Stochastically Timed Competition Between Division and Differentiation Fates Regulates the Transition From B Lymphoblast to Plasma Cell.

In response to external stimuli, naïve B cells proliferate and take on a range of fates important for immunity. How their fate is determined is a topic of much recent research, with candidates including asymmetric cell division, lineage priming, stochastic assignment, and microenvironment instruction. Here we manipulate the generation of plasmablasts from B lymphocytes in vitro by varying CD40 stimulation strength to determine its influence on potential sources of fate control. Using long-term live cell imaging, we directly measure times to differentiate, divide, and die of hundreds of pairs of sibling cells. These data reveal that while the allocation of fates is significantly altered by signal strength, the proportion of siblings identified with asymmetric fates is unchanged. In contrast, we find that plasmablast generation is enhanced by slowing times to divide, which is consistent with a hypothesis of competing timed stochastic fate outcomes. We conclude that this mechanistically simple source of alternative fate regulation is important, and that useful quantitative models of signal integration can be developed based on its principles.

Delineating the distinct role of AKT in mediating cell survival and proliferation induced by CD154 and IL-4/IL-21 in chronic lymphocytic leukemia.

The functional significance of AKT in chronic lymphocytic leukemia (CLL) remains unclear. Given the importance of non-malignant T cells in regulating clonal expansion in CLL, we investigated the role of AKT in T cell-mediated cytoprotection and proliferation using an established co-culture system in which primary CLL cells were incubated on a monolayer of transfected mouse fibroblasts expressing human CD40L (CD154). Stimulation of CLL cells via CD40 induced activation of AKT, which was closely associated with downregulation of its negative regulator PTEN, and protected CLL cells from killing by bendamustine. This cytoprotective effect of CD40 stimulation was prevented by a selective inhibitor of AKT. Stimulation of CLL cells with CD154 + IL-4 or IL-21 induced proliferation detected as reduced fluorescence of cells pre-stained with CFSE. AKT inhibition produced a significant, consistent reduction in proliferation induced by CD154 + IL-4 and a reduction in proliferation induced by CD154 + IL-21 in most but not all cases. In contrast, AKT inhibition had no effect on the proliferation of normal B cells induced by CD154 + IL-4 or IL-21. These findings indicate that AKT contributes in a significant way to T-cell mediated survival and proliferation signalling in CLL and support the clinical evaluation of AKT inhibitors in this disease.

The expression of endothelin-1 in chronic lymphocytic leukemia is controlled by epigenetic mechanisms and extracellular stimuli.

Endothelin-1 (ET-1) is a hormone peptide widely expressed and is involved in several biological processes, important not only for normal cell function but also for tumor development, including cell proliferation, invasion, metastasis, angiogenesis and osteogenesis. In accordance, ET-1 was already shown to contribute to the growth and progression of many different solid cancers. We recently demonstrated that ET-1 has a role in the pathogenesis of chronic lymphocytic leukemia (CLL) where it is abnormally expressed. In the context of this malignancy, ET-1 is able to mediate survival, drug-resistance and growth signals in leukemic cells. Previous studies, not conducted in CLL, have shown that ET-1 regulatory mechanisms are numerous and cell specific. Here, we valued the expression of ET-1 in CLL, in relation to DNA methylation but also in response to stimulation of some important pathways for the dialogue between CLL and microenvironment. We found that a high methylation of ET-1 first intron affects the basal expression of ET-1 in CLL. Moreover, we showed that the activation of CD40 or Toll-like receptor (TLR) by extracellular stimuli produces an augment of ET-1 level in CLL cells. Finally, we demonstrated the fundamental role of NF-kB signalling pathway in promoting and maintaining ET-1 expression in CLL cells, both in basal conditions and after CD40 activation.

Selective IAP inhibition results in sensitization of unstimulated but not CD40-stimulated chronic lymphocytic leukaemia cells to TRAIL-induced apoptosis.

Despite recent advances in therapy, chronic lymphocytic leukaemia (CLL) remains incurable and new treatment strategies are therefore urgently required. Inhibitor of apoptosis proteins (IAPs) are over-expressed in CLL, suggesting both a role in disease pathogenesis and the potential for therapeutic targeting. To explore these questions, we evaluated the effects on primary CLL cells of AZD5582, a novel potent and selective inhibitor of IAPs. AZD5582 at nanomolar concentrations induced extensive degradation of cIAP-1 and cIAP-2, but minimally of X chromosome-linked IAP (XIAP). However, these effects of AZD5582 produced little or no direct cytotoxicity, nor did they sensitize CLL cells to p53-dependent killing by fludarabine or p53-independent killing by dexamethasone. In contrast, AZD5582 significantly enhanced apoptosis induced by the death receptor (DR) agonist tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, killing by TRAIL plus AZD5582 was independent of adverse prognostic features including TP53 deletion which is strongly associated with chemoresistance in CLL. Coculture experiments involving transfected mouse fibroblasts expressing human CD40L (CD154) to mimic the effect of T cells at sites of tissue involvement showed that CD40 stimulation almost completely prevented the killing of CLL cells by TRAIL plus AZD5582 despite up-regulating TRAIL receptors 1 and 2. In conclusion, our findings confirm the rate-limiting, upstream involvement of IAPs in the extrinsic but not intrinsic apoptotic pathway of CLL cells and suggest that drug combinations that simultaneously activate DRs and inhibit IAPs may have therapeutic potential in patients with CLL who have failed T-cell-depleting chemotherapy.