ALBERT

Description: Genome-wide gene expression profiling of chronic lymphocytic leukemia (CLL) cells in comparison to healthy donor CD5-positive B-cells revealed deregulated expression of lipase-associated genes. A set of 19 lipase activity defined genes, e.g. LPL, phospholipases A1, −A2, −C and −D2 family members and other lipase-associated genes were overexpressed in CLL. Recently lipoprotein lipase (LPL) was identified as prognostic factor in CLL. Here we show that the expression of LPL in CLL is induced by B-cell receptor (BCR) stimulus both in mutated and unmutated CLL samples. In native and BCR-stimulated CD5+ healthy B-cells no LPL-expression was detected. Antigenic stimulation via the BCR is thought to be functionally and prognostically relevant in CLL pathogenesis, LPL expression in CLL cells could reflect a permanent BCR-stimulus due to autoantigens including also unmutated IgVH cases. We hypothesized that the overexpression of lipases and especially of LPL reveals a putative therapeutic target by lipase inhibition through the the FDA-approved obesity drug and lipase inhibitor orlistat (tetrahydrolipstatin), which is known to inhibit LPL as well. Treatment of CLL cells with orlistat in vitro revealed significant cytotoxicity and induction of apoptosis in primary CLL cells with an IC50 of 5.48 μM (n=18). In comparison, no significant cytotoxicity was seen with healthy PBMC’s (n=12; p

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: Resistance towards apoptotic stimuli mediated by overexpression of antiapoptotic factors or extracellular survival signals like B-cell receptor stimulation (BCR) are considered to be responsible for accumulation of malignant B cells in CLL . TOSO, also known as Fas-inhibitory molecule 3 (FAIM3), was identified as overexpressed candidate gene in CLL based on re-evaluation of publicly available microarray data sets. Based on primary CLL samples from 106 patients, TOSO expression was compared to healthy donor B cells using quantitative real-time PCR, western-blot, flow cytometry and immunohistochemistry. To reveal underlying mechanisms of TOSO overexpression, B-cell receptor (BCR) and CD40Ligand stimulation as well as bone marrow stroma cell co-incubation was performed. Apoptotic resistance was assessed by annexin V/7-AAD flow cytometry in context of CH11-Fas-agonistic antibody. TOSO was identified to exhibit elevated relative expression of 6.8 compared to healthy donor B cells using quantitative real-time PCR (p=0.004). High levels of TOSO expression in CLL correlated with high leukocyte count, advanced Binet stage, previous need for chemotherapy and unmutated IgVH status. CD38+ CLL subsets harboring proliferative activity showed significantly enhanced TOSO expression. Immunohistochemistry revealed upregulation of TOSO in lymph nodes of CLL patients. In lymph nodes derived from healthy donors TOSO was detected in single plasmocytoid cells within the germinal center and in the marginal zone. No specific staining was seen in follicular lymphomas. CLL-specific upregulation of TOSO was confirmed by RT-PCR, samples of follicular lymphomas, DLBCL, marginal zone lymphoma and Hodgkin cell lines did not reveal TOSO up-regulation. We evaluated functional mechanisms of aberrant TOSO expression in CLL cells and identified TOSO expression significantly being induced by BCR-stimulation compared to control cells (relative expression (RE) 8.25 vs. 4.86, p=0.013). In contrast, CD40L signaling significantly reduced TOSO expression (RE 2.60; p=0.007). Spontaneous apoptosis of CLL cells was significantly reduced by BCR-stimulus, in CD40Ligandstimulated CLL samples a slight sensitization towards Fas-mediated apoptosis was seen. In summary, we show that the anti-apoptotic factor TOSO is associated with progressive disease and enhanced in the proliferative CD38+ cell subset. Both association with unmutated IgVH and the specific induction of TOSO via the BCR suggest autoreactive BCR signaling as a key mediator of apoptosis resistance in CLL. Down-regulation of TOSO by CD40Ligand in the context of CD40Ligand-mediated Fas-sensitization of CLL reveal TOSO as a new anti-apoptotic factor in CLL. CLL-specific over-expression of the transmembrane protein might further offer new therapeutic strategies in CLL treatment.

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: 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: Tumor metabolism and its specific alterations have become an integral part of understanding functional alterations leading to malignant transformation and maintaining cancer progression. Here, we review the metabolic changes in B-cell neoplasia, focusing on the effects of tumor metabolism on the tumor microenvironment (TME). Particularly, innate and adaptive immune responses are regulated by metabolites in the TME such as lactate. With steadily increasing therapeutic options implicating or utilizing the TME, it has become essential to address the metabolic alterations in B-cell malignancy for therapeutic approaches. In this review, we discuss metabolic alterations of B-cell lymphoma, consequences for currently used therapy regimens, and novel approaches specifically targeting metabolism in the TME.

Description: Macrophage polarity has recently been shown to play a pivotal role in progression and prognosis of human malignancies. In CLL the high dependence of the malignant cell to the tumor microenvironment has revealed macrophages as major mediators of leukemia cell survival. In contrast, macrophage activation also offers novel therapeutic strategies for leukemia cell targeting. Here we analyze the reciprocal relationship of leukemia cells and macrophages and the specific functional impact of phagocytosis in leukemia progression and therapy. We employed the humanized hMB-Lymphoma and the Eµ-TCL1 CLL mouse model. Thioglycollate-induced macrophages, bone marrow derived murine macrophages and human monocyte derived macrophages were used for in vitroevaluation of phagocytosis either by bead based approaches or by ADCC. Applying gene-expression profiling of macrophages in Eµ-TCL1 mice we could identify profound transcriptional alterations indicating a re-programming during leukemogenesis. Functional genomic analysis particularly revealed impaired phagocytic function induced during leukemia onset. In vivo and ex vivo phagocytosis assays of primary macrophages showed a significant reduced phagocytic activity during CLL progression. Human macrophages in co-culture with CLL cells in vitro compared to healthy B-cells similarly showed defects in phagocytosis. In the humanized leukemia model we similarly observed impaired phagocytosis resulting in resistance towards therapeutic antibody/macrophage mediated therapy. Resistance was actively induced by increasing leukemia cell infiltration in the bone marrow. Impaired macrophage function could be identified being mediated by secretory crosstalk such as release of PGE2 by leukemia cells and Cholecalciferol. We could restore phagocytic function by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, an acute secretory activating phenotype (ASAP), releasing cytokines from leukemia cells induces macrophage infiltration and phagocytic activity in the bone marrow. Thus, malignant cells can be effectively targeted by modulation of macrophage polarization and function. In conclusion, we have identified decreased phagocytic activity of macrophages as key functional aspect in leukemia and lymphoma associated macrophages. Inversely, enhancing phagocytosis rendered essential for the re-sensitization of refractory niches treatment towards monoclonal antibodies defined by macrophage polarity. Overall macrophage function represents a key therapeutic target in CLL and other B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1375 Background: Since aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukemia (CLL), alternative therapeutic approaches are needed. Moreover, there is an essential need to improve specific therapeutic regimes for “non-fit” patients, which cannot receive myeloablative therapies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce tumor-specific apoptosis. However, apoptosis might be inhibited by elevated X-linked inhibitor of apoptosis (XIAP) level, the only cellular protein capable to bind to and effectively inhibit caspases. Use of XIAP-inhibiting compounds might sensitize primary CLL cells towards TRAIL-induced lysis. Experimental design: We compared XIAP protein levels between freshly purified CD5+CD19+ primary CLL cells (n=28) and CD19+ B cells from healthy donors (n=16) by western blotting. In a knockdown approach, specific siRNAs against XIAP were nucleofected to check whether XIAP expression prevents TRAIL-mediated apoptosis in CLL. After proof of concept, we applied the novel small molecule IAP antagonizing compound (IAC), an inhibitor of XIAP, in combination with TRAIL to induce apoptosis in primary CLL cells (n=48). Compound A (CA) was developed based on the crystal structure of four amino acids of SMAC, which enabling SMAC to efficiently bind the BIR3 domain of XIAP. In contrast to the active compound CA, which consists of an amino terminal methyl alanine, the inactive compound CB used in our studies as a negative control has an amino terminal methyl glycine. This specific substitution results in a significant reduction of IAP binding capability of CB as CA has binding affinity to XIAP in the picomolar range and CB is a weak binder with micromolar binding affinity to XIAP. Results: XIAP is significantly higher expressed in primary CLL cells (n=28) compared to healthy B cells (n=16) (P=0.02). Our data obtained by specific knockdown of XIAP via siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Based on these results we used IAC in combination with TRAIL. Combined treatment with both drugs significantly increased apoptosis compared to untreated (P=8.5×10-10), solely IAC (P=4.1×10-12) or TRAIL treated (P=4.8×10-10) CLL cells. As a potent cellular caspase inhibitor, we also examined the involvement of caspases in CA/TRAIL-mediated apoptosis. Not surprisingly, co-application of pan-caspase inhibitor zVAD.fmk inhibited cell death induced by CA/TRAIL underscoring the apoptotic caspase-dependent cytotoxicity of CA/TRAIL treatment in CLL cells. IAC rendered 40 of 48 (83.3%) primary CLL samples susceptible towards TRAIL-mediated apoptosis. Especially cells derived from patients with poor prognosis (ZAP-70+, IGHV unmutated, 17p-) were highly responsive to this drug combination. Furthermore, this study reveals that TRAIL application alone induces apoptosis in poor-prognosis CLL samples (13,8% in ZAP-70+ (n=10) vs. 2,3 in ZAP-70- (n=9); P=0.0008), which correlates with the elevated expression levels of TRAIL-R1 and –R2 on ZAP-70+ CLL cells. To assess whether TRAIL treatment is CLL cell specific, healthy B cells (n=4) were exposed to TRAIL alone or CA(CB)/TRAIL and showed significantly lower susceptibility towards CA/TRAIL administration than CLL cells. Conclusion: XIAP is over-expressed in CLL and displays a suitable target to induce TRAIL-mediated apoptosis. The novel XIAP inhibitor used in our study was able to inhibit XIAP function at a concentration of 0,1μM. CA/TRAIL administration was also shown not to induce apoptosis in healthy donor B cells and might therefore also display an attractive option for “non-fit” CLL patients. Our highly effective XIAP inhibitor CA, in concert with TRAIL, shows potential for treatment of CLL of those cases with poor prognosis and therefore warrants further clinical investigation. Disclosures: No relevant conflicts of interest to declare.