B-cell receptor signaling contributes to apoptosis resistance in chronic lymphocytic leukemia (CLL), limiting the efficacy of current therapeutic approaches. In this study, we investigated the expression of spleen tyrosine kinase (SYK), a key component of the B-cell receptor signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared with healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLC; 2 , signal transducers and activators of transcription 3, and extracellular signal regulated kinase 1/2 in CLL compared with healthy B cells, suggesting enhanced activation of these mediators in CLL. SYK inhibitors reduced phosphorylation of SYK downstream targets and induced apoptosis in primary CLL cells. With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70 + cases. Cytotoxic effects of SYK inhibitors also associated with SYK protein expression, potentially predicting response to therapy. Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared with fludarabine therapy alone. We observed no stroma-contact-mediated drug resistance for SYK inhibitors as described for fludarabine treatment. CD40 ligation further enhanced efficacy of SYK inhibition. Our data provide mechanistic insight into the recently observed therapeutic effects of the SYK inhibitor R406 in CLL. Combination of SYK inhibitors with fludarabine might be a novel treatment option particularly for CLL patients with poor prognosis and should be further evaluated in clinical trials. [Cancer Res 2009;69(13):5424-32]

B-cell chronic lymphoid leukemia (B-CLL) is a highly heterogeneous human malignancy, presumably reflecting specific molecular alterations in gene expression and protein activity that are thought to underlie the variable disease outcome. Most B-CLL cell samples undergo apoptotic death in response to DNA damage. However, a clinically distinct aggressive subset of B-CLL is completely resistant in vitro to irradiation-induced apoptosis. We therefore addressed 2 series of microarray analyses on 4 sensitive and 3 resistant B-CLL cell samples and compared their gene expression patterns before and after apoptotic stimuli. Data analysis pointed out 16 genes whose expression varied at least 2-fold specifically in resistant cells. We validated these selected genes by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) on 7 microarray samples and confirmed their altered expression level on 15 additional B-CLL cell samples not included in the microarray analysis. In this manner, in 11 sensitive and 11 resistant B-CLL cell samples tested, 13 genes were found to be specific for all resistant samples: nuclear orphan receptor TR3, major histocompatibility complex (MHC) class II glycoprotein HLA-DQA1, mtmr6, c-myc, c-rel, c-IAP1, mat2A, and fmod were up-regulated, whereas MIP1a/ GOS19-1 homolog, stat1, blk, hsp27, and ech1 were down-regulated. In some cases, the expression profile may be dependent on the status of p53. Some of these genes encode general apoptotic factors but also exhibit lymphoid cell specificities that could potentially be linked to the development of lymphoid malignancies (MIP1␣, blk, TR3, mtmr6). Taken IntroductionB-cell chronic lymphoid leukemia (B-CLL) results in an accumulation of mature CD5 ϩ /CD23 ϩ B cells in peripheral blood, not due to excessive proliferation (at the early stage of disease, cells are mainly arrested in G 0 /G 1 of the cell cycle), but rather to decreased cell mortality resulting from an uncharacterized defect in apoptotic cell death. 1 The most revealing alteration of gene expression that may explain the prolonged survival of malignant B-cells is the overexpression of the bcl-2 gene resulting from its hypomethylation. 2 Although this overexpression as well as deregulated expression of some other bcl-2 family members 1 is consistent with prolonged cell survival, it does not clearly correlate with disease aggressiveness, clinical outcome, or cell sensitivity to apoptosis induced in vitro. Another gene involved in the apoptotic process through its transducing role in the cellular response to DNA damage is ATM, which is deleted or mutated in an aggressive form of B-CLL. [3][4][5] These alterations can lead to dysfunction of p53, whose direct inactivation by mutation is a relatively rare molecular event in B-CLL. 6 Deregulation of p53 can also result from an altered function of the proteasome system, which we observed in all B-CLL samples tested. 7 Together, these observations make deregulated control of the apoptotic death pathway more easily conceivable. Becau...