A Complex Molecular Network Controls Palmitoylation-Dependant CD95 Sensitivity Of CLL Cells
Authors: Berg Valeska , Vartak Nachiket , Claus Rainer , Claasen Julia , Rusch Marion , Pallasch Christian P. , Waldmann Herbert , Plass Christopher , Bastiaens Philippe , Hallek Michael , Hedberg Christian , Wendtner Clemens , Frenzel Lukas P. 
Published: October 2019

Abstract

Introduction
CD95-mediated apoptosis is a central physiologic mechanism to eliminate e.g. auto-reactive and malignant cells. However, its mode of action remains still not fully understood. Recently, it could be shown that palmitoylation of CD95 alters its apoptotic function. However, the role, regulation and precise molecular function of palmitoylated-CD95 need to be determined.

Methods and Results
Applying acyl-biotin exchange (ABE) assays and click chemistry we uncovered, that CD95 is palmitoylated in weakly palmitoylated in primary CLL cells and other malignant cell types. Via mutational analysis and ABE assays we identified the palmitoylation site of CD95 and applied a mutant as control in further experiments. Interestingly, we could show that the de-palmitoylating enzymes LYPLA1 and LYPLA2 are significantly over-expressed on gene and protein level in primary CLL cells. Importantly, FLIM-FRET experiments (Fluorescence Lifetime Imaging Microscopy - Fluorescence Resonance Energy Transfer) reveal direct interactions between LYPLAs and CD95 for the first time. To uncover how LYPLA1 and LYPLA2 are regulated, we determined differentially expressed miRNAs between CLL cells and normal B cells via bead chip arrays, confirmed their expression via qPCR and checked their binding to both enzymes via luciferase reporter-assays. Over-expression of those finally four miRNAs lead to down-regulation of both enzymes in malignant cells on protein level. Moreover, our data reveal, that these miRNAs are down-regulated due to epigenetics, as these miRNAs were up-regulated after 5-AZA treatment and in DNMT knockout cells. Most remarkable, pharmacological inhibition and siRNA-mediated knockdown of LYPLA1 and LYPLA2 resulted in increased CD95 palmitoylation and subsequently in increased CD95-mediated apoptosis. Interestingly, also over-expression of miRNAs increased susceptibility towards CD95-mediated apoptosis significantly. These results show that the interaction between LYPLA1/LYPLA2 and CD95 is essential for a proper apoptotic signaling. To understand the functional relevance of the palmitoylation site during the apoptotic process, we analyzed the receptor by FACS and microscopy (FRAP, Fluorescence Recovery After Photobleaching) and revealed that the precise localization of CD95 on the plasma membrane might be responsible for the effects observed on CLL cells and other tumor cells.

Conclusion
Here we uncovered the complexity of CD95 signaling in CLL and malignant cells in general. We identified novel interaction partners of CD95, which account for the molecular switch between survival and apoptosis mediated by CD95. Moreover, our data reveal that susceptibility towards CD95 is dramatically altered by a molecular network of epigenetics, miRNAs and de-palmitoylating enzymes. Importantly, we can show that de-palmitoylating enzymes are drugable and their inhibition restores CD95 apoptotic signaling and improves thereby immunogenicity of CLL cells.
L.P.F. and C-M.W. contributed equally to this work.

Disclosures:
No relevant conflicts of interest to declare.