Mirs-138 and -424 Control Palmitoylation-Dependent CD95-Mediated Cell Death By Targeting Acyl Protein Thioesterases 1 and 2 in Chronic Lymphocytic Leukemia

Introduction: Resistance towards CD95-mediated apoptosis is a hallmark of many different malignancies, like it is known from primary chronic lymphocytic leukemia (CLL) cells. Moreover, apoptosis mediated through CD95 is an essential mechanism to eliminate e.g. auto-reactive or virally infected cells. However, its mode of action is still not fully understood. Recently, it could be shown that palmitoylation of CD95 can influence its signaling properties. Nevertheless, the role and regulation of palmitoylated CD95 still needs to be determined.

Methods and results: Previously, we could show that miR-138 and -424 are down-regulated in CLL cells. By applying luciferase reporter assays, mutations of the binding sites qRT-PCR and immunoblots after transfection of both miRs, we identified two new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby are significantly over-expressed in CLL cells. Interestingly, our data reveal that expression of APTs is already controlled by miRs on mRNA level. This way APT1 is regulated by miR-138 and expression of APT2 is controlled by miR-424. So far, 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 as we determined by click-chemistry, which is a non-radioactive method to determine palmitoylated proteins. Importantly, via acyl-biotin exchange assays with subsequent immunoprecipitation of CD95 and fluorescence lifetime imaging microscopy (FLIM) to Foerster resonance energy transfer (FRET) in living cells we identified APTs to directly interact with CD95 to promote de-palmitoylation, thus impairing apoptosis mediated through CD95. As proof of concept APTs were inhibited specifically by siRNAs, miRs-138/-424 or our pharmacological inhibitor Palmostatin B. Thereby we could restore CD95-mediated apoptosis in CLL cells and other cancers, pointing to a central regulatory role of APTs in CD95 apoptosis.

Conclusion: The identification of the de-palmitoylation 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. In light of global palmitoylome studies, which show that potentially palmitoylated proteins are involved in all central cellular processes, such as protein transport, survival, migration, apoptosis and B-cell receptor signaling, this emphasizes the importance of palmitoylation and might put it on par with modifications like phosphorylation.