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The use of the anti-CD20 antibody rituximab has improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab was shown to act via various mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct apoptosis, or sensitization to chemotherapy (reviewed in ref. [1]). However, the biological function of CD20 and the reasons for the impressive activity of rituximab and other anti-CD20 antibodies remain elusive. It has been suggested, but remains controversial, whether CD20 functions as a calcium channel, couples with CD40 and MHCII, or B-cell receptor (BCR), and whether it has a role in T cell-dependent and -independent immunity [1,2,3,4]. This is also of great clinical interest for the design of combinatorial treatment of rituximab with BCR inhibitors, DNA damaging or immunomodulatory drugs, or CAR T cells. We have previously shown that microenvironmental interactions upregulate the CD20 levels on CLL cells through the CXCR4/SDF-1 axis [5]. Here we describe that higher CD20 expression has a direct role in the BCR signaling in CLL cells, and a BCR-proficient intra-clonal CLL cell subpopulation is more efficiently eliminated by rituximab in vivo due to higher CD20 levels.

The variable clinical course in chronic lymphocytic leukaemia (CLL) largely depends on p53 functionality and B-cell receptor (BCR) signalling propensity; however, it is unclear if there is any crosstalk between these pathways. We show that DNA damage response (DDR) activation leads to down-modulating the transcriptional factor FOXP1, which functions as a positive BCR signalling regulator and its high levels are associated with worse CLL prognosis. We identified microRNA (miRNA) miR-34a as the most prominently upregulated miRNA during DDR in CLL cells in vitro and in vivo during FCR therapy (fludarabine, cyclophosphamide, rituximab). MiR-34a induced by DDR activation and p53 stabilization potently represses FOXP1 expression by binding in its 3'-UTR. The low FOXP1 levels limit BCR signalling partially via derepressing BCR-inhibitory molecule CD22. We also show that low miR-34a levels can be used as a biomarker for worse response or shorter progression free survival in CLL patients treated with FCR chemoimmunotherapy, and shorter overall survival, irrespective of TP53 status. Additionally, we have developed a method for the absolute quantification of miR-34a copies and defined precise prognostic/predictive cutoffs. Overall, herein, we reveal for the first time that B cells limit their BCR signalling during DDR by down-modulating FOXP1 via DDR-p53/miR-34a axis.

MicroRNAs (miRNAs) represent important regulators of gene expression besides transcriptional control. miRNA regulation can be involved in the cell developmental fate decisions, but can also have more subtle roles in buffering stochastic fluctuations in gene expression. They participate in pathways fundamental to B-cell development like B-cell receptor (BCR) signalling, B-cell migration/adhesion, cell-cell interactions in immune niches, and the production and class-switching of immunoglobulins. miRNAs influence B-cell maturation, generation of pre-, marginal zone, follicular, B1, plasma and memory B cells. In this review, we discuss miRNAs with essential functions in malignant B-cell development (such as miR-150, miR-155, miR-21, miR-34a, miR-17-92 and miR-15-16). We also put these miRNAs in the context of normal B-cell differentiation, as this is intimately connected to neoplastic B-cell development. We review miRNAs' role in the most common B-cell malignancies, including chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). We focus on miR-contribution to the regulation of important signalling pathways (such as NF-κB, PI3K/AKT and TGF-β), BCR signalling and its modulators (such as PTEN, SHIP-1, ZAP-70, GAB1 and BTK), anti- and pro-apoptotic proteins (such as BCL2, MCL1, TCL1, BIM, p53 and SIRT1) and transcription factors (such as MYC, MYB, PU.1, FOXP1 and BCL6). We also discuss the association of miRNAs' expression levels with the patients' survival and response to therapy, summarizing their potential use as predictive and prognostic markers. Importantly, the targeting of miRNAs (like use of anti-miR-155 or miR-34a mimic) could provide a novel therapeutic approach as evidenced by tumour regression in xenograft mouse models and initial promising data from clinical trials.

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In chronic lymphocytic leukemia (CLL), the worst prognosis is associated with TP53 defects with the affected patients being potentially directed to alternative treatment. Therapy administration was shown to drive the selection of new TP53 mutations in CLL. Using ultra-deep next-generation sequencing (NGS), we performed a detailed analysis of TP53 mutations' clonal evolution. We retrospectively analyzed samples that were assessed as TP53-wild-type (wt) by FASAY from 20 patients with a new TP53 mutation detected in relapse and 40 patients remaining TP53-wt in relapse. Minor TP53-mutated subclones were disclosed in 18/20 patients experiencing later mutation selection, while only one minor-clone mutation was observed in those patients remaining TP53-wt (n=40). We documented that (i) minor TP53 mutations may be present before therapy and may occur in any relapse; (ii) the majority of TP53-mutated minor clones expand to dominant clone under the selective pressure of chemotherapy, while persistence of minor-clone mutations is rare; (iii) multiple minor-clone TP53 mutations are common and may simultaneously expand. In conclusion, patients with minor-clone TP53 mutations carry a high risk of mutation selection by therapy. Deep sequencing can shift TP53 mutation identification to a period before therapy administration, which might be of particular importance for clinical trials.