LYN kinase programs stromal fibroblasts to facilitate leukemic survival via regulation of c-JUN and THBS1.

Microenvironmental bystander cells are essential for the progression of chronic lymphocytic leukemia (CLL). We have discovered previously that LYN kinase promotes the formation of a microenvironmental niche for CLL. Here we provide mechanistic evidence that LYN regulates the polarization of stromal fibroblasts to support leukemic progression. LYN is overexpressed in fibroblasts of lymph nodes of CLL patients. LYN-deficient stromal cells reduce CLL growth in vivo. LYN-deficient fibroblasts show markedly reduced leukemia feeding capacity in vitro. Multi-omics profiling reveals that LYN regulates the polarization of fibroblasts towards an inflammatory cancer-associated phenotype through modulation of cytokine secretion and extracellular matrix composition. Mechanistically, LYN deletion reduces inflammatory signaling including reduction of c-JUN expression, which in turn augments the expression of Thrombospondin-1, which binds to CD47 thereby impairing CLL viability. Together, our findings suggest that LYN is essential for rewiring fibroblasts towards a leukemia-supportive phenotype.

Distinct Genetically Determined Origins of Myd88/BCL2-Driven Aggressive Lymphoma Rationalize Targeted Therapeutic Intervention Strategies.

Genomic profiling revealed the identity of at least 5 subtypes of diffuse large B-cell lymphoma (DLBCL), including the MCD/C5 cluster characterized by aberrations in MYD88, BCL2, PRDM1, and/or SPIB. We generated mouse models harboring B cell-specific Prdm1 or Spib aberrations on the background of oncogenic Myd88 and Bcl2 lesions. We deployed whole-exome sequencing, transcriptome, flow-cytometry, and mass cytometry analyses to demonstrate that Prdm1- or Spib-altered lymphomas display molecular features consistent with prememory B cells and light-zone B cells, whereas lymphomas lacking these alterations were enriched for late light-zone and plasmablast-associated gene sets. Consistent with the phenotypic evidence for increased B cell receptor signaling activity in Prdm1-altered lymphomas, we demonstrate that combined BTK/BCL2 inhibition displays therapeutic activity in mice and in five of six relapsed/refractory DLBCL patients. Moreover, Prdm1-altered lymphomas were immunogenic upon transplantation into immuno-competent hosts, displayed an actionable PD-L1 surface expression, and were sensitive to antimurine-CD19-CAR-T cell therapy, in vivo. SIGNIFICANCE: Relapsed/refractory DLBCL remains a major medical challenge, and most of these patients succumb to their disease. Here, we generated mouse models, faithfully recapitulating the biology of MYD88-driven human DLBCL. These models revealed robust preclinical activity of combined BTK/BCL2 inhibition. We confirmed activity of this regimen in pretreated non-GCB-DLBCL patients. See related commentary by Leveille et al., p. 8. This article is highlighted in the In This Issue feature, p. 1.

Spleen tyrosine kinase mediates innate and adaptive immune crosstalk in SARS-CoV-2 mRNA vaccination.

Durable cell-mediated immune responses require efficient innate immune signaling and the release of pro-inflammatory cytokines. How precisely mRNA vaccines trigger innate immune cells for shaping antigen specific adaptive immunity remains unknown. Here, we show that SARS-CoV-2 mRNA vaccination primes human monocyte-derived macrophages for activation of the NLRP3 inflammasome. Spike protein exposed macrophages undergo NLRP3-driven pyroptotic cell death and subsequently secrete mature interleukin-1ß. These effects depend on activation of spleen tyrosine kinase (SYK) coupled to C-type lectin receptors. Using autologous cocultures, we show that SYK and NLRP3 orchestrate macrophage-driven activation of effector memory T cells. Furthermore, vaccination-induced macrophage priming can be enhanced with repetitive antigen exposure providing a rationale for prime-boost concepts to augment innate immune signaling in SARS-CoV-2 vaccination. Collectively, these findings identify SYK as a regulatory node capable of differentiating between primed and unprimed macrophages, which modulate spike protein-specific T cell responses.

The scaffold protein NEDD9 is necessary for leukemia-cell migration and disease progression in a mouse model of chronic lymphocytic leukemia.

The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in cancers, and is associated with poor clinical outcome. NEDD9 promotes B-cell adhesion, migration and chemotaxis, pivotal processes for malignant development. We show that global or B-cell-specific deletion of Nedd9 in chronic lymphocytic leukemia (CLL) mouse models delayed CLL development, markedly reduced disease burden and resulted in significant survival benefit. NEDD9 was required for efficient CLL cell homing, chemotaxis, migration and adhesion. In CLL patients, peripheral NEDD9 expression was associated with adhesion and migration signatures as well as leukocyte count. Additionally, CLL lymph nodes frequently expressed high NEDD9 levels, with a subset of patients showing NEDD9 expression enriched in the CLL proliferation centers. Blocking activity of prominent NEDD9 effectors, including AURKA and HDAC6, effectively reduced CLL cell migration and chemotaxis. Collectively, our study provides evidence for a functional role of NEDD9 in CLL pathogenesis that involves intrinsic defects in adhesion, migration and homing.

Constitutive activation of Lyn kinase enhances BCR responsiveness, but not the development of CLL in Eµ-TCL1 mice.

The treatment of chronic lymphocytic leukemia (CLL) has been improved dramatically by inhibitors targeting B-cell receptor (BCR)-associated kinases. The tyrosine kinase Lyn is a key modulator of BCR signaling and shows increased expression and activity in CLL. To evaluate the functional relevance of Lyn for CLL, we generated a conditional knockin mouse model harboring a gain-of-function mutation of the Lyn gene (LynY508F), which was specifically expressed in the B-cell lineage (Lynup-B). Kinase activity profiling revealed an enhanced responsiveness to BCR stimulation in Lynup-B B cells. When crossing Lynup-B mice with Eµ-TCL1 mice (TCL1tg/wt), a transgenic mouse model for CLL, the resulting TCL1tg/wt Lynup-B mice showed no significant change of hepatomegaly, splenomegaly, bone marrow infiltration, or overall survival when compared with TCL1tg/wt mice. Our data also suggested that TCL1 expression has partially masked the effect of the Lynup-B mutation, because the BCR response was only slightly increased in TCL1tg/wt Lynup-B compared with TCL1tg/wt. In contrast, TCL1tg/wt Lynup-B were protected at various degrees against spontaneous apoptosis in vitro and upon treatment with kinase inhibitors targeting the BCR. Collectively, and consistent with our previous data in a Lyn-deficient CLL model, these data lend further suggest that an increased activation of Lyn kinase in B cells does not appear to be a major driver of leukemia progression and the level of increased BCR responsiveness induced by Lynup-B is insufficient to induce clear changes to CLL pathogenesis in vivo.

Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides.

BACKGROUND: Approximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3',5'-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced. RESULTS: To quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity. CONCLUSIONS: We found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses.