Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.

Key Points• APTs as miRNA targets provide a novel molecular mechanism for how primary CLL cells escape from CD95-mediated apoptosis.• Palmitoylation as a novel posttranslational modification in CLL might also impact on survival signaling, proliferation, and migration.Resistance toward CD95-mediated apoptosis is a hallmark of many different malignancies, as it is known from primary chronic lymphocytic leukemia (CLL) cells. Previously, we could show that miR-138 and -424 are downregulated in CLL cells. Here, we identified 2 new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby significantly overexpressed in CLL cells. APTs are the only enzymes known to promote depalmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared with normal B cells. We identified APTs to directly interact with CD95 to promote depalmitoylation, thus impairing apoptosis mediated through CD95. Specific inhibition of APTs by siRNAs, treatment with miRs-138/-424, and pharmacologic 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 microRNA (miRNA) target provides a novel molecular mechanism for how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel posttranslational modification in CLL, which might impact on localization, mobility, and function of molecules, survival signaling, and migration. (Blood. 2015;125(19):2948-2957 IntroductionChronic lymphocytic leukemia (CLL) cells are characterized by a distinct resistance toward CD95 (Fas/APO-1).1-4 CD95, as prototypical member of the tumor necrosis factor superfamily, is able to transmit a death signal to the cell upon binding of the CD95 ligand (CD95L). 5CD95 is involved in fundamental processes like the maturation or homeostasis of lymphocytes, as well as the elimination of virus-infected or malignant cells. Malignant cells might develop resistance mechanisms to escape from CD95-mediated apoptosis, either by silencing its expression, 6 by mutation, 7 or by overexpression of antiapoptotic proteins.2 Prior studies emphasized that proper CD95-mediated apoptotic signaling might depend on posttranslational modifications of CD95. [8][9][10][11] This is of particular interest because the nature of molecular events downstream of the death-inducing signaling complex is well investigated, but the knowledge of initial events, especially at the plasma membrane, remains poorly defined. It was shown that palmitoylation, a common posttranslational modification, is crucial for localization of CD95 to specialized membrane microdomains and for formation of stable receptor aggregates. 8,9 Palmitoylation is the only reversible lipid modification, indicating that it is involved in dynamic processes.12-14 Overall, proteome analyses identified .1000 proteins to be palmitoylated. These proteins are invol...

BTK inhibitors are highly active drugs for the treatment of CLL. To understand the response to BTK inhibitors on a molecular level, we performed (phospho)proteomic analyses under ibrutinib treatment. We identified 3466 proteins and 9184 phosphopeptides (representing 2854 proteins) in CLL cells exhibiting a physiological ratio of phosphorylated serines, threonines and tyrosines (pS:pT:pY). Expression of 83 proteins differed between unmutated IGHV (UM)-CLL and mutated IGHV (M)-CLL. Strikingly, UM-CLL cells showed higher basal phosphorylation levels than M-CLL samples. Effects of ibrutinib on protein phosphorylation levels were stronger in UM-CLL, especially on phosphorylated tyrosines. The differentially regulated phosphopeptides and proteins clustered in pathways regulating cell migration, motility, cytoskeleton composition and survival. One protein, MARCKS, showed striking differences in expression and phosphorylation level in UM-CLL versus M-CLL. MARCKS sequesters PIP2, thereby affecting central signaling pathways and clustering of the B cell receptor. Genetically induced loss of MARCKSignificantly increased AKT signaling and the migratory capacity. CD40L stimulation increased expression of MARCKS. BCR stimulation induced phosphorylation of MARCKS, which was reduced by BTK inhibitiors. In line with our in vitro findings, low MARCKS expression is associated with significantly higher treatment-induced leukocytosis and more pronounced decrease of nodal disease in CLL patients treated with acalabrutinib.