No abstract

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF). KAT6A has essential roles in normal haematopoietic stem cells and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus, a function that requires its KAT activity. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

LAQ824 and LBH589 (panobinostat) are histone deacetylase inhibitors (HDACi) developed as cancer therapeutics and we have used the E-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death was necessary for these agents to mediate therapeutic responses in vivo and both HDACi engaged the intrinsic apoptotic cascade that did not require p53. Death receptor pathway blockade had no effect on the therapeutic activities of LAQ824 and LBH589; however, overexpression of Bcl-2 or Bcl-X L protected lymphoma cells from HDACi-induced killing and suppressed their therapeutic activities. Deletion of Apaf-1 or Caspase-9 delayed HDACi-induced lymphoma killing in vitro and in vivo, associated with suppression of many biochemical indicators of apoptosis, but did not provide long-term resistance to these agents and failed to inhibit their therapeutic activities. E-myc lymphomas lacking a functional apoptosome displayed morphologic and biochemical features of autophagy after treatment with LAQ824 and LBH589, indicating that, in the absence of a complete intrinsic apoptosis pathway involving apoptosome formation, these HDACi can still mediate a therapeutic response. Our data indicate that damage to the mitochondria is the key event necessary for LAQ824 and LBH589 to mediate tumor cell death and a robust therapeutic response. (Blood. 2009;114:380-393)

No abstract

Granzyme B, a protease released from cytotoxic lymphocytes, has been proposed to induce target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid. It has also been proposed that granzyme B can induce target cell death by activating caspases directly, by cleaving caspase substrates, and/or by cleaving several non-caspase substrates. The relative importance of Bid in granzyme B-induced cell death has therefore remained unclear. Here we report that cells isolated from various tissues of Bid-deficient mice were resistant to granzyme B-induced cell death. Consistent with the proposed role of Bid in regulating mitochondrial outer membrane permeabilization, cytochrome c remained in the mitochondria of Bid-deficient cells treated with granzyme B. Unlike wild type cells, Biddeficient cells survived and were then able to proliferate normally, demonstrating the critical role for Bid in mediating granzyme B-induced apoptosis.Granzyme B is a serine protease contained within the granules of cytotoxic lymphocytes (CLs).1 Upon conjugation with their targets, CLs release their granule contents into the synaptic cleft. Granzyme B then enters the target cell by endocytosis and induces apoptotic death via a perforin-dependent mechanism. The importance of CL-mediated killing in the immune response to various pathogens has made it imperative to understand the mechanism of action of granzyme B.Overexpression of the oncogene Bcl-2 renders cells resistant to granzyme B-induced apoptosis (1, 2), and the cells maintain their ability to proliferate (2, 3). Bcl-2 is one of a large family of proteins that regulate mitochondrial outer membrane permeabilization (MOMP) during apoptosis (4). Pro-apoptotic Bcl-2 family members (such as Bid, Bax, and Bak) induce MOMP (5), whereas anti-apoptotic members (e.g. Bcl-2 and Bcl-XL) prevent MOMP (6). Following MOMP, several pro-apoptotic proteins are released from the mitochondrial intermembrane space. In the cytosol, these proteins facilitate the activation of caspases, proteases that orchestrate the death of a cell by apoptosis. One of these proteins, cytochrome c, initiates a complex with dATP, apoptotic protease-activating factor (APAF-1), and pro-caspase-9. This results in the activation of caspase-9, which in turn activates caspase-3 (7). A second protein SMAC/ Diablo that is also released from the mitochondrial intermembrane space displaces inhibitor of apoptosis proteins (IAPs) from caspases, allowing them to become activated by autoprocessing (8, 9).Granzyme B has been reported to induce MOMP by cleaving and activating the pro-apoptotic Bcl-2 family member Bid after residue [10][11][12]. Granzyme B has also been shown to cleave caspase-3 directly when mixed with cytosolic lysates (13, 14); however, in intact cells, granzyme B only appears to be capable of partially processing procaspase-3 to a p20 form that shows little activity in a cellular context (15). SMAC/Diablo, released following MOMP, then displaces the IAPs from the p20 form of caspase-3, allowing auto-proc...

The apoptotic and therapeutic activities of the histone deacetylase inhibitor (HDACi) vorinostat are blocked by overexpresssion of Bcl-2 or Bcl-X L . Herein, we used the small molecule inhibitor ABT-737 to restore sensitivity of E-myc lymphomas overexpressing Bcl-2 or Bcl-X L to vorinostat and valproic acid (VPA). Combining low-dose ABT-737 with vorinostat or VPA resulted in synergistic apoptosis of these cells. ABT-737 was ineffective against E-myc/Mcl-1 and E-myc/A1 cells either as a single agent or in combination with HDACi. However, in contrast to the reported binding specificity data, E-myc/Bcl-w lymphomas were insensitive to ABT-737 used alone or in combination with HDACi, indicating that the regulatory activity of ABT-737 is restricted to Bcl-2 and Bcl-X L . E-myc lymphomas that expressed Bcl-2 throughout the tumorigenesis process were especially sensitive to ABT-737, while those forced to overexpress Mcl-1 were not. This supports the notion that tumor cells "addicted" to ABT-737 target proteins (ie, Bcl-2 or Bcl-X L ) are likely to be the most sensitive target cell population. Our studies provide important preclinical data on the binding specificity of ABT-737 and its usefulness against primary hematologic malignancies when used as a single agent and in combination with HDACi. (Blood.

No abstract

It is now well recognized that mutations, deregulated expression, and aberrant recruitment of epigenetic readers, writers, and erasers are fundamentally important processes in the onset and maintenance of many human tumors. The molecular, biological, and biochemical characteristics of a particular class of epigenetic erasers, the histone deacetylases (HDACs), have been extensively studied and small-molecule HDAC inhibitors (HDACis) have now been clinically approved for the treatment of human hemopoietic malignancies. This review explores our current understanding of the biological and molecular effects on tumor cells following HDACi treatment. The predominant responses include induction of tumor cell death and inhibition of proliferation that in experimental models have been linked to therapeutic efficacy. However, tumor cell-intrinsic responses to HDACi, including modulating tumor immunogenicity have also been described and may have substantial roles in mediating the antitumor effects of HDACi. We posit that the field has failed to fully reconcile the biological consequences of exposure to HDACis with the molecular events that underpin these responses, however progress is being made. Understanding the pleiotrophic activities of HDACis on tumor cells will hopefully fast track the development of more potent and selective HDACi that may be used alone or in combination to improve patient outcomes.