BackgroundCancer cells display widespread changes in DNA methylation that may lead to genetic instability by global hypomethylation and aberrant silencing of tumor suppressor genes by focal hypermethylation. In turn, altered DNA methylation patterns have been used to identify putative tumor suppressor genes.MethodsIn a methylation screening approach, we identified ECRG4 as a differentially methylated gene. We analyzed different cancer cells for ECRG4 promoter methylation by COBRA and bisulfite sequencing. Gene expression analysis was carried out by semi-quantitative RT-PCR. The ECRG4 coding region was cloned and transfected into colorectal carcinoma cells. Cell growth was assessed by MTT and BrdU assays. ECRG4 localization was analyzed by fluorescence microscopy and Western blotting after transfection of an ECRG4-eGFP fusion gene.ResultsWe found a high frequency of ECRG4 promoter methylation in various cancer cell lines. Remarkably, aberrant methylation of ECRG4 was also found in primary human tumor tissues, including samples from colorectal carcinoma and from malignant gliomas. ECRG4 hypermethylation associated strongly with transcriptional silencing and its expression could be re-activated in vitro by demethylating treatment with 5-aza-2'-deoxycytidine. Overexpression of ECRG4 in colorectal carcinoma cells led to a significant decrease in cell growth. In transfected cells, ECRG4 protein was detectable within the Golgi secretion machinery as well as in the culture medium.ConclusionsECRG4 is silenced via promoter hypermethylation in different types of human cancer cells. Its gene product may act as inhibitor of cell proliferation in colorectal carcinoma cells and may play a role as extracellular signaling molecule.

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 UDPglucose ceramide glucosyltransferase (UGCG). Besides specific UGCG inhibitors, our data demonstrate that IgMmediated UGCG expression was inhibited by the novel and highly effective PI3K␦ 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 PI3K␦ 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 UGCGmediated ceramide/glucosylceramide equilibrium as a downstream molecular switch of BCR signaling, also providing novel targeted treatment options beyond current chemotherapy-based regimens. (Blood. 2012;120(19):3978-3985) IntroductionDespite recent advances in the treatment of chronic lymphocytic leukemia (CLL) by use of modern chemoimmunotherapies, 1,2 the disease remains incurable for most patients with the exception of those who have the option of an allogeneic transplantation. 3 Moreover, most chemotherapeutic regimens require a certain physical fitness of the patient. Because CLL is a disease of the elderly, there is a need for novel therapeutic concepts, which are able to disrupt resistance to cytostatic drugs. Chemoresistance is thought to be partially the result of malignant cell clones that find a niche within the microenvironment. Resistance might be mediated at least by 3 major stimuli: (1) by engagement of the B-cell receptor (BCR), (2) by CD40 ligand (CD40L)-CD40 interaction, and (3) by stimulation via interleukin-4 (IL-4). 4 Those signals lead via downstream pathways to reduced susceptibility of CLL cells toward chemotherapy within the microenvironment. To some extent, these stimuli share common pathways to mediate survival. 5 As a consequence, the balance between proapoptotic and antiapoptotic signals is disrupted toward pro-survival signals. BCR signaling has been identified as the central and determining factor in CLL. Therefore, novel compounds, which target this pathway, have been developed: CAL-101 as PI3K␦ inhibitor and PCI-32765 as inhibitor of Bruton tyrosine kinase (BTK).Recent data from first trials using CAL-101 and PCI-32765 ind...

1766 Introduction: Survival of CLL cells is triggered by the B-cell receptor (BCR). However, little is known about metabolic processes, which are influenced by the BCR and which are essential for survival of malignant cells such as sphingolipid metabolism. Certain sphingolipids are considered as bioeffector signaling molecules since they regulate several pathways involved in cell metabolism and survival (e.g. mitochondria). For instance ceramide, as the central molecule in sphingolipid metabolism, contributes to apoptosis and growth inhibition. In contrast, glucosylceramide, generated out of ceramide, is responsible for proliferative attributes such as resistance to apoptosis and to several chemotherapeutics. We therefore investigated the role of sphingolipid metabolism in survival and apoptosis-resistance of CLL cells. Methods and Results: We performed liquid chromatography electrospray ionization tandem mass spectrometry of 8 CLL samples in order to determine sphingolipid levels. Prior analysis, cells were either incubated with anti-IgM immunobeads for 24h or were left native. IgM stimulation significantly increased survival of primary CLL cells (n=9; p=0.0246) shown by flow cytometry. Our mass spectrometric analysis revealed a significant decrease of apoptosis-inducing ceramide in BCR-stimulated CLL cells compared to native controls (16:0 p<0.0001, 22:0 p=0.0325, 24:0 p<0.0001, 24:1 p=0.0010). Simultaneously, glucosylceramide synthesis was significantly increased after BCR engagement pointing out its pro-survival effect (16:0 p=0.0004, 18:0 p=0.0343, 24:1 p=0.0012, 26:1 p=0.0027). The total amount of ceramide and glucosylceramide did not change after IgM stimulation. Most importantly, the ratio between pro-apoptotic ceramide and pro-survival glucosylceramide became almost completely reverted towards glucosylceramide after IgM stimulation. Via PCR, we could identify the enzyme UDP-glucose ceramide glucosyltransferase (UGCG) to catalyze the synthesis of glucosylceramide out of ceramide after BCR engagement (p=0.0126). In order to investigate the functional impact of this observation, we tested whether inhibition of UGCG (UGCGi) in combination with a ceramide-inducing drug might lead to increased apoptosis during IgM stimulation. Thereby, we identified ABT-737 as agent that induces apoptosis through up-regulation of ceramide. As UGCG enzyme inhibitor, we used N-(n-Butyl)deoxygalactonojirimycin (OGB-1) and N-(n-Nonyl)deoxygalactonojirimycin (OGB-2). While IgM stimulation protected CLL cells partly from ABT-737-induced apoptosis as determined by AnnexinV-7AAD and JC-1 staining (mitochondrial outer membrane permeabilization) and subsequent flow cytometry, UGCGi reverted this effect leading to a significantly higher amount of apoptotic cells (n=9; p=0.0021). In order to prove that ABT-737-induced apoptosis influenced the ratio of ceramide:glucosylceramide in primary CLL cells, we performed additional mass spectrometric analyses. Most importantly, we could show that UGCGi reverted the ratio between ceramide:glucosylceramide towards ceramide after IgM stimulation. Protection from ABT-737 by IgM stimulation was also measurable by glucosylceramide-dominated ratio. Finally, inhibition of UGCG during IgM stimulation and ABT-737 treatment resulted in higher apoptosis accompanied by ceramide-dominated ratio. Conclusion: Here we identified how BCR engagement controls lipid metabolism and thereby survival and apoptosis-resistance of primary CLL cells. Our findings suggest that ceramide and glucosylceramide may be mediators of survival of CLL cells upon BCR stimulation. The ratio between ceramide and glucosylceramide seems to be crucial to induce resistance to apoptosis. This study provides potential targets for treatment of CLL beyond current concepts. C.M.W. and L.P.F contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

3905 Background: Apoptosis resistance of chronic lymphocytic leukemia (CLL) cells is mediated by several pro-survival stimuli. In particular, engagement of the B-cell receptor (BCR), CD40-CD40 ligand (CD40L) interaction or stimulation by interleukin-(IL)-4 were identified as major factors to regulate chemoresistance. Sphingolipids are known to be involved in several metabolic pathways involved in chemoresitance. Therefore, we focused on ceramide as pro-apoptotic molecule and its counterpart glucosylceramide, which rather contributes to proliferation and survival. Methods and Results: Applying liquid chromatography electrospray ionization tandem mass spectrometry, we revealed a significant decrease of pro-apoptotic ceramide in BCR/IL-4/CD40L-stimulated primary CLL cells compared to untreated controls (p=0.0258, p=0.0478, p=0.0114). Anti-apoptotic glucosylceramide levels were significantly increased after BCR cross-linking (p=0.0435) while other stimuli caused no relevant change in glucosylceramide expression. We identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide via the enzyme UDP-glucose ceramide glucosyltransferase (UGCG) (p=0.0001). Besides specific UGCG inhibitors, we could show for the first time that IgM-mediated UGCG expression was significantly inhibited by the novel and highly effective PI3Kδ and BTK inhibitors CAL-101 and PCI-32765, which were able to revert IgM-induced apoptosis resistance of CLL cells. Recently published data revealed sphingolipids to be essential for mediation of apoptosis via mitochondria. Therefore, we chose ABT-737 – a well-known and also mitochondria-targeting drug – as candidate partner for PI3Kδ and BTK inhibition. When combining each tyrosine kinase inhibitor with ABT-737, a synergistic apoptotic effect could be documented, even under protection by BCR stimulation. Conclusion: In summary, we could demonstrate that sphingolipids are critically involved in CLL pathogenesis. UGCG could be identified as drugable target by the novel kinase inhibitors CAL-101 and PCI-32765 resulting in even synergistic apoptosis following additional application of ABT-737. Sphingolipids seem to offer further targets providing novel treatment options in CLL. C.M.W. and L.P.F. contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.