B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of several B-cell malignancies, including chronic lymphocytic leukemia (CLL), and can be targeted by inhibitors of BCRassociated kinases, such as Bruton tyrosine kinase (Btk). PCI-32765, a selective, irreversible Btk inhibitor, is a novel, molecularly targeted agent for patients with B-cell malignancies, and is particularly active in patients with CLL. In this study, we analyzed the mechanism of action of PCI-32765 in CLL, using in vitro and in vivo models, and performed correlative studies on specimens from patients receiving therapy with PCI-32765. PCI-32765 significantly inhibited CLL cell survival, DNA synthesis, and migration in response to tissue homing chemokines (CXCL12, CXCL13 IntroductionChronic lymphocytic leukemia (CLL), the most common leukemia in western societies, is characterized by the accumulation of mature, CD5 ϩ CD23 ϩ monoclonal B lymphocytes in the blood, secondary lymphatic tissues, and the bone marrow. 1 Proliferating CLL cells, which account for approximately 0.1% to 1% of the CLL clone, 2 are typically found within microanatomical structures called proliferation centers or pseudofollicles, 3 where CLL cells interact with accessory cells (ie, stromal cells or T cells), thereby receiving survival and growth signals. 4 Such external signals from the leukemia microenvironment can supplement intrinsic oncogenic lesions, thereby promoting maintenance and expansion of the CLL clone. 3,5,6 Among the various external stimuli in the tissue microenvironments, B-cell receptor (BCR) activation and signaling, particularly in lymphatic tissues, 6 is a central pathologic mechanism, even though the precise mechanism of BCR stimulation and the nature of the antigen(s) that activate the BCRs remain obscure. 1,7 The most direct evidence for the importance of BCR signaling in CLL comes from recent comparative gene expression profiling (GEP) data that revealed BCR signaling as the most prominent pathway activated in CLL cells isolated from lymphatic tissues. 6 These GEP changes displayed remarkable similarity to GEP changes of CLL cells cocultured with monocyte-derived nurselike cells (NLC), 8 a system for studying the impact of the lymphatic tissue microenvironment in CLL in vitro. Additional evidence for the importance of BCR signaling in CLL comes from the observation that important CLL risk factors have functional links to the BCRs. The mutation status of the IgV H segments of the BCR distinguishes "mutated" (M-CLL) from "unmutated" CLL (U-CLL), with a low or high risk for disease progression, respectively, each accounting for approximately 50% of the patients. ZAP-70 is predominantly expressed in U-CLL cases, 9 and ZAP-70 expression is associated with enhanced BCR signaling. 10 Furthermore, CLL patients express restricted sets of BCRs, as determined by BCR sequencing. These BCRs have immunoglobulin (Ig) heavy-chain variable (V) gene sequences that are identical or stereotyped in subsets of patients, 11,12 suggesting that these BCR...

SummaryThe heritability of B cell chronic lymphocytic leukemia (CLL) is relatively high; however, no predisposing mutation has been convincingly identified. We show that loss or reduced expression of death-associated protein kinase 1 (DAPK1) underlies cases of heritable predisposition to CLL and the majority of sporadic CLL. Epigenetic silencing of DAPK1 by promoter methylation occurs in almost all sporadic CLL cases. Furthermore, we defined a disease haplotype, which segregates with the CLL phenotype in a large family. DAPK1 expression of the CLL allele is downregulated by 75% in germline cells due to increased HOXB7 binding. In the blood cells from affected family members, promoter methylation results in additional loss of DAPK1 expression. Thus, reduced expression of DAPK1 can result from germline predisposition, as well as epigenetic or somatic events causing or contributing to the CLL phenotype.

Functional loss of CCAAT/enhancer binding protein a (C/ EBPa), a master regulatory transcription factor in the hematopoietic system, can result in a differentiation block in granulopoiesis and thus contribute to leukemic transformation. Here, we show the effect of epigenetic aberrations in regulating C/EBPA expression in acute myeloid leukemia (AML). Comprehensive DNA methylation analyses of the CpG island of C/EBPa identified a densely methylated upstream promoter region in 51% of AML patients. Aberrant DNA methylation was strongly associated with two generally prognostically favorable cytogenetic subgroups: inv(16) and t(15;17). Surprisingly, while epigenetic treatment increased C/EBPa mRNA levels in vitro, C/EBPA protein levels decreased. Using a computational microRNA (miRNA) prediction approach and functional studies, we show that C/EBPa mRNA is a target for miRNA-124a. This miRNA is frequently silenced by epigenetic mechanisms in leukemia cell lines, becomes upregulated after epigenetic treatment, and targets the C/EBPa 3 ¶ untranslated region. In this way, C/EBPA protein expression is reduced in a posttranscriptional manner. Our results indicate that epigenetic alterations of C/EBPa are a frequent event in AML and that epigenetic treatment can result in downregulation of a key hematopoietic transcription factor. [Cancer Res 2008;68(9):3142-51]

B-cell chronic lymphocytic leukemia (CLL) is characterized by a clonal accumulation of mature neoplastic B cells indicating disruption of apoptosis. Restriction Landmark Genome Scanning was done to identify novel target genes silenced by CpG island methylation in CLL. Secreted frizzled-related protein 4 (SFRP4), a negative regulator of the Wnt signaling pathway, was found to be frequently methylated in CLL samples. Wnt signaling has been shown to control normal apoptotic behavior and is required for normal B-cell development whereas aberrant activation of this pathway has been observed in CLL. We show aberrant DNA methylation and silencing of SFRP4, as well as of additional SFRP family members, in primary CLL samples. Induction of their expression in a dose-dependent manner following treatment with a demethylating agent, 5-aza-2V-deoxycytidine, was shown. Of the five SFRP family members studied in detail, SFRP1 was hypermethylated and down-regulated in all CLL patient samples studied, suggesting that this epigenetic event is a critical step during leukemogenesis. Our results suggest that silencing of SFRPs by CpG island methylation is one possible mechanism contributing to aberrant activation of