Nonselective inhibitors of human histone deacetylases (HDAC) are known to have antitumor activity in mice in vivo, and several of them are under clinical investigation. The first of these, Vorinostat (SAHA), has been approved for treatment of cutaneous T-cell lymphoma. Questions remain concerning which HDAC isotype(s) are the best to target for anticancer activity and whether increased efficacy and safety will result with an isotypeselective HDAC inhibitor. We have developed an isotypeselective HDAC inhibitor, MGCD0103, which potently targets human HDAC1 but also has inhibitory activity against HDAC2, HDAC3, and HDAC11 in vitro. In intact cells, MGCD0103 inhibited only a fraction of the total HDAC activity and showed long-lasting inhibitory activity even upon drug removal. MGCD0103 induced hyperacetylation of histones, selectively induced apoptosis, and caused cell cycle blockade in various human cancer cell lines in a dose-dependent manner. MGCD0103 exhibited potent and selective antiproliferative activities against a broad spectrum of human cancer cell lines in vitro, and HDAC inhibitory activity was required for these effects. In vivo, MGCD0103 significantly inhibited growth of human tumor xenografts in nude mice in a dose-dependent manner and the antitumor activity correlated with induction of histone acetylation in tumors. Our findings suggest that the isotype-selective HDAC inhibition by MGCD0103 is sufficient for antitumor activity in vivo and that further clinical investigation is warranted. [Mol Cancer Ther 2008;7(4):759 -68]

Tyrosine protein phosphorylation is necessary for antigen receptor-mediated activation of T lymphocytes. This signal is generated at least in part by the Src-related tyrosine protein kinases p56lck and p59fynT (refs 2, 3). The activity of these two enzymes is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory phosphorylation may be caused by p50csk (for C-terminal Src kinase), a tyrosine protein kinase which accumulates most abundantly in thymus and spleen. To investigate the function of Csk in T lymphocytes and characterize the processes regulating T-cell receptor (TCR) signalling, we examined the effects of overexpression of Csk on the physiology of an antigen-specific mouse T-cell line. We report here that p50csk negatively regulates TCR-induced tyrosine protein phosphorylation and lymphokine production. This provides evidence for the involvement of Csk in the regulation of T-cell activation.

Significant effort is being made to understand the role of HDAC isotypes in human cancer and to develop antitumor agents with better therapeutic windows. A part of this endeavor was the exploration of the 14 A internal cavity adjacent to the enzyme catalytic site, which led to the design and synthesis of compound 4 with the unusual bis(aryl)-type pharmacophore. SAR studies around this lead resulted in optimization to potent, selective, nonhydroxamic acid HDAC inhibitors.

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SllnunaryRecent observations suggest that the src-related tyrosine protein kinase p59~ may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59~ exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59~ isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. To further understand the role of p59~ in T cell activation and to test the hypothesis that p59~ T serves a tissue-specific function in T lymphocytes, we have examined the effects of expression of activated versions (tyrosine 528 to phenylalanine 528 mutants) of either form of p59~ on the physiology of an antigen-spedfic mouse T cell hybridoma. Our results demonstrated that the two forms offyn, expressed in equivalent amounts, efficiently enhanced antibody-induced T cell receptor (TCR)-mediated signals. In contrast, only p59~ T increased interleukin 2 production in response to antigen stimulation. This finding implies that the distinct p59~ isoform expressed in T lymphocytes regulates the coupling of TCtL stimulation by antigen/major histocompatibility complex to lymphokine production.ntigen-induced T lymphocyte activation is primarily mediated by changes in intracellular tyrosine protein phosphorylation (for review, see reference 1). Although the cellular machinery regulating this biochemical signal remains poorly characterized, recent observations suggest that the srerelated nonreceptor tyrosine protein kinases p56 ~ and p59~ are involved in this process, p56 lck is a lymphocyte-specific tyrosine protein kinase associated with and regulated by the CD4 and CD8 T cell surface antigens (for review, see reference 2). Increasing evidence demonstrates that a large part of the accessory function provided by CD4 or CD8 during T cell activation is mediated through p561r Contrary to p56 lCk, p59~ is expressed in most cell types (3-6). It is interesting that as a result of alternative splicing of mutually exclusive exons 7, p59~ exists as two isoforms differing solely within a stretch of 51 amino acids spanning the end of the SH21 region and the beginning of an otherwise classical tyrosine protein kinase domain (see Fig. 1 A) (7). It has been postulated that as a consequence of different substrate specificities and/or catalytic efl~ciencies, these two enzymes regulate distinct cellular processes.Whereas onefyn isoform 0CynB) accumulates highly in brain, the other (fynT) is expressed predominantly in T lymphocytes (7; L. M. L. Chow, A. Veillette, D. Davidson, unpublished data). The limited homology (53% or less) between the exon 7-encoded sequences offynT and those off, B, as well as of all other src-related tyrosine protein kinases, fostered the early view that p59~ T plays a specialized role in T lymphocyte physiology. The subsequent demonstration that a fraction of p59~ T is physically associated with elements of the TCR complex ...

T-cell antigen receptor (TCR) signalling has been shown to involve two classes of tyrosine protein kinases: the Src-related kinases p56lck and p59 Stimulation of T cells by antigen or anti-T-cell receptor (TCR)antibodies results in the rapid tyrosine phosphorylation of several intracellular proteins (for reviews, see references 26, 38, and 40). This signal predicates all subsequent biochemical and biological events of T-cell activation. Since the TCR and the associated CD3 and zeta () chains are devoid of intrinsic tyrosine protein kinase (TPK) activity, antigen receptor-mediated tyrosine protein phosphorylation is postulated to require the participation of cellular TPKs. Over the past several years, evidence has accumulated that p56 lck and p59 fynT , two members of the Src family of enzymes, are responsible for initiating this response (for reviews, see references 25 and 26). This property relates to the ability of Lck and FynT to phosphorylate conserved tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) of CD3 and . Phosphorylation of these sites allows the recruitment of Src homology 2 (SH2) domain-containing molecules involved in signal amplification and diversification.The Syk/Zap-70 family of cytoplasmic TPKs comprises two known members termed Zap-70 and Syk (for reviews, see references 36 and 38). Zap-70 is solely expressed in T cells and natural killer cells, whereas Syk accumulates in most hemopoietic cell types, including B cells, mast cells, and platelets. Syk is also contained in several T-cell types, including all major thymocyte populations, intraepithelial ␥␦ T lymphocytes (21), and various T-cell lines. Additionally, a recent study suggested that Syk is expressed in naive, but not in proliferating, mature T cells (24). The structure of Zap-70 and Syk is highly conserved. It includes from the amino terminus to the carboxy terminus (i) two SH2 domains, which bind tyrosine-phosphorylated ITAMs; (ii) a linker region of 80 to 110 amino acids, postulated to bind and recruit cellular effectors; (iii) a catalytic domain, including sites of ATP binding and tyrosine phosphorylation; and (iv) a short carboxy-terminal extension of as yet undetermined function.Currently available data show that following their phosphorylation by Lck and/or FynT, doubly phosphorylated ITAMs bind Zap-70 and Syk via their tandem SH2 domains (reviewed in reference 38). This association results in tyrosine phosphorylation of Zap-70 and Syk, which leads to their enzymatic activation and creates docking sites for the recruitment of other SH2 domain-bearing signalling molecules such as phospholipase C-␥ and Vav. Genetic evidence supports the notion that Zap-70 provides an essential function during T-cell activation. Indeed, expression of dominant-interfering versions of Zap-70 in Jurkat T cells resulted in a marked reduction in TCR-mediated responses (27). Zap-70 is also necessary for normal T-cell development, as demonstrated by the finding that humans and mice lacking Zap-70 have markedly abnormal T-...

The design, synthesis, and biological evaluation of N-(2-aminophenyl)-4-[(4-pyridin-3-ylpyrimidin-2-ylamino)methyl]benzamide 8 (MGCD0103) is described. Compound 8 is an isotype-selective small molecule histone deacetylase (HDAC) inhibitor that selectively inhibits HDACs 1-3 and 11 at submicromolar concentrations in vitro. 8 blocks cancer cell proliferation and induces histone acetylation, p21 (cip/waf1) protein expression, cell-cycle arrest, and apoptosis. 8 is orally bioavailable, has significant antitumor activity in vivo, has entered clinical trials, and shows promise as an anticancer drug.

SummaryAccumulating data show that the tyrosine protein kinase Zap-70 plays an essential role in T cell receptor-mediated signal transduction. However, the mode of action, as well as the physiologically relevant substrates of Zap-70, have not been determined. We have attempted to identify a 120-kD tyrosine-phosphorylated protein (p120) that associates with Zap-70 in activated T lymphocytes. The results of our analyses showed that p120 is largely encoded by the c-cbl protooncogene. Furthermore, the association of Zap-70 with c-Cbl was shown to be induced by T cell receptor stimulation, implying that it required posttranslational modification of one or both of these products. FynT, but not Lck, also associated with c-Cbl in activated T cells. Finally, using a heterologous system, it was demonstrated that the ability of Zap-70 to cause tyrosine phosphorylation of p120 c-cbl was dependent on Lck-or FynT-mediated signals. As c-Cbl can associate with several other signaling molecules, it may couple Zap-70 to downstream es lectors during T cell activation.