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BACKGROUND Studies show that proallergic TH 2 cells decrease after successful allergen-specific immunotherapy (AIT). It is likely that iatrogenic administration of allergens drives these cells to exhaustion due to chronic T-cell receptor stimulation. This study aimed to investigate the exhaustion of T cells in connection with allergen exposure during AIT in mice and two independent patient cohorts. METHODS OVA-sensitized C57BL/6J mice were challenged and treated with OVA, and the development of exhaustion in local and systemic TH 2 cells was analyzed. In patients, the expression of exhaustion-associated surface markers on TH 2 cells was evaluated using flow cytometry in a cross-sectional grass pollen allergy cohort with and without AIT. The treatment effect was further studied in PBMC collected from a prospective long-term AIT cohort. RESULTS The exhaustion-associated surface markers CTLA-4 and PD-1 were significantly upregulated on TH 2 cells upon OVA aerosol exposure in OVA-allergic compared to non-allergic mice. CTLA-4 and PD-1 decreased after AIT, in particular on the surface of local lung TH 2 cells. Similarly, CTLA-4 and PD-1 expression was enhanced on TH 2 cells from patients with allergic rhinitis with an even stronger effect in those with concomitant asthma. Using an unbiased Louvain clustering analysis, we discovered a late-differentiated TH 2 population expressing both markers that decreased during up-dosing but persisted long term during the maintenance phase. CONCLUSIONS This study shows that allergen exposure promotes CTLA-4 and PD-1 expression on TH 2 cells and that the dynamic change in frequencies of exhausted TH 2 cells exhibits a differential pattern during the up-dosing versus the maintenance phases of AIT.

The severe‐acute‐respiratory‐syndrome‐coronavirus‐2 (SARS‐CoV‐2) is the causative agent of COVID‐19, but host cell factors contributing to COVID‐19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS‐CoV‐2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID‐19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS‐CoV‐2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease‐targeted inhibitors severely impair lung cell infection by the SARS‐CoV‐2 variants of concern alpha, beta, delta, and omicron and also reduce SARS‐CoV‐2 infection of primary human lung cells in a TMPRSS2 protease‐independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.

SARS-CoV-2 has evolved to enter the host via the ACE2 receptor which is part of the kinin-kallikrein pathway. This complex pathway is only poorly understood in context of immune regulation but critical to control infection. This study examines SARS-CoV-2-infection and epithelial mechanisms of the kinin-kallikrein-system at the kinin B

Biosensors such as toll-like receptors (TLR) induce the expression of interferons (IFNs) after viral infection that are critical to the first step in cell-intrinsic host defense mechanisms. Their differential influence on epithelial integrity genes, however, remains elusive. A genome-wide gene expression biosensor chip for gene expression sensing was used to examine the effects of type-I, -II, and -III IFN stimulation on the epithelial expression profiles of primary organotypic 3D air-liquid interface airway cultures. All types of IFNs induced similar interferon-stimulated genes (ISGs): OAS1, OAS2, and IFIT2. However, they differentially induced transcription factors, epithelial modulators, and pro-inflammatory genes. Type-I IFN-induced genes were associated with cell–cell adhesion and tight junctions, while type-III IFNs promoted genes important for transepithelial transport. In contrast, type-II IFN stimulated proliferation-triggering genes associated and enhanced pro-inflammatory mediator secretion. In conclusion, with our microarray system, we provide evidence that the three IFN types exceed their antiviral ISG-response by inducing distinct remodeling processes, thereby likely strengthening the epithelial airway barrier by enhancing cross-cell-integrity (I), transepithelial transport (III) and finally reconstruction through proliferation (II).

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'‐O‐ribose cap needed for viral immune escape. We find that the host cap 2'‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.

Abstract SARS-CoV-2 has evolved to enter the host via the ACE2 receptor which is part of the kinin-kallikrein pathway. This complex pathway is only poorly understood in context of immune regulation but critical to control infection. This study examines SARS-CoV-2-infection and epithelial mechanisms of the kinin-kallikrein-system at the kinin B2 receptor level in SARS-CoV-2-infection that is of direct translational relevance. From acute SARS-CoV-2-positive study participants and -negative controls, transcriptomes of nasal curettages were analyzed. Primary airway epithelial cells (NHBEs) were infected with SARS-CoV-2 and treated with the approved B2R-antagonist icatibant. SARS-CoV-2 RNA RT-qPCR, cytotoxicity assays, plaque assays, and transcriptome analyses were performed. The treatment effect was further studied in a murine airway inflammation model in vivo. Here, we report a broad and strong upregulation of kallikreins and the kinin B2 receptor (B2R) in the nasal mucosa of acutely symptomatic SARS-CoV-2-positive study participants. A B2R-antagonist impeded SARS-CoV-2 replication and spread in NHBEs, as determined in plaque assays on Vero-E6 cells. B2R-antagonism reduced the expression of SARS-CoV-2 entry receptor ACE2, G protein–coupled receptor signaling, and ion transport in vitro and in a murine airway inflammation in vivo model. In summary, this study provides evidence that treatment with B2R-antagonists protects airway epithelial cells from SARS-CoV-2 by inhibiting its replication and spread, through the reduction of ACE2 levels and the interference with several cellular signaling processes. Future clinical studies need to shed light on the airway protection potential of approved B2R-antagonists, like icatibant, in the treatment of early-stage COVID-19. Graphical Abstract Key messages Induction of kinin B2 receptor in the nose of SARS-CoV-2-positive patients. Treatment with B2R-antagonist protects airway epithelial cells from SARS-CoV-2. B2R-antagonist reduces ACE2 levels in vivo and ex vivo. Protection by B2R-antagonist is mediated by inhibiting viral replication and spread.

Studies show that proallergic TOVA-sensitized C57BL/6J mice were challenged and treated with OVA, and the development of exhaustion in local and systemic TThe exhaustion-associated surface markers CTLA-4 and PD-1 were significantly upregulated on TThis study shows that allergen exposure promotes CTLA-4 and PD-1 expression on T

Background While several systemic immunomodulatory effects of allergen-specific immunotherapy (AIT) have been discovered, local anti-inflammatory mechanisms in the respiratory tract are largely unknown. We sought to elucidate local and epithelial mechanisms underlying allergen-specific immunotherapy in a genome-wide approach. Methods We induced sputum in hay fever patients and healthy controls during the pollen peak season and stratified patients by effective allergen immunotherapy or as untreated. Sputum was directly processed after induction and subjected to whole transcriptome RNA microarray analysis. Nasal secretions were analyzed for Secretoglobin1A1 (SCGB1A1) and IL-24 protein levels in an additional validation cohort at three defined time points during the 3-year course of AIT. Subsequently, RNA was extracted and subjected to an array-based whole transcriptome analysis. Results Allergen-specific immunotherapy inhibited pro-inflammatory CXCL8, IL24, and CCL26mRNA expression, while SCGB1A1, IL7, CCL5, CCL23, and WNT5BmRNAs were induced independently of the asthma status and allergen season. In our validation cohort, local increase of SCGB1A1 occurred concomitantly with the reduction of local IL-24 in upper airways during the course of AIT. Additionally, SCGB1A1 was identified as a suppressor of epithelial gene expression. Conclusions Allergen-specific immunotherapy induces a yet unknown local gene expression footprint in the lower airways that on one hand appears to be a result of multiple regulatory pathways and on the other hand reveals SCGB1A1 as novel anti-inflammatory mediator of long-term allergen-specific therapeutic intervention in the local environment.

BACKGROUND Several microRNAs (miRs) have been described as potential biomarkers in liquid biopsies and in the context of allergic asthma, while therapeutic effects on the airway expression of miRs remain elusive. In this study, we investigated epigenetic miR-associated mechanisms in the sputum of grass pollen-allergic patients with and without allergen-specific immunotherapy (AIT). METHODS Induced sputum samples of healthy controls (HC), AIT-treated and -untreated grass pollen-allergic rhinitis patients with (AA) and without asthma (AR) were profiled using miR microarray and whole-transcriptome microarray analysis of the same samples. miR targets were predicted in silico and used to identify inverse regulation. Local PGE2 levels were measured using ELISA. RESULTS Two hundred and fifty nine miRs were upregulated in the sputum of AA patients compared with HC, while only one was downregulated. The inverse picture was observed in induced sputum of AIT-treated patients: while 21 miRs were downregulated, only 4 miRs were upregulated in asthmatics upon AIT. Of these 4 miRs, miR-3935 stood out, as its predicted target PTGER3, the prostaglandin EP3 receptor, was downregulated in treated AA patients compared with untreated. The levels of its ligand PGE2 in the sputum supernatants of these samples were increased in allergic patients, especially asthmatics, and downregulated after AIT. Finally, local PGE2 levels correlated with ILC2 frequencies, secreted sputum IL-13 levels, inflammatory cell load, sputum eosinophils and symptom burden. CONCLUSIONS While profiling the sputum of allergic patients for novel miR expression patterns, we uncovered an association between miR-3935 and its predicted target gene, the prostaglandin E3 receptor, which might mediate AIT effects through suppression of the PGE2 -PTGER3 axis.

The crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70-positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70-positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70-positive CLL.

Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL has an even worse prognosis and new treatment options are clearly needed. We analysed the effects of BL22, an immunotoxin composed of the Fv portion of an anti- CD22 antibody fused to a 38-kDa Pseudomonas exotoxin-A fragment on four MCL cell lines as well as on primary cells of four MCL patients. Apoptosis induction by BL22 was much more pronounced in MCL cell lines with low Bcl-2 expression (NCEB-1, JeKo-1 and JVM-2) compared to Granta-519 cells with high Bcl-2 expression. While the expression of the antiapoptotic protein Mcl-1 declined (NCEB-1, Granta-519), Bcl-2 levels remained unchanged in Granta-519 cells. However transfection of BCL2 cDNA into NCEB-1, JeKo-1 and JVM-2 cells significantly reduced BL22-mediated toxicity. Accordingly we examined the effects of Bcl-2 inactivation in Granta-519 cells using siRNA. Indeed, apoptosis induction was strongly enhanced in Granta-519 cells with silenced Bcl-2. Our results were confirmed in freshly isolated MCL-cells from patients with leukaemic MCL. We conclude that Bcl-2 expression is important for mediating resistance against the immunotoxin BL22 in MCL cells.

International audience; Although B-cell chronic lymphocytic leukemia (CLL) is treatable, it remains an incurable disease and most patients inevitably suffer relapse. Many therapeutic options exist for those requiring therapy, including monoclonal antibodies and stem cell transplantation, but remissions tend to last shorter in the course of the disease. Targeting the cell cycle has recently been realized to be an attractive therapeutic approach in solid and hematological malignancies, and the proliferative nature of B-CLL is increasingly accepted. Here, we report data on a phase II pilot trial with the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 5 mg/daily in patients with advanced B-CLL who had progressive disease after at least two lines of treatment. After treatment of seven patients, this trial was stopped because of toxicity concerns, although some degree of activity was observed (one partial remission, three patients with stable disease). Interestingly, cyclin E expression decreased in responding patients. Further strategies of mTOR inhibition by RAD001 in B-CLL should focus on different treatment schedules, adequate anti-infectious prophylaxis, or combinations with cytotoxic drugs.

International audience; Although B chronic lymphocytic leukemia (B-CLL) cells express the alpha chain of the interleukin-2 (IL-2) receptor CD25, little is known about the effect of IL-2 on apoptosis in B-CLL cells. We have shown previously that stimulation of B-CLL cells with a CpG-oligonucleotide induces IL-2 high affinity receptors. In our current work, we analyzed the effect of IL-2 on apoptosis in resting B-CLL cells and in our model of activated B-CLL cells (CD25 high cells). IL-2 had modest antiapoptotic activity in resting B-CLL cells. In contrast, IL-2 was much more potent to prevent apoptosis in activated cells. Prevention of cell death was also associated with the maintenance of the mitochondrial membrane potential. While only limited regulation of apoptosis controlling proteins was observed in resting B-CLL cells, IL-2 had strong effects on MCL-1, Bcl-xl, and survivin expression and inhibited Bax cleavage in CD25 high cells. Interestingly, expression of Bcl-2 was reduced. Addition of IL-2 to activated B-CLL cells caused rapid phosphorylation of Akt, while IL-2 failed to significantly phosphorylate Akt in resting B-CLL cells. Pharmacological inhibition of Akt by LY294002 restored sensitivity of activated B-CLL cells to fludarabine. IL-2 might be an important survival factor in activated B-CLL cells and might contribute to disease progression by upregulation of several critical antiapoptotic proteins.

To assess whether the poor prognosis of ZAP70-positive B-cell chronic lymphocytic leukemia (CLL) is associated with the overexpression of ABC transporter genes that are responsible for pleiotropic drug resistance. The transcript level of ten drug transporters was analyzed using semiquantitative and quantitative RT-PCR in control hematopoietic cells, in 41 CLL patient samples and in 5 lymphoma cell lines. ZAP70 status was determined by immunoblotting. Of all analyzed transporters, MDR1, MDR2, MRP1, MRP4, MRP5, and MRP7 were expressed at a significantly higher level in B lymphocytes when compared with other hematopoietic cells in peripheral blood. A subgroup of 41 CLL patient samples showed similar or higher expression of these genes than control B cells, and CLL cells exhibited high expression when compared with multiple lymphoma cell lines. No significant correlation between ZAP70 expression and ABC transporter expression was observed. The ZAP70 status is independent of the multidrug resistance phenotype in CLL.

The recent clinical success with small molecule inhibitors targeting the B cell receptor (BCR) pathway provides unambiguous evidence that signals engaging the BCR play a key role in the pathogenesis of CLL. The binding of CLL cells to a proposed auto-antigen has encouraged many researchers to identify these antigens. This has more recently been challenged by the identification of an internal epitope within the BCR leading to a cell autonomous, antigen-independent activation of the BCR. Overall, the response of CLL cells to a continuous activation of the BCR is anergy. Notably, with respect to different subgroup of patients, M-CLL/ZAP70 negative cases appear to be more "locked" in an anergic state than UM/ZAP70 positive cases. Since investigations in non-malignant B cells suggest that innate signaling-pathways integrate into the BCR-signaling cascade to overcome anergy, we investigated the responses to TLR9 ligation in 57 purified CLL patient samples with regard to the expression of ZAP70, which is considered to amplify signals funneled through the BCR: Here we report that ZAP70 positive CLL shows an increased proliferative response (p=0.005) and enhanced survival (p=0.001) upon TLR9 stimulation when compared to ZAP70 negative CLL. Notably, compared to untreated controls, TLR9 ligation induced apoptosis in ZAP70 negative clones, as opposed to ZAP70 positive cells, in which TLR9 agonists inhibit cell death. TLR9-conditioned media from ZAP70 positive cells completely inhibited the pro-apoptotic effect of TLR9 on ZAP70 negative cells, indicating that soluble factors protect cells from TLR9 induced cell death. Analyzing the expression and modulation of BH3 proteins in TLR9 activated CLL cells, we observed a loss of the pro-apoptotic protein BIM in ZAP70 positive cells, strongly correlating with prolonged survival. Notably, increased BIM expression upon TLR9 ligation was observed in ZAP70 negative cells. In non-malignant B cells BIM is degraded upon activation of the BCR, but not due to the ligation of TLR9. Therefore, we hypothesized that BCR-mediated signals are involved in the TLR9 response of ZAP70 positive CLL. This hypothesis is further supported by the finding that TLR9 mediated loss of BIM in ZAP70 positive CLL was inhibited by the highly specific Syk inhibitor P505-15. Accordingly, TLR9 mediated survival of CLL cells was decreased by P505-15. Since the anti-apoptotic effect of TLR9 stimulation is mediated by soluble factors, we analyzed the secretom of TLR9-conditioned media from CLL samples by mass spectrometry. Surprisingly, we found secreted IgM in conditioned media from TLR9 stimulated, ZAP70 positive, but not negative CLL. TLR9 induced secretion of IgM could also be antagonized with P505-15, further supporting our finding that TLR9 ligation impinges on BCR signaling in ZAP70 positive CLL. In addition, TLR9 conditioned media depleted of soluble IgM, failed to protect cells from apoptosis, indicating that IgM is essential to rescue TLR9 stimulated cells from cell death. In summary, our data strongly indicate that -in ZAP70 positive CLL- TLR9 ligation induces the secretion of IgM and down-regulation of BIM, both features of BCR activation. We propose the idea that the soluble IgM binds to the surface BCR through an internal epitope interaction, engaging a positive feed-back loop and further promoting the survival of ZAP70 positive CLL. Thus, ZAP70 may operate as an amplifier for TLR9 mediated activation of the BCR signaling-cascade. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other.

Heterotypic interactions between bone marrow stromal cells (BMSCs) and malignant B cells contribute to apoptosis-resistance of tumour cells, based on the provision of anti-apoptotic factors by stromal cells. For this reason, interference with the microenvironment may offer an alternative approach to chemotherapy to target B cell lymphomas/ leukaemias. However, the success of such treatments critically relies on specific targets expressed in the lymphoma microenvironment. We have previously reported that monoclonal B cells from patients with CLL, MCL und ALL activate and reprogram BMSCs. This activation of stromal cells is an essential prerequisite for microenvironment-mediated survival of malignant B cells and requires the induction and activation of protein-kinase C-β in stromal cells in vitro and in vivo. This is underscored by a complete resistance of PKC-β knockout mice to adoptively transferred B cell lymphomas from TCL1 transgenic mice (Lutzny et al. Cancer Cell. 2013 Jan 14; 23(1):77-92.). Analyses of primary CLL cells co-cultured on human or mouse BMSCs indicate that stromal cells protect malignant B cells from apoptosis primarily by enhancing the expression of the anti-apoptotic proteins Mcl1, XIAP, BclXL and Bcl2A1. Knockdown of Mcl1 expressed in CLL cells further demonstrates that its expression is crucial for stroma-mediated survival even in the presence of BMSCs. Since BMSC-mediated survival and propagation of CLL depends on the kinase activity of PKC-β, we hypothesized that pharmacological inhibition of the activation of stromal cells using small molecule inhibitors against PKC-β may display anti-leukemic effects. Here we report that the PKC-β inhibitor enzastaurin enhances the cytotoxic effects of standard chemotherapeutic drugs and of the BCL2-inhibitor ABT737. Dose-response analyses indicate that enzastaurin potentiates the cytotoxic effects of ABT737 in a synergistic manner, rapidly causing caspase-mediated apoptosis of malignant B cells. This drug-sensitising effect of enzastaurin is mediated by inhibition of PKC-β induced and expressed in activated stromal cells and not related to a direct cytotoxic effect on malignant B cells: PKC-β deficient BMSCs fail to maintain high expression levels of the anti-apoptotic proteins Mcl1, XIAP and Bcl2A1 in CLL cells, which show a significantly enhanced sensitivity to ABT737 compared to CLL cells cultured on PKC-β proficient stromal cells. These data provide evidence that PKC-β inhibitors can therapeutically be used to abolish microenvironment-mediated survival of malignant B cells. In order to assess whether drug combinations of enzastaurin and BCL2 inhibitors demonstrate synergistic anti-lymphoma effects in vivo, we adoptively transferred malignant B cells from diseased TCL1 mice into wild-type recipient mice. Tumour-bearing mice were treated with enzastaurin, ABT199 or a combination of both drugs. Response to treatment, assessed by PET-CT scan, indicates that the combination of enzastaurin and ABT199 is superior to single agent treatment. These data indicate that malignant B cells can be sensitized to cytotoxic drugs by inhibiting the tumour-promoting effects of the lymphoma microenvironment with PKC-β inhibitors. Our preclinical in vitro and in vivo experiments justify testing this drug combination as a new approach to leukaemia/ lymphoma therapy in a phase I/II clinical trial. Disclosures: No relevant conflicts of interest to declare.

Abstract 2354 Poster Board II-331 A defect in the programmed cell death, apoptosis, is implemented in the pathogenesis of CLL. About ten years ago, it became evident that patients with CLL can be divided into those with an indolent course of the leukaemia and those which suffer from a more aggressive disease, typically requiring frequent chemotherapy and ultimately develop a chemotherapy-refractory state. The latter group of patients aberrantly express the T-cell associated protein ZAP-70. The object of this study was to identify the molecular differences underlying the pathogenesis of these two CLL subgroups. To study differences in the apoptotic program we used primary CLL cells derived from untreated ZAP-70 negative and positive patients. Here we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor (BCR) and recruits protein kinase C-beta (PKC-beta) into lipid raft domains only in patients with an aggressive variant of the disease. Subsequently, PKC-beta is activated and shuttles from the plasma membrane into the mitochondria. By using co-immunoprecipitation experiments and PKC-beta specific small molecule inhibitors we unravel that the anti-apoptotic protein Bcl-2 and its antagonistic BH3-protein Bim are putative substrates for PKC-beta. PKC-beta mediated phosphorylation of Bcl-2 augments its anti-apoptotic function by increasing its ability to sequester more pro-apoptotic Bim. In addition, the phosphorylation of Bim by PKC-beta leads to its proteasomal degradation. Therefore, high levels of phospho-Bcl-2 and low levels of Bim are a hallmark of ZAP-70 positive, aggressive CLL. Importantly, posttranscriptional modifications of Bcl-2 seem to outweigh the absolute expression of Bcl-2 with respect to the suppression of apoptosis. We demonstrate that these cells are strongly protected from chemotherapy-induced cytotoxic stress. Our data indicate that the constitutive activation of PKC-beta is directly involved in the apoptotic defect in ZAP-70 positive CLL. We finally show that targeting PKC-beta is an attractive approach to the treatment of CLL patients. Enzastaurin is a PKC-beta specific inhibitor and currently tested in clinical phase I/II trials for cancer patients. Our data demonstrate that this compound is highly active in CLL cells and augments the cytotoxic effects of standard chemotherapeutic drugs. Our results provide evidence that the constitutive activation of PKC-beta is directly implicated in the pathogenesis of aggressive CLL by altering the function of the apoptosis-regulating proteins Bcl-2 and Bim. These changes confer cells to a more anti-apoptotic state with aggressiveness of the disease. Targeting PKC-beta with small-molecule inhibitors like Enzastaurin might offer a new therapeutic strategy to control or even cure CLL. Disclosures: No relevant conflicts of interest to declare.