Src family kinases (SFKs) were described to be overexpressed in chronic lymphocytic leukemia (CLL). We wished to examine the effects of the Src and Abl kinase inhibitor dasatinib on the intracellular signaling and survival of CLL cells. Dasatinib showed a dose-and time-dependent reduction of global tyrosine phosphorylation and of activating phosphotyrosine levels of SFKs. Treatment with 100 nM dasatinib led to decreased levels of the activated, phosphorylated forms of Akt, Erk1/2, and p38, and induced PARP cleavage through caspase activity. In Mec1 and JVM-3 cell lines, dasatinib increased p53 protein levels and inhibited proliferation.

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SummaryThe effects of two CD20 antibodies, namely rituximab, the current standard for treatment of chronic lymphocytic leukaemia (CLL) in combination with chemotherapy, and GA101, a glyco-engineered type II antibody were compared on CLL cells ex vivo. Antibody-induced phosphatidylserine exposure was examined in isolated CLL cells. For a more comprehensive assessment of antibody-mediated cell killing including Fc-mediated mechanisms, B cell depletion from whole blood samples was monitored. Treatment with rituximab or GA101 reduced the average viability of isolated CLL cells by 6% or 11%, and the ratio of B to T cells in whole blood samples by 12% or 33%, respectively. Combination with GA101 enhanced the cytotoxicity of the chemotherapeutic agent chlorambucil on isolated CLL cells. CD20 surface expression on CLL cells correlated with GA101-induced B cell depletion, but not with direct cell death induction. Treatment of whole blood samples from CLL patients with a CpG-containing oligonucleotide increased CD20 expression on CLL cells and GA101-dependent B cell depletion. Despite the variable responses of individual CLL samples, the CLL cell depletion from whole blood by GA101 was consistently much stronger than by rituximab, which argues for clinical investigation of GA101 in CLL patients.

IntroductionChronic lymphocytic leukemia (CLL) is a clonal B-cell disorder that is not curable by conventional chemoimmunotherapies. The leukemic transformation may be initiated by specific genomic alterations (eg, del13q) that may cause the deletion of specific micro-RNA genes (eg, miR15 and miR16) and increase the resistance of B cells toward apoptosis. 1,2 Survival of CLL cells depends on a permissive microenvironment composed of cellular components, such as macrophages, T cells, or stromal follicular dendritic cells. [3][4][5] This microenvironment provides various chemokines and angiogenic factors, which interact with leukemic cells via appropriate surface receptors and adhesion molecules. 2,5 Macrophage migration inhibitory factor (MIF) is a proinflammatory and immunoregulatory cytokine that seems to be involved in the pathogenesis of various malignant diseases. 6-9 MIF was identified as a product of T cells 10 but also other cells of the immune system (B cells, monocytes/macrophages). 11 Later, MIF was found to be an almost ubiquitous mediator secreted by a wide variety of cells in the mammalian organism, such as endothelial cells, epithelial cells, or fibroblasts. 12 Macrophages are considered to be a prime source for MIF, as they are able to secrete large amounts of MIF in response to various stimuli. 13 MIF binds to the surface receptors CD74 and CXCR2/CXCR4, thereby stimulating signaling pathways, such as MAPK, NF-B, and AKT. [14][15][16] In B cells, activation of the surface receptor complex CD74/CD44 by MIF induces the proteolytic release of the intracellular domain of CD74, which in turn initiates a signaling cascade composing Syk, AKT, and NF-B; this leads to the production of IL-8 and to an increased resistance to apoptosis via the up-regulation of BCL-2. 17,18 Thus, the MIF-MIF receptor system may be seen as a part of the B-cell costimulatory signals that are required for full B-cell activation and maturation. MIF-deficient mice do not show developmental abnormalities and appear to have normal numbers of B cells. 6 However, they exhibit a number of immune dysfunctions when challenged by antigens or infectious agents. [19][20][21][22] Even more importantly, MIF seems to be required for bone marrow-derived dendritic cells to maintain mature B cells in the bone marrow compartment. 23 Submitted May 22, 2012; accepted October 14, 2012. Prepublished online as Blood First Edition paper, November 1, 2012; DOI 10.1182/blood-2012-05-431452.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. MIF is overexpressed in a variety of malignancies compared with the respective primary tissues (eg, prostate, 24 colon, 25 melanoma, 26 glioblastoma, 27 breast cancer 28,29 ). This overexpression might be caused by the tumor-activated HSP90 chaperone complex that protects MIF from degradation, a...