p66Shc deficiency in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia enhances leukemogenesis by altering the chemokine receptor landscape

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 have analyzed the impact of p66Shc ablation on disease severity and progression in the mouse model of chronic lymphocytic leukemia Eμ-TCL1. We show that Eμ-TCL1/p66Shc-/- mice develop an aggressive disease that has an earlier onset, a higher incidence and leads to earlier death compared to Eμ-TCL1 mice. Eμ-TCL1/p66Shc-/- mice display substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlates with an upregulation of chemokine receptors whose ligands are expressed therein. This also applies to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to inversely correlate with their p66Shc expression levels. 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 chronic lymphocytic leukemia progression and severity and underscore p66Shc expression as a relevant therapeutic target

Wiskott-Aldrich syndrome protein-mediated actin dynamics control type-I interferon production in plasmacytoid dendritic cells

Mutations in Wiskott-Aldrich syndrome (WAS) protein (WASp), a regulator of actin dynamics in hematopoietic cells, cause WAS, an X-linked primary immunodeficiency characterized by recurrent infections and a marked predisposition to develop autoimmune disorders. The mechanisms that link actin alterations to the autoimmune phenotype are still poorly understood. We show that chronic activation of plasmacytoid dendritic cells (pDCs) and elevated type-I interferon (IFN) levels play a role in WAS autoimmunity. WAS patients display increased expression of type-I IFN genes and their inducible targets, alteration in pD

The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells

Sustained engagement of the B-cell receptor (BCR) increases apoptosis resistance in chronic lymphocytic leukemia (CLL) B cells, whereas transient stimulation usually has an opposite effect. The antiapoptotic BCR signal has been associated with prolonged activation of the PI3K/Akt and MEK/ERK pathways, which are key regulators of survival and proliferation in various cell types. To further define the relative contribution of the Akt and ERK kinases in regulating CLL B-cell survival, we introduced constitutively active mutants of Akt and MEK in primary CLL B cells and evaluated changes in the expression of relevant pro- and antiapoptotic proteins. Sustained activation of Akt resulted in increased leukemic cell viability and increased expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and X-linked inhibitor of apoptosis protein (XIAP), thus largely recapitulating the effects of sustained BCR stimulation. Constitutively active MEK2 also up-regulated XIAP, but did not show a significant impact on leukemic cell survival. Down-regulation of Mcl-1 by siRNA treatment induced rapid and potent apoptosis in CLL B cells and blocked the antiapoptotic effect of sustained BCR stimulation, whereas down-regulation of Bcl-xL and XIAP did not affect leukemic cell viability. These data demonstrate that Akt and Mcl-1 are major components of a survival pathway that can be activated in CLL B cells by antigen stimulation

BCR signaling inhibitors differ in their ability to overcome Mcl-1-mediated resistance of CLL B cells to ABT-199

The Bcl-2 antagonist ABT-199 has demonstrated promising clinical activity in patients with CLL. ABT-199 is strongly cytotoxic against unstimulated peripheral blood CLL cellsin vitro, but is much less effective against CLL cells that have received survival signals from the microenvironment. In particular, stimulation of CLL cells with CD40L results in substantial resistance that is mediated by induction of the antiapoptotic Bcl-2 family proteins Bcl-xLand Bfl-1. In the present study we investigated whether resistance to ABT-199 can be conferred by B-cell receptor (BCR) stimulation, which is another important survival signal from the leukemic microenvironment. We show that sustained BCR stimulation results in significant ABT-199-resistance, which correlates with induction of the antiapoptotic protein Mcl-1 and less consistently with downregulation of proapoptotic Bmf, Hrk and BimEL A major role for Mcl-1 in conferring ABT-199 resistance is additionally supported by knockdown and enforced expression experiments with primary CLL cells. We further show that SYK, BTK and PI3K\u3b4 inhibitors significantly downregulate Mcl-1, but with different efficacy. Complete Mcl-1 downregulation was consistently achieved only with the SYK inhibitors R406 and GS-9973, whereas the BTK inhibitor ibrutinib and the PI3K\u3b4 inhibitor idelalisib in more than half of the cases had only a partial effect. The greater ability of SYK inhibitors to downregulate Mcl-1 correlated with their greater capacity to block BCR-mediated inactivation of GSK-3, a major negative regulator of Mcl-1. The finding that BCR-signaling inhibitors differ in their ability to target Mcl-1 is relevant for the design of clinical trials combining these agents with ABT-199

Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL)

Chronic Lymphocytic Leukemia (CLL) demonstrates variable reactivity of the B cell receptor (BCR) to antigen ligation, but constitutive pathway activation. Bruton's Tyrosine Kinase (BTK) shows constitutive activity in CLL, and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we utilized patient samples and the E\u3bc-TCL1 (TCL1) transgenic mouse model of CLL which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by siRNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL

Inhibition of thioredoxin-dependent H2O2 removal sensitizes malignant B-cells to pharmacological ascorbate

L-ascorbate (L-ASC) is a widely-known dietary nutrient which holds promising potential in cancer therapy when given parenterally at high doses. The anticancer effects of L-ASC involve its autoxidation and generation of H2O2, which is selectively toxic to malignant cells. Here we present that thioredoxin antioxidant system plays a key role in the scavenging of extracellularly-generated H2O2 in malignant B-cells. We show that inhibition of peroxiredoxin 1, the enzyme that removes H2O2 in a thioredoxin system-dependent manner, increases the sensitivity of malignant B-cells to L-ASC. Moreover, we demonstrate that auranofin (AUR), the inhibitor of the thioredoxin system that is used as an antirheumatic drug, diminishes the H2O2-scavenging capacity of malignant B-cells and potentiates pharmacological ascorbate anticancer activity in vitro and in vivo. The addition of AUR to L-ASC-treated cells triggers the accumulation of H2O2 in the cells, which results in iron-dependent cytotoxicity. Importantly, the synergistic effects are observed at as low as 200 µM L-ASC concentrations. In conclusion, we observed strong, synergistic, cancer-selective interaction between L-ASC and auranofin. Since both of these agents are available in clinical practice, our findings support further investigations of the efficacy of pharmacological ascorbate in combination with auranofin in preclinical and clinical settings

Overexpression of the autoimmunity-associated phosphatase PTPN22 promotes survival of antigen-stimulated CLL cells by selectively activating AKT

A polymorphic variant of the phosphatase PTPN22 has been associated with increased risk for multiple autoimmune diseases. The risk allele is thought to function by diminishing antigen-receptor signals responsible for negative selection of autoreactive lymphocytes. We now show that PTPN22 is markedly overexpressed in chronic lymphocytic leukemia (CLL), a common malignancy of autoreactive B lymphocytes. We also show that overexpression of PTPN22 significantly inhibits antigen-induced apoptosis of primary CLL cells by blocking B-cell receptor (BCR) signaling pathways that negatively regulate lymphocyte survival. More importantly, we show that PTPN22 positively regulates the antiapoptotic AKT kinase, which provides a powerful survival signal to antigen-stimulated CLL cells. This selective uncoupling of AKT from other downstream BCR signaling pathways is a result of inhibition of a negative regulatory circuit involving LYN, CD22, and SHIP. Finally, we show that PTPN22 can be effectively down-regulated by the PKC inhibitors ruboxistaurin and sotrastaurin, resulting in enhanced killing of CLL cells exposed to proapoptotic BCR stimuli. Collectively, these data suggest that PTPN22 overexpression represents a protective mechanism that allows autoantigen-activated CLL cells to escape from negative selection and indicate that this mechanism could be exploited for therapeutic purposes by targeting PTPN22 with PKC inhibitors