated on Tyr³⁵² by both mAbs, whereas SYK Tyr^525/526 phosphorylation, essential for normal kinase function, was detectable at a low level only with rituximab. Phospho-SYK Tyr³⁵² in the absence of phospho-SYK Tyr^525/526 has been associated with autoimmune checkpoint activation and B-cell apoptosis. Both mAbs phosphorylated the key BCR kinase BTK on Tyr⁵⁵¹ but not on Tyr²²³. The latter site was essential for full kinase activity and, in line with incomplete BTK activation, downstream PCLγ2 phosphorylation was delayed. No evidence was found for PLCγ2 dependent de-phosphorylation of NFAT2/NFAT3 and PKCβ activation. Instead, we observed NFAT1 de-phosphorylation and PKCδ activation, both of which have been associated with an anergic B-cell reaction. The MAPK pathway and MYK were strongly activated by both mAbs serving as potent inducers of cell death in the absence of pro-survival signals. To determine if pro-survival signals were generated, we assessed PI3K and NF-κB activation. While we found no evidence for NF-κB activation, differences in the phosphorylation status of PI3K binding sites on CD19 and BCAP suggest stronger PI3K activation by rituximab than obinutuzumab. In keeping with this, the PI3K effector AKT was much more strongly activated by rituximab. AKT activity was likely also increased by Ca²⁺-flux following rituximab but not obinutuzumab treatment and modified by differential SHIP1 phosphorylation, a phosphatase that hydrolyzes PI3K-generated PIP3. A central AKT target in apoptosis regulation is BAD. We found increased BAD Ser⁹⁹ and Ser¹¹⁸ phosphorylation only after rituximab treatment. Rituximab thereby sequestered BAD in the cytosol and impaired its inhibitory effects on anti-apoptotic BCL-2 and BCL-xL. Moreover, signaling events protecting against caspase 2 activation were exclusively found for rituximab. In summary, here we show that rituximab and obinutuzumab both induce BCR signaling capable of promoting B-cell apoptosis. However, rituximab generates signals that increase anti-apoptotic BCL-2 effects and diminish B-cell death, whereas obinutuzumab more readily overcomes resistance through BCL-2 overexpression characteristic for FL and CLL. The differences found between rituximab and obinutuzumab are likely less pronounced in cases with strong tonic BCR signaling, which is more prevalent in DLBCL. Thus, our findings provide a mechanistic rationale for the observations made in clinical trials comparing rituximab and obinutuzumab head-to-head. We believe that our results will help to identify new patient subgroups benefitting from obinutuzumab and to better assess the role of anti-CD20 treatment in combination therapies.