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.

Survival of chronic lymphocytic leukemia (CLL) cells is triggered by several stimuli, such as the B-cell receptor (BCR), CD40 ligand (CD40L), or interleukin-4 (IL-4).We identified that these stimuli regulate apoptosis resistance by modulating sphingolipid metabolism. Applying liquid chromatography electrospray ionization tandem mass spectrometry, we revealed a significant decrease of proapoptotic ceramide in BCR/IL-4/CD40L-stimulated primary CLL cells compared with untreated controls. Antiapoptotic glucosylceramide levels were significantly increased after BCR cross-linking. We identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide via UDPglucose ceramide glucosyltransferase (UGCG). Besides specific UGCG inhibitors, our data demonstrate that IgMmediated UGCG expression was inhibited by the novel and highly effective PI3K␦ and BTK inhibitors CAL-101 and PCI-32765, which reverted IgM-induced resistance toward apoptosis of CLL cells. Sphingolipids were recently shown to be crucial for mediation of apoptosis via mitochondria. Our data reveal ABT-737, a mitochondria-targeting drug, as interesting candidate partner for PI3K␦ and BTK inhibition, resulting in synergistic apoptosis, even under protection by the BCR. In summary, we identified the mode of action of novel kinase inhibitors CAL-101 and PCI-32765 by controlling the UGCGmediated ceramide/glucosylceramide equilibrium as a downstream molecular switch of BCR signaling, also providing novel targeted treatment options beyond current chemotherapy-based regimens. (Blood. 2012;120(19):3978-3985) IntroductionDespite recent advances in the treatment of chronic lymphocytic leukemia (CLL) by use of modern chemoimmunotherapies, 1,2 the disease remains incurable for most patients with the exception of those who have the option of an allogeneic transplantation. 3 Moreover, most chemotherapeutic regimens require a certain physical fitness of the patient. Because CLL is a disease of the elderly, there is a need for novel therapeutic concepts, which are able to disrupt resistance to cytostatic drugs. Chemoresistance is thought to be partially the result of malignant cell clones that find a niche within the microenvironment. Resistance might be mediated at least by 3 major stimuli: (1) by engagement of the B-cell receptor (BCR), (2) by CD40 ligand (CD40L)-CD40 interaction, and (3) by stimulation via interleukin-4 (IL-4). 4 Those signals lead via downstream pathways to reduced susceptibility of CLL cells toward chemotherapy within the microenvironment. To some extent, these stimuli share common pathways to mediate survival. 5 As a consequence, the balance between proapoptotic and antiapoptotic signals is disrupted toward pro-survival signals. BCR signaling has been identified as the central and determining factor in CLL. Therefore, novel compounds, which target this pathway, have been developed: CAL-101 as PI3K␦ inhibitor and PCI-32765 as inhibitor of Bruton tyrosine kinase (BTK).Recent data from first trials using CAL-101 and PCI-32765 ind...