Palmitoylation Controls Death Receptor Function in Chronic Lymphocytic Leukemia
Authors: Frenzel Lukas P. , Feldhaus Valeska , Rusch Marion , Brinker Reinhild , Claasen Julia , Pallasch Christian P. , Waldmann Herbert , Hallek Michael , Hedberg Christian , Wendtner Clemens-Martin 
Published: October 2019

Abstract
Abstract 2829

Introduction:
Post-translational modifications are important fine-tuning elements for controlling protein activity and signaling. Palmitoylation is a common post-translational modification and defined as the addition of palmitic acid to internal cysteins. Interestingly, in contrast to other lipid modifications, it is reversible. Control over the palmitoylation cycle therefore provides indirect control over protein localization and function. While a number of proteins with palmitoyl transferase activity are known, LYPLA1 (lysophospholipase 1) is the only enzyme known to be responsible for the process of depalmitoylation. CLL cells are known to be resistant to TRAIL-mediated apoptosis. While TRAIL-R1 is reported to be palmitoylated, TRAIL-R2 seems to contain a region with basic amino acids in its membrane-proximal cytoplasmatic domain. Some studies showed that palmitoylation is crucial for several steps of death receptor signaling. Therefore, regulation of depalmitoylation by LYPLA1 seems to be an important tool for the regulation of death receptor function.
Methods and Results: Global palmitoylation in CLL cells was investigated by screening for all palmitoylated proteins via a click chemistry assay. There, cells were metabolically labeled, coupled to a specific reporter group and then analyzed by in-gel fluorescence. Comparison of healthy B cells, healthy PBMCs and CLL cells revealed a significant difference in global palmitoylation (+38.5 % in B cells, n=6, p<0.001; +57.8 % in healthy PBMCs, n=6, p<0.001 compared to CLL cells, n=10). We identified LYPLA1 as overexpressed in CLL compared to healthy controls on both protein and mRNA level. We generated a potent LYPLA1 inhibitor. We could show, that inhibition of LYPLA1 led to a significant increase of the overall protein palmitoylation level in CLL cells (+24.7 % n=6, p=0.0118). Ours and other groups have shown, that treatment of cancer cells with TRAIL and X-linked inhibitor of apoptosis protein (XIAP)-inhibition lead to apoptosis in otherwise TRAIL resistant CLL cells. Since death receptors might be palmitoylated, we extended these studies. Treatment of CLL cells with TRAIL, XIAP- and LYPLA1-inhibition led to significantly increased apoptosis compared to TRAIL treatment and XIAP-inhibition alone (+43.2 %, n=12, p=0.0089). Palmitoylation of death receptors was investigated with the help of acyl-biotin exchange chemistry. We could show that palmitoylation of TRAIL-R1 was significantly increased after LYPLA1-inhibiton (+58.7 %, n=3, p=0.0169). It could be demonstrated, that inhibition of LYPLA1 in combination with death receptor stimulation increased the amount of activated caspase-8 in comparison to solely TRAIL and DMSO treated cells (+41.8 %, n=3, p=0.0199), indicating that palmitoylation plays a crucial role in apoptotic signaling far from XIAP. In addition to that, we could show that inhibition of depalmitoylation of TRAIL-R1 led to more death receptor located to lipid rafts. To understand how LYPLA1 is regulated, we investigated two highly conserved miRNAs which were predicted as key regulators of LYPLA1 and which are significantly downregulated in CLL. Indeed, luciferase assays revealed that both miRNAs were able to downregulate LYPLA1 expression.

Conclusion:
We show for the first time, that LYPLA1 is a central enzyme which regulates the apoptotic signaling of TRAIL. Furthermore, we identified LYPLA1 to be regulated by miRNAs, which are deregulated in CLL. These novel findings allow speculation, that LYPLA1 inhibitors could be used for the treatment of CLL. Future experiments should therefore aim at investigating the LYPLA1 signaling pathway as a potential target for CLL/ cancer therapy.
L.P.F. and V.F. contributed equally to this work.

Disclosures:
No relevant conflicts of interest to declare.