ALBERT

Description: Background The recovery of the host immune system after allogeneic hematopoietic stem cell transplantation is pivotal to prevent infections, relapse and secondary malignancies. In particular, numerical CD4 T-cell reconstitution is delayed and CD4-helper cell function considered impaired as consequence of the transplant procedure and concommitant immunosuppressive medication. From HIV/AIDS patients it is known that numerical and functional CD4 defects increase the risk of opportunistic infections. Therefore, even in the absence of immunosuppressants and graft-vs-host disease, anti-infective prophylaxis is usually given for at least six months. We hypothesized that the numerical CD4 defect in patients may be reflected by immunosuppressive RNA fingerprints previously established for certain immuno-inhibitory molecules and tested whether the functional CD4 capacity was different according to the CD4 cell number. Methods RNA was separated from CD4 T-cells of 10 patients with CD4 counts 〉500/µl, 10 patients with CD4 counts 500/µl were demonstrated to be under the influence of PGE2, whereas patients with CD4 T-cells

Description: Survival of chronic lymphocytic leukemia (CLL) cells is triggered by several stimuli, such as the B-cell receptor (BCR), CD40 ligand (CD40L), or interleukin-4 (IL-4). We identified that these stimuli regulate apoptosis resistance by modulating sphingolipid metabolism. Applying liquid chromatography electrospray ionization tandem mass spectrometry, we revealed a significant decrease of proapoptotic ceramide in BCR/IL-4/CD40L–stimulated primary CLL cells compared with untreated controls. Antiapoptotic glucosylceramide levels were significantly increased after BCR cross-linking. We identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide via UDP-glucose ceramide glucosyltransferase (UGCG). Besides specific UGCG inhibitors, our data demonstrate that IgM-mediated UGCG expression was inhibited by the novel and highly effective PI3Kδ and BTK inhibitors CAL-101 and PCI-32765, which reverted IgM-induced resistance toward apoptosis of CLL cells. Sphingolipids were recently shown to be crucial for mediation of apoptosis via mitochondria. Our data reveal ABT-737, a mitochondria-targeting drug, as interesting candidate partner for PI3Kδ and BTK inhibition, resulting in synergistic apoptosis, even under protection by the BCR. In summary, we identified the mode of action of novel kinase inhibitors CAL-101 and PCI-32765 by controlling the UGCG-mediated ceramide/glucosylceramide equilibrium as a downstream molecular switch of BCR signaling, also providing novel targeted treatment options beyond current chemotherapy-based regimens.

Description: Abstract 3597 Introduction: The microenvironment and especially the antigenic stimulation of the B-cell receptor on the surface of the malignant cell play a crucial role in the pathogenesis of chronic lymphocytic leukemia (CLL). Aberrant Nuclear Factor kappa B (NFκB) activity is another major hallmark of B-cell malignancies as well as of CLL. NFκB-dependent genes are involved in anti-apoptotic regulation, cell proliferation and metastasis and are responsible for survival and proliferation of tumors. However, the mechanisms of NFκB over-expression in CLL still remain to be elucidated. Prior studies revealed that cylindromatosis (CYLD) function might be of special interest in CLL since it inhibits signaling via TRAF2 and c-IAP1/2, which are known to be over-expressed in CLL. CYLD inactivation might therefore result in sustained NFκB signaling. The enzyme CYLD, a tumor suppressor that functions as a deubiquitinase, plays a role in other physiological aspects such as cell cycle response, inflammatory and immune processes. Moreover, it could be shown that impaired CYLD activity leads to increased NFκB activity in multiple myeloma cells demonstrating the negative regulatory function of CYLD regarding NFκB. Aside from CYLD, which is constitutively active preventing uncontrolled transcription factor activation, the enzyme A20, a key player in negative feedback loop regulation of NFκB, operates via induction, supposing that both enzymes might proceed at different phases of NFκB signaling. A20, also known as tumor necrosis factor alpha-induced protein 3 (TNFAIP3), acts as an ubiquitin-editing enzyme. Its inactivation is involved in immunopathologies (e.g. Crohn's disease, rheumatoid arthritis, systemic lupus erythematodes, psoriasis and type 1 diabetes mellitus) and in tumorigenesis. Frequent mutations in the A20 locus – leading to sustained NFκB activity – could be shown to play a dominant role in development of different B-cell malignancies. Experimental design and results: Based on genome-wide gene expression profiling analysis of CLL samples (n=8) compared to healthy donor B-cells (n=5), CYLD is expressed and its expression was reduced following B-cell receptor cross-linking (24 hours) (p=0,0036) contrary to A20 that could be induced after receptor stimulation (p=0,044). These results underline the role of B-cell receptor signaling in survival regulation of CLL cells and also its in-direct influence on NFκB activity. Recently, our report revealed by methylation analysis and additional sequence analysis that the A20 region neither contains any methylation (64 CLL patients versus 10 healthy donors) nor mutation (55 CLL patients with sequence analysis of exons 2–9 of the tnfaip3 gene) contrary to reports from other B-cell malignancies. Moreover, A20 expression could be confirmed by immunoblotting showing comparable results to healthy B-cells. In order to check if such alterations in the enzyme CYLD might occur in CLL leading to sustained activity of NFκB similar to other B-cell entities, we performed analysis of the methylation status of the promoter region of CYLD in 64 CLL patients compared to 10 DNAs of CD19-selected B-cells from healthy donors. Epigenetic alterations of the CYLD promoter could not be identified. Conclusions: Here we present the first report of epigenetic and mRNA expression analysis concerning the deubiquitinase CYLD in CLL. We identified that CYLD as well as A20 are regulated by B-cell receptor signaling. The opposed expression of CYLD and A20 after stimulation of the receptor might contribute to an almost balanced and well-adjusted NFκB activity. Our results of lacking epigenetic alteration in both proteins (A20 and CYLD) and absence of mutations in A20 indicate that malignant development in CLL differs from most of other B-cell malignancies, which show frequent inactivation of either CYLD or A20. Disclosures: No relevant conflicts of interest to declare.

Description: Post-translational modifications are important fine-tuning elements for controlling protein activity and signaling. Regulation of phosphorylation events of the BCR is critical for survival and proliferation of CLL cells. Palmitoylation, a common post-translational modification defined as the addition of palmitic acid to internal cysteins, was shown recently to regulate phosphorylation of proteins by controlling their localization and activity. Many proteins in the B cell receptor (BCR) signaling pathway in CLL cells are primarily cytosolic, but upon activation transiently located to the plasma membrane to fulfill their functions. Some of these proteins, like Src kinase family members Lyn, Yes and Fyn, are already reported to be palmitoylated. Previous studies by our group showed that global protein palmitoylation is deregulated in CLL cells and primarily caused by overexpression of the depalmitoylating enzyme APT1. To investigate, if overexpressed APT1 directly targets BCR signaling, we inhibited (genetically and pharmacologically) APT1 in CLL cells and analyzed occurring changes in 45 different phosphorylation-sites of major signaling pathways. Interestingly, we found that APT1 controls the central phosphorylation events within Akt/mTOR/p70S6 signaling. For example, phosphorylation of Akt (T308, S473) and p70S6 (T389, T421, S424) was significantly decreased after interference with protein depalmitoylation. By biochemical dissection of these pathways with acyl-biotin exchange (ABE) assays we identified novel palmitoylation candidates particularly within the PI3K/Akt axis, which are indispensable for phosphorylation of kinases of the Akt/mTOR/p70S6 axis. Functionally, pharmacological inhibition of APTs and genetic knockdown of APT1 sensitizes CLL cells towards BCR-associated KIs like Ibrutinib and Idelalisib. Our data uncovers that central phosphorylation events within the BCR pathway are dependent on palmitoylation controlled by APT1. Future studies should therefore investigate more in detail how addition of APT1 inhibitors can improve clinical outcome of patients treated with Idelalisib or Ibrutinib-based regimens. Disclosures Wendtner: Hoffmann-La Roche, Mundipharma, Janssen, Gilead, Abbvie, Servier, Morphosys: Consultancy, Other: Travle grants, Research Funding. Hallek:Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau.

Description: 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. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: Abstract 1335 Therapy-resistant microenvironments represent a major barrier to the effective elimination of disseminated malignancies. However, microenvironment dependent resistance mechanisms as potential synergistic drug interactions particularly for biologicals and monoclonal therapeutic antibodies are not completely understood. Here, we used the hMB humanized lymphoma mouse model as primary human B-ALL xenograft mice to address mechanisms of resistance and potential synergy of the clinical grade antibodies alemtuzumab and rituximab. Response to antibody therapy was shown to be mediated by macrophages as effector cells by direct phagocytosis - indicated by an abrogated response in macrophage-depleted mice. Antibody mono-therapy however still showed limited response in the bone marrow as a site of a primarily resistant microenvironment. In order to overcome therapeutic resistance and generate a functional state of the tumor microenvironment allowing effective antibody-mediated phagocytosis of lymphoma cells we applied a series of combinatorial regimens. Supplementing treatment with GM-CSF in order to improve the effector to target ratio slightly enhanced the therapeutic response as only modest additive affects were seen with total body irradiation. By combining alemtuzumab and cyclophosphamide in the hMB model as rituximab and cyclophosphamide respectively in CD20+ B-ALL xenografts we identified a striking synergy leading to profound depletion of malignant cells from bone marrow and spleen. Mice treated in the combinatorial arm survived significantly longer (Median survival 7 weeks vs. 28 weeks, p

Description: MicroRNAs (miRNA) play a key role in cellular regulation and, if deregulated, in the development of neoplastic disorders including chronic lymphocytic leukemia (CLL). RNAs from primary cells of 50 treatment-naive CLL patients and peripheral B cells of 14 healthy donors were applied to miRNA expression profiling using bead chip technology. In CLL cells, a set of 7 up- and 19 down-regulated miRNAs was identified. Among the miRNAs down-regulated in CLL cells, 6 of 10 miRNA promoters examined showed gain of methylation compared with normal B-cell controls. Subsequent target prediction of deregulated miRNAs revealed a highly significant binding prediction at the 3′ untranslated region of the pleomorphic adenoma gene 1 (PLAG1) oncogene. Luciferase reporter assays including site-directed mutagenesis of binding sites revealed a significant regulation of PLAG1 by miR-181a, miR-181b, miR-107, and miR-424. Although expression of PLAG1 mRNA was not affected, PLAG1 protein expression was shown to be significantly elevated in CLL cells compared with the levels in healthy donor B cells. In summary, we could demonstrate disruption of miRNA-mediated translational control, partly due to epigenetic transcriptional silencing of miRNAs, with subsequent overexpression of the oncogenic transcription factor PLAG1 as a putative novel mechanism of CLL pathogenesis.

Description: Ibrutinib is especially effective in CLL patients with unmutated IgHV (UM-CLL), although presence of unmutated IgHV is usually associated with poor clinical outcome and shorter PFS. Sustained BCR signaling is one major reason for this. The BCL2 protein BIM plays a pivotal role in regulating apoptosis and high expression of BIM has been associated with presence of unmutated IgHV. This led us to investigate if there is a causality between BIM regulation and ibrutinib sensitivity. Methods: We used quantitative mass spectometry (MS) to analyze the (phospho) proteome of CLL patients with unmutated and mutated IgHV (n=3 respectively) upon BCR stimulation and ibrutinib treatment. BTKC481S overexpressing cells were used to validate our findings from MS. Results: Among all identified (phospho)peptides we found a striking phosphorylation pattern of BIM when comparing UM-CLL and M-CLL. Interestingly, we found that only phosphorylation of S77 was significantly altered, but not phosphorylation of S69 as previously reported. Phosphorylation of BIM S77 was upregulated upon BCR stimulation only in UM-CLL, but not in M-CLL. It is known, that BIM phosphorylation of both S69 and S77 leads to degradation of the protein by ubiquitination. In line with this and with the increase in BIM phosphorylation, total level of BIM decreased upon BCR stimulation. Furthermore we identified BIM as a target of ibrutinib. The BTK inhibitor was able to reduce phosporylation of BIM S77 both in UM-CLL and M-CLL. In accord with the observations of decreased BIM expression upon BCR stimulation, the decrease in phosphorylation of BIM induced by ibrutinib led to higher total levels of BIM. In order to investigate if phosphorylation of BIM is BTK dependent we generated an ibrutinib-resistant BTK mutant (C481S) that was overexpressed in Ramos cells. While ibrutinib treatment led to a decrease of phosphorylation of BIM S77 in wildtype cells, no change in phosphorylation could be observed in cells expressing the BTKC481S mutant, which suggests that BIM regulation by ibrutinib is indeed BTK dependent. Similar to ibrutinib, SYK inhibitor entospletinib leads to reduction of BIM phoshorylation in wildtype Ramos. But in contrast to ibrutinib, both BTK and BIM phosphorlyation were also reduced upon entospletinib treatment in Ramos cells expressing the BTKC481S mutant, which indicates that entospletinib can overcome ibrutinib resistance caused by BTKC481S mutation. Conclusion: For the first time we found that BIM S77 is a target of ibrutinib. Whereas BCR stimulation led to a differential response dependent on IgHV, ibrutinib was able to target BIM both in UM-CLL and M-CLL in a BTK dependent manner. Furthermore, ibrutinib can regulate total BIM levels by preventing protein degradation. Interestingly, the SYK inhibitor entospletinib can also target BIM, but is able to overcome ibrutinib resistance mediated by BTKC481S mutation. Disclosures Hallek: Roche, Gilead Sciences, Inc., Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie: Honoraria, Research Funding, Speakers Bureau. Frenzel:Gilead: Research Funding; Abbvie: Consultancy.

Description: Background: The immunoglobulin-like protein TOSO, which has been found to serve as Fc receptor for IgM (FcµR), was shown by us and others to be overexpressed on CLL cells and only weakly expressed on more aggressive B-NHL. However the functional role of TOSO on lymphomagenesis has not been explored so far. Methods: To determine the role of TOSO on lymphoma development, we took advantage of the Eµ-TCL1 transgenic mice, which usually end up with an aggressive (IgVH unmutated) CLL-like phenotype. We generated a novel B cell-specific conditional knockout (KO) mouse model in which EµTCL1 mice (TC or control in the following) were crossbred with TOSO-floxed mice, expressing Cre recombinase under the control of the CD19 promoter (EµTCL1;Tosofl/fl;Cd19cre/wtor TCT in the following). TCT mice were further compared with p53 conditional knockout (EµTCL1;Tp53fl/fl;Cd19cre/wt or TCP). Results: In this study, we compared kinetics, overall survival and phenotype of lymphoma/CLL in TC, TCT and TCP mice. Interestingly, TCTmice developed a very aggressive phenotype and resulted in significantly shorter overall survival compared to TC mice (TCT 274 days vs. TC 346 days; p