miRs-138 and -424 control palmitoylation-dependent CD95-mediated cell death by targeting acyl protein thioesterases 1 and 2 in chronic lymphocytic leukemia

Resistance towards CD95-mediated apoptosis is a hallmark of many different malignancies, like it is known from primary chronic lymphocytic leukemia (CLL) cells. Previously, we could show that miR-138 and -424 are down-regulated in CLL cells. Here, we identified two new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby significantly over-expressed in CLL cells. APTs are the only enzymes known to promote de-palmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared to normal B cells. We identified APTs to directly interact with CD95 to promote de-palmitoylation, thus impairing apoptosis mediated through CD95. Specific inhibition of APTs by siRNAs, treatment with miRs-138/-424 and pharmacological approaches restore CD95-mediated apoptosis in CLL cells and other cancer cells, pointing to an important regulatory role of APTs in CD95 apoptosis. The identification of the depalmitoylation reaction of CD95 by APTs as a miRNA target provides a novel molecular mechanism how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel post-translational modification in CLL, which might impact on localization, mobility and function of molecules, survival signaling and migration. we expected interactions between APTs and their substrates to be transient and hence performed fluorescence lifetime imaging microscopy - Förster resonance energy transfer (FLIM-FRET) assays. Since APTs are partially cytosolic proteins and therefore might interact with CD95 either at the membrane or in the cytosol, we performed experiments in HeLa cells, which are much larger than small lymphocytes and easier to transfect. Analyses of the FLIM-FRET data indentified a direct interaction of both APT1 and APT2 with CD95, but not with the CD95 (C199S) palmitoylation-mutant (Figure 4A-C) . These results reveal an interaction taking place directly at the palmitoylation site of CD95 with most significance at the plasma membrane, but also in the cytoplasm. So far this is the first report, proofing a direct interaction between de-palmitoylating enzymes and their substrate.