No abstract

IntroductionProtective immunity against pathogens relies on the production of high-affinity antibodies by long-lived plasma cells (PCs). Moreover, the ability to respond faster and with more potent antibodies to subsequent encounters with the same infectious agent depends on the generation of long-lived memory B cells. Both, high-affinity memory B cells and PCs differentiate from antigenspecific B cells that are recruited into the GC reaction during T cell-dependent immune responses (1). In GCs, B cells undergo clonal expansion, a process during which they accumulate mutations at high frequency within the Ig heavy and light chain variable (V) region genes. The highly dynamic nature of the GC reaction is characterized by repeated cycles of cell division, Ig somatic mutation, and strict selection based on the ability of B cells to capture and present antigen to T follicular helper cells (2). These processes occur within distinct areas of the GC reached by B cells through migratory paths regulated by chemokine gradients (1). The molecular determinants enabling cyclic reentry of B cells into the proliferating and mutating compartment of centroblasts, preventing terminal differentiation and the ensuing exit from the GC, remain poorly characterized.

T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110δ, and class IB, p110γ catalytic subunits. However, the receptors on immature T cells by which each of these PI3Ks are activated have not been identified, nor has the mechanism behind their functional redundancy in the thymus. Here, we show that PI3K signaling from the preTCR requires p110δ, but not p110γ. Mice deficient for the class IB regulatory subunit p101 demonstrated the requirement for p101 in T cell development, implicating G protein–coupled receptor signaling in β-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of β-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos. Finally, we demonstrate that CXCL12, the ligand for CXCR4, allows for Notch-dependent differentiation of DN3 thymocytes in the absence of supporting stromal cells. These findings establish a role for CXCR4-mediated PI3K signaling that, together with signals from Notch and the preTCR, contributes to continued T cell development beyond β-selection.

Similar to resting mature B cells, where the B-cell antigen receptor (BCR) controls cellular survival, surface BCR expression is conserved in most mature B-cell lymphomas. The identification of activating BCR mutations and the growth disadvantage upon BCR knockdown of cells of certain lymphoma entities has led to the view that BCR signalling is required for tumour cell survival. Consequently, the BCR signalling machinery has become an established target in the therapy of B-cell malignancies. Here we study the effects of BCR ablation on MYC-driven mouse B-cell lymphomas and compare them with observations in human Burkitt lymphoma. Whereas BCR ablation does not, per se, significantly affect lymphoma growth, BCR-negative (BCR) tumour cells rapidly disappear in the presence of their BCR-expressing (BCR) counterparts in vitro and in vivo. This requires neither cellular contact nor factors released by BCR tumour cells. Instead, BCR loss induces the rewiring of central carbon metabolism, increasing the sensitivity of receptor-less lymphoma cells to nutrient restriction. The BCR attenuates glycogen synthase kinase 3 beta (GSK3β) activity to support MYC-controlled gene expression. BCR tumour cells exhibit increased GSK3β activity and are rescued from their competitive growth disadvantage by GSK3β inhibition. BCR lymphoma variants that restore competitive fitness normalize GSK3β activity after constitutive activation of the MAPK pathway, commonly through Ras mutations. Similarly, in Burkitt lymphoma, activating RAS mutations may propagate immunoglobulin-crippled tumour cells, which usually represent a minority of the tumour bulk. Thus, while BCR expression enhances lymphoma cell fitness, BCR-targeted therapies may profit from combinations with drugs targeting BCR tumour cells.

The synergism between c-MYC and miR-17-19b, a truncated version of the miR-17-92 cluster, is well-documented during tumor initiation. However, little is known about miR-17-19b function in established cancers. Here we investigate the role of miR-17-19b in c-MYC-driven lymphomas by integrating SILAC-based quantitative proteomics, transcriptomics and 3′ untranslated region (UTR) analysis upon miR-17-19b overexpression. We identify over one hundred miR-17-19b targets, of which 40% are co-regulated by c-MYC. Downregulation of a new miR-17/20 target, checkpoint kinase 2 (Chek2), increases the recruitment of HuR to c-MYC transcripts, resulting in the inhibition of c-MYC translation and thus interfering with in vivo tumor growth. Hence, in established lymphomas, miR-17-19b fine-tunes c-MYC activity through a tight control of its function and expression, ultimately ensuring cancer cell homeostasis. Our data highlight the plasticity of miRNA function, reflecting changes in the mRNA landscape and 3′ UTR shortening at different stages of tumorigenesis.