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Acetylation site specificities of lysine deacetylase inhibitors in human cells.

Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases.

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Fractional killing arises from cell-to-cell variability in overcoming a caspase activity threshold.

When cells are exposed to death ligands such as TRAIL, a fraction undergoes apoptosis and a fraction survives; if surviving cells are re-exposed to TRAIL, fractional killing is once again observed. Therapeutic antibodies directed against TRAIL receptors also cause fractional killing, even at saturating concentrations, limiting their effectiveness. Fractional killing arises from cell-to-cell fluctuations in protein levels (extrinsic noise), but how this results in a clean bifurcation between life and death remains unclear. In this paper, we identify a threshold in the rate and timing of initiator caspase activation that distinguishes cells that live from those that die; by mapping this threshold, we can predict fractional killing of cells exposed to natural and synthetic agonists alone or in combination with sensitizing drugs such as bortezomib. A phenomenological model of the threshold also quantifies the contributions of two resistance genes (c-FLIP and Bcl-2), providing new insight into the control of cell fate by opposing pro-death and pro-survival proteins and suggesting new criteria for evaluating the efficacy of therapeutic TRAIL receptor agonists.
© 2015 The Authors. Published under the terms of the CC BY 4.0 license.

In vivo anti-tumor efficacy of afucosylated anti-CS1 monoclonal antibody produced in glycoengineered Pichia pastoris.

Monoclonal antibody (mAb) therapy has been successfully used for the treatment of B-cell lymphomas and is currently extended for the treatment of multiple myeloma (MM). New developments in MM therapeutics have achieved significant survival gains in patients but the disease still remains incurable. Elotuzumab (HuLuc63), an anti-CS1 monoclonal IgG1 antibody, is believed to induce anti-tumor activity and MM cytotoxicity through antibody dependent cellular cytotoxicity (ADCC) and inhibition of MM cell adhesion to bone marrow stromal cells (BMSCs). Modulations of the Fc glycan composition at the N297 site by selective mutations or afucosylation have been explored as strategies to develop bio-better therapeutics with enhanced ADCC activity. Afucosylated therapeutic antibodies with enhanced ADCC activity have been reported to possess greater efficacy in tumor growth inhibition at lower doses when compared to fucosylated therapeutic antibodies. The N-linked glycosylation pathway in Pichia pastoris has been engineered to produce human-like N-linked glycosylation with uniform afucosylated complex type glycans. The purpose of this study was to compare afucosylated anti-CS1 mAb expressed in glycoengineered Pichia pastoris with fucosylated anti-CS1 mAb expressed in mammalian HEK293 cells through in vitro ADCC and in vivo tumor inhibition models. Our results indicate that Fc glycosylation is critical for in vivo efficacy and afucosylated anti-CS1 mAb expressed in glycoengineered Pichia pastoris shows a better in vivo efficacy in tumor regression when compared to fucosylated anti-CS1 mAb expressed in HEK293 cells. Glycoengineered Pichia pastoris could provide an alternative platform for generating homogeneous afucosylated recombinant antibodies where Fc mediated immune effector function is important for efficacy.
Copyright © 2015 Elsevier B.V. All rights reserved.

MAFG is a transcriptional repressor of bile acid synthesis and metabolism.

Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis, and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG(+/-) mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR.
Copyright © 2015 Elsevier Inc. All rights reserved.

Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2.

Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the β3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active &quot;in&quot; conformation, imparting resistance to inhibitors like vemurafenib that bind the αC &quot;out&quot; conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy.
Copyright © 2016 Elsevier Inc. All rights reserved.