IntroductionChronic myelogenous leukemia (CML) is a hematopoietic stem cell malignant disease 1 that arises from a reciprocal translocation between chromosomes 9 and 22. 2 It is characterized by a massive expansion of myeloid progenitors as well as more differentiated cells originating from the malignant clone. The disease progresses from an initial chronic phase through an accelerated phase followed by an inevitable acute leukemia or blast crisis as the malignant cells lose their ability to differentiate. The translocation (9;22)(q34;q11) fuses a truncated BCR gene (chromosome 9) to sequences upstream of the second exon of ABL (chromosome 22). The resulting fusion gene encodes a protein tyrosine kinase, Bcr-Abl, with more elevated and disregulated activity compared with c-abl. Expression of Bcr-Abl in mice was found to induce a CML-like myeloproliferative disorder, [3][4][5][6][7] indicating that Bcr-Abl plays a key role in leukemic transformation. Additional studies have shown that the tyrosine kinase activity is critical to the transforming ability of 7,8 making it an attractive target for drug development.Imatinib mesylate (STI571; formerly CGP571418B; Gleevec; Novartis Pharmaceuticals, East Hanover, NJ) is a 2-phenylaminopyrimidine derivative developed as a potent inhibitor of the Abl protein tyrosine kinases (v-Abl, Bcr-Abl, and c-Abl). 9,10 It also has activity against the platelet-derived growth-factor receptor (PDGF-R), 10 c-Kit, 11 ARG, 12 and their fusion proteins, Tel-Abl and Tel-PDGF-R, 13 but does not affect other kinases.Phase I and phase II clinical trials of imatinib mesylate in the treatment of chronic-phase CML showed that it is well tolerated, with few adverse effects. 14 Complete hematologic responses and cytogenetic responses, including complete cytogenetic responses, were reported in a significant proportion of patients. Activity against more advanced accelerated and blast-crisis phases of CML was also observed. 15 Imatinib mesylate is currently approved for the treatment of patients with chronic-phase CML in whom interferon therapy has failed and for accelerated-phase and blast-crisis disease. However, the durability of responses to imatinib mesylate, the agent's impact on long-term survival and normal hematopoiesis, and its role in the treatment of CML compared with other modalities remain unclear.In vitro studies have shown that imatinib mesylate inhibits growth of cell lines expressing Bcr-Abl. 10,13,16,17 Additionally, the numbers of colony-forming cells (CFCs) in peripheral blood or bone marrow from patients with CML have been found to be 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 U.S.C. section 1734. For personal use only. on May 9, 2018. by guest www.bloodjournal.org From reduced, with minimal inhibition of normal cells. 10,17 One study showed that imatinib mesylate inhibited the enhanced replating ability of granulocyte-macroph...

The sirtuin SIRT1 is a NAD(+)-dependent histone deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPARγ, NFκB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241-516) bound to NAD(+) and the 27 analogue compound 35. 35 binds deep in the catalytic cleft, displacing the NAD(+) nicotinamide and forcing the cofactor into an extended conformation. The extended NAD(+) conformation sterically prevents substrate binding. The SIRT1/NAD(+)/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.

The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.

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Endowing T lymphocytes with novel functional attributes by genetic modification is under development for a broad range of clinical cellular immunotherapy applications. To circumvent many of the limitations associated with viral vector systems, a plasmid-based electroporation system that reliably generates G418-resistant primary human T lymphocyte clones was developed. TCR alpha/beta+ CD4+CD8-, and CD4-CD8+ T lymphocyte clones can be routinely isolated from OKT3-stimulated peripheral blood mononuclear cells electroporated with linear plasmid DNA in a limiting dilution drug selection format. Fluorescence in situ hybridization (FISH) studies performed on T cell metaphase spreads using a probe specific for plasmid sequence demonstrated a single FISH signal doublet that varied in chromosomal location from clone to clone. Southern blot analysis using a Neo-specific probe verified chromosomal integration of plasmid vector at a single site. Band intensity quantitation of blots developed with a zeta-specific probe capable of annealing to both endogenous TCR-zeta and the introduced chimeric zeta sequence demonstrated that integrated plasmid was present at a single copy number. Expression levels of the CD20-specific chimeric immunoreceptor construct from a CMV immediate/early promoter present in the plasmid vector varied widely from clone to clone but remained stable during ex vivo expansion to cell numbers in excess of 10(10). This T lymphocyte genetic modification strategy is currently being piloted in a FDA-sanctioned adoptive therapy trial for recurrent lymphoma.