Glucosinolates (GS) are important plant secondary metabolites in plant resistance to herbivores, bacteria, and fungi, which have been shown to be accumulating in different organs and tissue types at varying concentrations. There are more than 200 GS species found in order Brassicales and presence of these compounds is well documented on organ-specific but not on cell-specific level. We used UPLC/ESI-QTOF-MS to measure the presence of GS and qRT-PCR to analyse the expression of GS biosynthetic and regulatory genes in isolated Arabidopsis thaliana trichomes. Trichomes of Arabidopsis are shown to synthesize chemoprotective aliphatic glucosinolates (AGS) and indolic glucosinolates (IGS), which are known for their biological activities against fungi, bacterial pathogens, or herbivores. UPLC/ESI-QTOF-MS analysis of various IGS mutants reveal increased or decreased levels of IGS in trichomes of gain- and loss-of-function mutants correspondingly. Using pMYB51/HIG1-uidA and pMYB28/PMG1/HAG1-uidA reporter plants we demonstrate that production of these important compounds is activated in trichomes of leaves or inflorescences in response to wounding. Since trichomes represent the first interface in plant-environment interactions, the possible role of GS containing trichomes in plant defense or signaling is discussed.

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene () has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.

Post-translational modifications are important fine-tuning elements for controlling protein activity and signaling. Regulation of phosphorylation events of the BCR is critical for survival and proliferation of CLL cells. Palmitoylation, a common post-translational modification defined as the addition of palmitic acid to internal cysteins, was shown recently to regulate phosphorylation of proteins by controlling their localization and activity. Many proteins in the B cell receptor (BCR) signaling pathway in CLL cells are primarily cytosolic, but upon activation transiently located to the plasma membrane to fulfill their functions. Some of these proteins, like Src kinase family members Lyn, Yes and Fyn, are already reported to be palmitoylated. Previous studies by our group showed that global protein palmitoylation is deregulated in CLL cells and primarily caused by overexpression of the depalmitoylating enzyme APT1. To investigate, if overexpressed APT1 directly targets BCR signaling, we inhibited (genetically and pharmacologically) APT1 in CLL cells and analyzed occurring changes in 45 different phosphorylation-sites of major signaling pathways. Interestingly, we found that APT1 controls the central phosphorylation events within Akt/mTOR/p70S6 signaling. For example, phosphorylation of Akt (T308, S473) and p70S6 (T389, T421, S424) was significantly decreased after interference with protein depalmitoylation. By biochemical dissection of these pathways with acyl-biotin exchange (ABE) assays we identified novel palmitoylation candidates particularly within the PI3K/Akt axis, which are indispensable for phosphorylation of kinases of the Akt/mTOR/p70S6 axis. Functionally, pharmacological inhibition of APTs and genetic knockdown of APT1 sensitizes CLL cells towards BCR-associated KIs like Ibrutinib and Idelalisib. Our data uncovers that central phosphorylation events within the BCR pathway are dependent on palmitoylation controlled by APT1. Future studies should therefore investigate more in detail how addition of APT1 inhibitors can improve clinical outcome of patients treated with Idelalisib or Ibrutinib-based regimens. Disclosures Wendtner: Hoffmann-La Roche, Mundipharma, Janssen, Gilead, Abbvie, Servier, Morphosys: Consultancy, Other: Travle grants, Research Funding. Hallek:Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau.