B cell receptor (BCR) signaling and T cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can utilize microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short non-coding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs. CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22), but also other candidates for a role in microenvironmental interactions. Notably, all three miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation, and significantly shorter overall survival of CLL patients. We identified Tumor-Necrosis Factor Receptor-Associated Factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream NFkB signaling. In CLL, BCR-represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40-NFkB signaling. This regulatory loop is disrupted by "BCR inhibitors" (BTK inhibitor ibrutinib or PI3K inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29-TRAF4-CD40 signaling axis modulated by the BCR activity.

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for BCR inhibitors such as ibrutinib or idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how precisely CLL cells regulate their homingintegrate multiple migratory signals while balancing survival in peripheral blood and the decision to return to immune niches. Here we provide evidence for the use of CXCR4/CD5 intraclonal subpopulations to study CLL cells migration regulation. We performed RNA profiling of CXCR4dimCD5bright versus CXCR4brightCD5dim cells and identified differential expression of dozens of molecules with a putative function cell migration. We have shown that GRB2 associated binding protein 1 (GAB1) positively regulates CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches is mediated by FoxO1-based transcriptional GAB1 activation. We also describe that upregulation of GAB1 plays an important role in maintaining basal PI3K activity and "tonic" AKT phosphorylation required to sustain survival of resting CLL cells. This is important during ibrutinib therapy since CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit "tonic" or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.