Activation of the B-cell antigen receptor (BCR) signaling pathway contributes to the initiation and maintenance of B-cell malignancies and autoimmune diseases. The Bruton tyrosine kinase (Btk) is specifically required for BCR signaling as demonstrated by human and mouse mutations that disrupt Btk function and prevent B-cell maturation at steps that require a functional BCR pathway. Herein we describe a selective and irreversible Btk inhibitor, PCI-32765, that is currently under clinical development in patients with B-cell nonHodgkin lymphoma. We have used this inhibitor to investigate the biologic effects of Btk inhibition on mature B-cell function and the progression of B cell-associated diseases in vivo. PCI-32765 blocked BCR signaling in human peripheral B cells at concentrations that did not affect T cell receptor signaling. In mice with collagen-induced arthritis, orally administered PCI-32765 reduced the level of circulating autoantibodies and completely suppressed disease. PCI-32765 also inhibited autoantibody production and the development of kidney disease in the MRL-Fas(lpr) lupus model. Occupancy of the Btk active site by PCI-32765 was monitored in vitro and in vivo using a fluorescent affinity probe for Btk. Active site occupancy of Btk was tightly correlated with the blockade of BCR signaling and in vivo efficacy. Finally, PCI-32765 induced objective clinical responses in dogs with spontaneous B-cell non-Hodgkin lymphoma. These findings support Btk inhibition as a therapeutic approach for the treatment of human diseases associated with activation of the BCR pathway.lymphoma | X-linked agammaglobulinemia

We have developed a potent, histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 with 4200-fold selectivity over the other HDAC isoforms. PCI-34051 induces caspase-dependent apoptosis in cell lines derived from T-cell lymphomas or leukemias, but not in other hematopoietic or solid tumor lines. Unlike broad-spectrum HDAC inhibitors, PCI-34051 does not cause detectable histone or tubulin acetylation. Cells defective in T-cell receptor signaling were still sensitive to PCI-34051-induced apoptosis, whereas a phospholipase C-c1 (PLCc1)-defective line was resistant. Jurkat cells showed a dosedependent decrease in PCI-34051-induced apoptosis upon treatment with a PLC inhibitor U73122, but not with an inactive analog. We found that rapid intracellular calcium mobilization from endoplasmic reticulum (ER) and later cytochrome c release from mitochondria are essential for the apoptotic mechanism. The rapid Ca 2 þ flux was dependent on PCI-34051 concentration, and was blocked by the PLC inhibitor U73122. Further, apoptosis was blocked by Ca 2 þ chelators (BAPTA) and enhanced by Ca 2 þ effectors (thapsigargin), supporting this model. These studies show that HDAC8-selective inhibitors have a unique mechanism of action involving PLCc1 activation and calcium-induced apoptosis, and could offer benefits including a greater therapeutic index for treating T-cell malignancies.

Chromatin structure is regulated, in part, by affecting the acetylation of lysine residues on the amino-terminal tails of nucleosomal histones. The acetylation state of histones is maintained by the opposing actions of histone acetyl transferase and histone deacetylase (HDAC) enzymes. There are 11 known isoforms in the classic HDAC family, denoted HDAC 1-11 (1). In addition to histones, HDAC enzymes are known to deacetylate other proteins, including ␣-tubulin (2), suggesting complex, multifunctional roles for HDACs in vivo.PCI-24781 (formerly CRA-024781) is a broad-spectrum phenyl hydroxamic acid HDAC inhibitor currently being evaluated in phase I clinical trials in patients with neoplastic disease (3). The compound is a specific inhibitor of multiple HDAC isoforms that potently inhibits tumor growth in vivo with acceptable toxicity. PCI-24781, along with other HDAC inhibitors also in clinical development, represent a promising class of anticancer therapy agents (4, 5). In addition to evidence of efficacy as a monotherapy, some HDAC inhibitors have been shown to inhibit tumor growth synergistically when administered together with ionizing radiation (IR) or with DNA-interacting cancer drugs in preclinical models (6-12). It has been suggested that the mechanism of the synergy may involve the inhibition of DNA double-strand break (DSB) repair, because after cellular irradiation, HDAC inhibition enhances and prolongs the phosphorylation of histone H2AX, a well characterized marker of DNA DSBs (13-17).In mammalian cells, DSBs are repaired by one of two genetically distinct processes, known as nonhomologous end joining (NHEJ) or homologous recombination (HR) (18). NHEJ is the simpler, but more error-prone mechanism, in which the DNA ends are recognized and bound by the Ku heterodimer, which recruits DNA-PK and other proteins to directly ligate the two DNA termini. By comparison, HR is a process of greater accuracy and complexity, requiring the presence of a sister chromatid to serve as a template for repair. HR begins with strand recognition and nucleolytic processing by the MRE11-RAD50-NBS1 (MRN) complex, followed by strand invasion, branch migration, and Holliday junction formation (18,19). Both strand invasion and branch migration are initiated by RAD51, a recA homolog that binds MRN-generated ssDNA, forming nucleoprotein filaments essential for recombinational repair. After exposure to IR, RAD51 rapidly forms a complex with BRCA2 and other proteins that stimulate RAD51-mediated strand exchange and the assembly of subnuclear foci characteristic of HR (20,21). Cells lacking functional RAD51 are unable to form foci and are significantly more sensitive to IR (22). In addition to repair of IR-induced DNA damage, recent evidence suggests that RAD51 (and HR) is involved in the repair of DNA DSBs produced by cisplatin and other platinum agents (23, 24) and also in the repair of DSBs produced by stalled replication forks (25), such as those produced by inhibitors of poly(ADP-ribose) polymerase (PARP) (26)(27)(28).In the pr...

Purpose: We investigated the cytotoxicity and mechanisms of cell death of the broadspectrum histone deacetylase (HDAC) inhibitor PCI-24781, alone and combined with bortezomib in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines and primary lymphoproliferative (CLL/SLL) cells. Experimental Design: Apoptosis, mitochondrial membrane potential, cell cycle analysis, and reactive oxygen species (ROS) were measured by flow cytometry, whereas caspase activation was determined by Western blot. Nuclear factor κB (NF-κB)-related mRNAs were quantified by reverse transcription-PCR, NF-κB-related proteins by Western blotting, and NF-κB DNA-binding activity by electromobility shift assay. Finally, gene expression profiling was analyzed. Results: PCI-24781 induced concentration-dependent apoptosis that was associated with prominent G 0 /G 1 arrest, decreased S-phase, increased p21 protein, and increased ROS in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. Dose-dependent apoptosis with PCI-24781 was also seen among primary CLL/SLL cells. PCI-24781-induced apoptosis was shown to be ROS-and caspase-dependent. Combined PCI-24781/bortezomib treatment resulted in strong synergistic apoptosis in all non-Hodgkin lymphoma lines (combination indices, 0.19-0.6) and was additive in Hodgkin lymphoma and primary CLL/SLL cells. Further, PCI-24781/bortezomib resulted in increased caspase cleavage, mitochondrial depolarization, and histone acetylation compared with either agent alone. Gene expression profiling showed that PCI-24781 alone significantly down-regulated several antioxidant genes, proteasome components, and NF-κB pathway genes, effects that were enhanced further with bortezomib. Reverse transcription-PCR confirmed down-regulation of NF-κB1 (p105), c-Myc, and IκB-kinase subunits, where NF-κB DNA binding activity was decreased. Conclusion: We show that PCI-24781 results in increased ROS and NF-κB inhibition, leading to caspase-dependent apoptosis. We also show that bortezomib is synergistic with PCI-24781. This combination or PCI-24781 alone has potential therapeutic value in lymphoma.

There is an emerging appreciation of the importance of zinc in regulating cancer cell growth and proliferation. Recently, we showed that the anticancer agent motexafin gadolinium (MGd) disrupted zinc metabolism in A549 lung cancer cells, leading, in the presence of exogenous zinc, to cell death. Here, we report the effect of MGd and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, and cell death in exponential phase human B-cell lymphoma and other hematologic cell lines. We find that increased levels of oxidative stress and intracellular free zinc precede and correlate with cell cycle arrest and apoptosis. To better understand the molecular basis of these cellular responses, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of metal response element-binding transcription factor-1 (MTF-1)-regulated and hypoxia-inducible transcription factor-1 (HIF-1)-regulated genes. Cultures cotreated with MGd and zinc acetate displayed further increases in the levels of MTF-1-and HIF-1-regulated transcripts as well as additional transcripts regulated by NF-E2-related transcription factor 2. These data provide insights into the molecular changes that accompany the disruption of intracellular zinc homeostasis and support a role for MGd in treatment of B-cell hematologic malignancies. (Cancer Res 2005; 65(24): 11676-88)