Although intrinsic apoptosis defects are causal to the extended survival of chronic lymphocytic leukemia (CLL) B cells, several lines of evidence support a contribution of the peripheral lymphoid organs and BM microenvironment to the extended lifespan of leukemic B cells. Lymphocyte trafficking is controlled by homing signals provided by stromal cellderived chemokines and egress signals provided by sphingosine-1-phosphate (S1P). In the present study, we show that expression of S1P1, the S1P receptor responsible for lymphocyte egress, is selectively reduced in CLL B cells with unmutated IGHV. Expression of S1P2, which controls B-cell homeostasis, is also impaired in CLL B cells but independently of the IGHV mutational status. We provide evidence herein that p66Shc, a Shc adaptor family member the deficiency of which is implicated in the apoptosis defects of CLL B cells, controls S1P1 expression through its pro-oxidant activity. p66Shc also controls the expression of the homing receptor CCR7, which opposes S1P1 by promoting lymphocyte retention in peripheral lymphoid organs. The results of the present study provide insights into the regulation of S1P1 expression in B cells and suggest that defective egress caused by impaired S1P1 expression contributes to the extended survival IntroductionAlthough progressive accumulation of monoclonal CD5 ϩ B cells in the blood, peripheral lymphoid organs, and BM is the hallmark of chronic lymphocytic leukemia (CLL), the clinical course of this disorder is highly variable, ranging from a stable disease that may only require monitoring over time to a progressive, severe disease. 1,2 Several markers have been associated with poor prognosis. Coupled to the cytogenetic abnormalities, the mutational status of immunoglobulin heavy chain variable region (IGHV) genes is the most valuable marker presently available, with unmutated IGHV found in patients who develop aggressive disease. 3 Nevertheless, the onset of disease progression and response to treatment are to date largely unpredictable.At variance with other hematologic malignancies, CLL B cells are usually arrested at G 0 /G 1 and their accumulation is the result of an abnormally prolonged survival rather than uncontrolled proliferation. 1,2 Intrinsic defects in the apoptotic machinery underlie the prolonged lifespan of CLL B cells, a major target being the Bcl-2 family, in which overexpression of antiapoptotic members (Bcl-2 and Mcl-1) or impaired expression of proapoptotic members (Bax and Bak) tilts the balance toward cell survival. 4 Extrinsic factors consisting mainly of stromal cell-derived chemokines (CXCL12, CXCL13, CCL19, and CCL21) also to contribute to the extended lifespan of CLL B cells by providing survival cues during their transit through peripheral lymphoid tissues and BM. 5 Lymphocyte trafficking is tightly controlled by the chemokines present in the lymphoid microenvironment and the chemokine receptors expressed by the lymphocyte itself. 6 CLL B cells express increased levels of CXCR4, CCR7, and CXCR5, which has...

IntroductionChronic lymphocytic leukemia (CLL) is the most common B-cell neoplasm in Europe and the United States, characterized by progressive accumulation of monoclonal CD5 ϩ B cells in peripheral blood, bone marrow, and peripheral lymphoid organs. The clinical course of CLL is highly variable, ranging from an indolent disease that may never require treatment to a rapidly progressive disease. 1 One of the principal prognostic features is the mutational status of the immunoglobulin heavy chain variable region genes (IGHV). CLL with poor prognosis has unmutated IGHV genes (U-CLL), whereas CLL with good prognosis carries somatic mutations in these genes (M-CLL). 2,3 Neoplastic CLL cells are typically arrested in the G 0 /G 1 phase of the cell cycle and accumulate in tissues because of prolonged survival. 1 Although extrinsic factors, such as escape from immune surveillance and chemotaxis to a favorable microenvironment, contribute to the extended survival of CLL B cells, 4 CLL is primarily a disease of defective apoptosis, and intrinsic defects in a number of components of the apoptotic circuitry have been identified, including overexpression of antiapoptotic proteins (eg, Bcl-2, Mcl-1, XIAP) and reduction in the expression of proapoptotic proteins (eg, Bax, DAPK-1). [5][6][7] Other apoptosis defects, including abnormalities in the p53 and ATM pathways in the most aggressive subsets, have also been implicated in the prolonged survival of malignant cells. [8][9][10] In this respect, apoptosis is emerging as a key therapeutic target in CLL, as witnessed by the ongoing clinical trials on Bcl-2 inhibitors. 11 p66Shc, a member of the Shc family of protein adapters, acts as antagonist of mitogenic signaling and positive regulator of oxidative stress-induced apoptosis in fibroblasts. 12,13 In T cells, where its expression is epigenetically controlled, 14,15 p66Shc uncouples the T-cell receptor from activation of the Ras/mitogen-activated protein kinase pathway by competitively inhibiting recruitment to the T-cell receptor of the mitogenic isoform, p52Shc. 16 Furthermore, p66Shc enhances T-cell susceptibility to apoptotic stimuli by increasing reactive oxygen species (ROS) production and impairing Ca 2ϩ homeostasis. 17 We have recently reported that p66Shc is expressed in murine B cells and that p66Shc deficiency results in enhanced proliferative responses of mouse B cells to B-cell receptor (BCR) engagement, 18 suggesting that p66Shc may display similar activities in T and B cells.Here, we have investigated the role of p66Shc in B-cell survival. We show that p66Shc promotes B-cell apoptosis by uncoupling the BCR from the survival pathways mediated by Akt and Erk. On the basis of these findings, we have investigated p66Shc expression and function in B cells from patients with CLL. The results identify a role for p66Shc in the imbalance among proapoptotic and antiapoptotic Bcl-2 family members in CLL and An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains...

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Lymphocyte trafficking is orchestrated by chemokine and sphingosine 1-phosphate (S1P) receptors that enable homing and egress from secondary lymphoid organs (SLO). These receptors undergo rapid internalization and plasma membrane recycling to calibrate cellular responses to local chemoattractants. Circulating chronic lymphocytic leukemia (CLL) cells display an abnormal increase in the surface levels of the homing receptors CCR7 and CXCR4 concomitant with low S1P receptor 1 (S1P1) expression. In this study, we investigated the role of receptor recycling on CXCR4/CCR7 surface levels in CLL cells and addressed the impact of quantitative alterations of these receptors and S1P1 on the ability of leukemic cells to accumulate in SLOs. We show that recycling accounts, to a major extent, for the high levels of surface CXCR4/CCR7 on CLL cells. In addition, increased expression of these receptors, together with S1P1 deficiency, is detectable not only in circulating leukemic cells, but also in SLOs of CLL patients with lymphoadenopathy. We further provide evidence that ibrutinib, a Btk inhibitor that promotes mobilization of leukemic cells from SLOs, normalizes the imbalance between CXCR4/CCR7 and S1P1. Taken together, our results highlight the relevance of chemokine and S1P receptor recycling in CLL pathogenesis and clinical outcome

The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eμ-TCL1/p66Shc−/− mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eμ-TCL1 mice. Eμ-TCL1/p66Shc−/− mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells’ level of p66Shc expression. p66Shc expression declined with disease progression in Eμ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.

The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2–driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4+ T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2–polarizing concentrations of ET and CyaA selectively inhibit TCR–dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR–signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3ζ phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4+ T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins.

Neoplastic cell traffic abnormalities are central to the pathogenesis of chronic lymphocytic leukemia (CLL). Enhanced CXC chemokine receptor-4 (CXCR4) and chemokine receptor-7 (CCR7) recycling contributes to the elevated surface levels of these receptors on CLL cells. Here we have addressed the role of p66Shc, a member of the Shc family of protein adaptors the expression of which is defective in CLL cells, in CXCR4/CCR7 recycling. p66Shc reconstitution in CLL cells reduced CXCR4/CCR7 recycling, lowering their surface levels and attenuating B-cell chemotaxis, due to their accumulation in Rab5 endosomes as serine-phosphoproteins bound to β-arrestin. This results from the ability of p66Shc to inhibit Ca and PP2B-dependent CXCR4/CCR7 dephosphorylation and β-arrestin release. We also show that ibrutinib, a Btk inhibitor that promotes leukemic cell mobilization from lymphoid organs, reverses the CXCR4/CCR7 recycling abnormalities in CLL cells by increasing p66Shc expression. These results, identifying p66Shc as a regulator of CXCR4/CCR7 recycling in B cells, underscore the relevance of its deficiency to CLL pathogenesis and provide new clues to the mechanisms underlying the therapeutic effects of ibrutinib.

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