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Despite several potent targeted treatments for chronic lymphocytic leukemia (CLL), the clinical challenge of treating drug-resistant disease is emerging. In this study, we discovered that the dual-specific phosphatases DUSP1 and DUSP6 are required to negatively regulate Mitogen-activated protein kinases (MAPK) and thus counterbalance excessive MAPK activity to prevent apoptosis in CLL. We show that DUSP1 and DUSP6 are widely expressed in CLL and high expression of DUSP6 in CLL correlates with a poor clinical prognosis, which may reflect high levels of MAPK activity. Importantly, genetic deletion of the inhibitory phosphatase DUSP1 or DUSP6 and blocking DUSP1/6 function using a small molecule are toxic for CLL cells in vitro and in vivo. Analyzing downstream effects using global phospho-proteome approaches, we observed that acute activation of MAPK signaling by DUSP1/6 inhibition induces DNA damage response and thereby apoptotic cell death in CLL cells. This cell death is mediated by CHK kinases and can function independent of p53 and ATM, both effectors of DNA damage response, which are frequently deleted in CLL. Finally, we observed that DUSP1/6 inhibition is particularly effective against treatment-resistant CLL and therefore suggest transient DUSP1/6 inhibition as a promising novel treatment concept to eliminate drug-resistant CLL cells.

Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet clinical need. Though BTK and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL treated with ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital PCR (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32/49 relapsing cases (65%) carried at least 1 hotspot BTK mutation and/or PLCG2 mutation(s); in 6/32, BTK mutations were only detected by ddPCR [variant allele frequency (VAF) 0.1-1.2%]. BTK/PLCG2 mutations were also identified in 6/49 responding patients (12%; 5/6 VAF

The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double–cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.

Acalabrutinib is a highly selective, potent, next-generation, covalent Bruton tyrosine kinase inhibitor with minimal off-target activity. Matching-adjusted indirect comparisons (MAICs) were performed to estimate the safety and efficacy of acalabrutinib compared to other targeted therapies for treatment-naïve patients with chronic lymphocytic leukemia (CLL). Individual patient data for acalabrutinib (ELEVATE-TN trial) were matched to aggregate baseline characteristics for comparators. After matching, acalabrutinib (with or without obinutuzumab) showed improved safety outcomes, except for increased risk of neutropenia (

Chronic lymphocytic leukemia (CLL) is an indolent B cell malignancy invariably infiltrating the bone marrow. Although treatment options for patients with advanced disease have significantly improved in the past years, the disease remains incurable and after emergence of therapy resistant disease patients succumb to infections due to secondary bone marrow failure. The underlying mechanisms impairing normal hematopoiesis in patients with CLL are poorly defined., We would like to express our deepest gratitude to patients who donated blood for this research. Samples were obtained with assistance from the Cambridge Blood and Stem Cell Biobank, funded by the Cambridge Cancer Centre and Cambridge Stem Cell Institute. This work was funded by the Cancer Research UK (CRUK; C49940/A17480). I.R. is a senior CRUK fellow. E.L. is supported by a Sir Henry Dale fellowship from Wellcome/Royal Society (107630/Z/15/Z). Research in E.L.’s laboratory is supported by Wellcome, BBSRC, EHA and Royal Society. Research in I.R. and E.L. laboratories is supported by core support grants by Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute.

Novel targeted therapies have substantially improved the prognosis of patients with B cell malignancies. However, a substantial fraction of patients still relapse, even after initially achieving deep remissions. Many studies have characterized the interactions between tumor cells and their microenvironment as integral to leukemia/ lymphoma homeostasis and for the provision of survival signals, also contributing to drug resistance (referred to as environment-mediated drug resistance (EMDR)). Therapeutic efforts to antagonize microenvironment-emanating survival cues have predominantly focused on perturbation of tumour cell adhesion enabling the physical displacement from protective niches (e.g. BCR-inhibitors). In an effort to address whether direct stromal targeting could more precisely mitigate EMDR, we recently characterised the molecular mechanisms underlying tumor-stroma interactions in B cell malignancies and identified a protein kinase C-β (PKC-β) as an essential kinase, required for activation of NF-κB in mesenchymal stromal cells (Lutzny et al Cancer Cell 2013). The dependency on stroma PKC-β was uniformly found for acute (ALL) and chronic (CLL, MCL) B cell malignancies. Importantly, our data further demonstrate that targeting stroma PKC-β is of key importance for multi-drug resistance of malignant B cells and can be used for therapeutic interventions (Park et al Science Trans Med 2020). Here we demonstrate novel mechanistic insights into stroma-mediated drug resistance in B cell malignancies. We identified that stroma PKC-β drives a transcriptional program in tumor cells, dependent on the activation of TGF-β and BMP-signaling, which ultimately leads to the stabilisation of BCL-XL. Our data show that BCL-XL expression in tumor cells is associated with SMAD1-induction by cytotoxic therapies, which simultaneously suppress SMAD4 expression. Importantly, SMAD1 expression was strictly dependent on stromal PKC-β activity. Antagonizing stroma signals with TGF-β inhibitors inhibits SMAD1 induction, abrogates the up-regulation of BCL-XL and overcomes stroma-dependent resistance to Venetoclax and conventional chemotherapy. The TGF-β pathway operates in parallel to the activation of the transcription factor EB (TFEB) as a down-stream target of PKC-β. Interference with these signaling pathways impairs plasma membrane integrity of stromal cells by down-regulation of numerous adhesion and signaling molecules, such as ADAM17, required for the reciprocal stabilization of BCL-XL in tumor cells. The significance of microenvironment PKC-β for drug resistance was demonstrated in vivo, using C57B/6 mice, diseased with EμTCL-1 driven B cell tumors and treated with Venetoclax in combination with or without PKC-β inhibitors. Combined treatment significantly prolonged survival, based on PKC-β mediated impairment of EMDR. Similarly, concurrent treatment of PKC-β inhibitors with chemotherapy also improved survival in an ALL-PDx model Our data demonstrate that mitigating EMDR with small molecule inhibitors of PKC-β or TGF-β signalling enhance the effectiveness of both targeted and non-targeted chemotherapies and moreover, has the ability to overcome Venetoclax resistance in B cell malignancies. Clinical trials with repurposed drugs inhibiting the here described pathways mediating EMDR are in planning. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Midostaurin as inhibitor of stroma PKC-β

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.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

Chronic lymphocytic leukemia (CLL) has a high prevalence in western countries and remains incurable to date. Here, we provide evidence that the multikinase inhibitor sorafenib induces apoptosis in primary CLL cells. This strong pro-apoptotic effect is not restricted to any subgroup of patients, based on Binet stage and the expression of ZAP70 or CD38. Mechanistically, sorafenib-induced cell death is preceded by a rapid downregulation of Mcl-1 through the inhibition of protein translation. Subsequently, the cell intrinsic apoptotic pathway is activated, indicated by destabilization of the mitochondrial membrane potential and activation of caspase-3 and -9. In contrast to sorafenib, the monoclonal vascular epidermal growth factor (VEGF)-antibody bevacizumab failed to induce apoptosis in CLL cells, suggesting that sorafenib induces cell death irrespectively of VEGF signalling. Notably, although sorafenib inhibits phosphorylation of the Scr-kinase Lck, knock-down of Lck did not induce apoptosis in CLL cells. Of note, the pro-apoptotic effect of sorafenib is not restricted to cell-cycle arrested cells, but is also maintained in proliferating CLL cells. In addition, we provide evidence that sorafenib can overcome drug resistance in CLL cells protected by microenvironmental signals from stromal cells. Conclusively, sorafenib is highly active in CLL and may compose a new therapeutic option for patients who relapse after immunochemotherapy.

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.

Dear Editor, A 44-year-old male presented with lower back pain radiating to both legs underwent magnetic resonance imaging (MRI) and was found to have a pathologic fracture of the L4 vertebra and an osteolytic lesion of the left sacrum with adjacent soft tissue infiltration. Apart from radiating back pain, the physical examination was unremarkable, the patient denied B-symptoms, and laboratory values including blood counts were within normal limits. CT-guided biopsy of the sacral lesion showed infiltrations of small blue round cells (Fig. 1a) with positive immunohistochemistry staining for CD99 (Fig. 1b), CD34, CD10, CD79a, CD56, nuclear PAX5 (Fig. 1c), and TdT (Fig. 1d); based on which, after initial suspicion of metastatic disease or Ewing sarcoma, B cell acute lymphoblastic leukemia was diagnosed. Karyotype was normal and molecular biology analysis did not detect any common ALL-associated aberration. Importantly, upon routine bonemarrow (BM) biopsy of the iliac crest, there was no evidence of malignancy, confirming localized disease. After spondylodesis between L3 and L5 for stabilization of the spine, staging was completed by positron emission tomography (PET)-MRI hybrid imaging, which we chose to screen for possible multifocal BM involvement, as seen in previous cases [1, 2]. T1(Fig. 1e) and T2-weighted short tau inversion recovery (STIR) MRI (Fig. 1f) revealed altered BM signal in the left proximal femur, acetabulum, and the left sacrum—with adjacent soft tissue infiltration, tumorous infiltration of the compressed L4 vertebra—with extra-osseous formations extending to the retroperitoneum and the aortic bifurcation-, as well as focal lesions in the right ischium and the transverse process of the T2 vertebra. [F] Fluorodeoxyglucose (FDG)–PET showed intense FDG uptake of the described lesions (Fig. 1g), with PET-MRI fusion images giving the best illustration of extent and localization of disease (Fig. 1h). Based on the immunohistochemistry findings, the mass lesions and a BM infiltration of

In the present work we investigated the role and biological significance of mitogen activated protein kinases (MAPK) in B-cell chronic lymphocytic leukaemia (B-CLL). The MAPK p38 was constitutively activated in B-CLL, but not in normal peripheral B cells. In addition, we demonstrated that the upstream kinases of p38, MKK3/6 were also constitutively activated in B-CLL cells. Furthermore, we determined by EMSA that the p38 MAP kinase pathway was not linked to the constitutive high expression of NF-kappaB, a critical survival factor of B-CLL cells. Recently, it has been shown that serum levels of angiogenic factors like VEGF, bFGF and MMP-9 are elevated in the serum of CLL patients and correlate with an unfavorable prognosis. We showed that the constitutive expression of MMP-9 was dependent on p38-activity and inhibition of p38 strongly downregulated MMP-9 expression. Coculture of B-CLL cells and stromal cells can protect spontaneous apoptosis of leukemic B cells. To determine the role of permanently activated p38 and MMP-9 expression, we cocultured B-CLL cells with bone marrow stromal cells. Survival of B-CLL cells on stroma was severely impaired when p38 was inhibited. Furthermore, blockade of MMP-9 activity also antagonized the antiapoptotic effect of stromal cells.

A new class of cell cycle inhibitors is currently entering clinical trials. These drugs exert their activity by inhibition of cyclin-dependent kinases (cdk) and induce cell cycle arrest and apoptosis in cancer cells. Roscovitine, a cdk2-inhibitor that is in preclinical evaluation, induced apoptosis in B-CLL cells at doses that were not cytotoxic for normal human B cells. At 20 microM, Roscovitine induced apoptosis in 21 of 28 B-CLL samples and was equally effective in zap-70-positive or -negative samples. Caspase-3 was cleaved in B-CLL cells exposed to Roscovitine and the pancaspase inhibitor z.VAD.fmk-blocked Roscovitine-induced apoptosis. Expression of the proapoptotic protein Bak was increased and Bax cleavage and conformational change was observed in Roscovitine-treated B-CLL cells. Antiapoptotic proteins Mcl-1 and XIAP were downregulated, but the expression of Bcl-2 remained unchanged. In contrast to previous reports in cancer cell lines, Roscovitine treatment was not accompanied by nuclear accumulation of p53. Cyc202 (R-Roscovitine) is in early clinical trials in cancer patients. Given its powerful effects on zap-70-positive and -negative B-CLL cells, but not on normal lymphocytes, Roscovitine might be an attractive drug to be tested in this incurable disease.

Mantle cell lymphoma (MCL) is a distinctive non-Hodgkin's lymphoma subtype, characterized by overexpression of cyclin D1 as a consequence of the chromosomal translocation t(11;14)(q13;q32). MCL remains an incurable disease, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL, which is often associated with additional cytogenetic alterations, has an even worse prognosis and new treatment options are clearly needed. The present study investigated the effect of a specific proteasome inhibitor, lactacystin, on cell cycle progression and apoptosis in two lymphoma cell lines harbouring the t(11;14)(q13;q32) and additional cytogenetic alterations, including p53 mutation (NCEB) and p16 deletion (Granta 519). Granta cells were more susceptible to inhibition of the proteasome with respect to inhibition of proliferation and apoptosis induction. No changes were observed in the expression levels of the G1 regulatory molecules cyclin D1 and cdk4, but cell cycle arrest and apoptosis induction was accompanied by accumulation of the cdk inhibitor p21 in both cell lines. Increased p53 expression was only observed in Granta cells with wild-type p53. Cleavage of procaspase-3 and -9 was observed but cleavage of procaspase-8 was not involved in apoptosis induction. The proapoptotic effect of lactacystin was reversed by pretreatment with the pancaspase inhibitor zVAD.fmk. Lactacystin was also effective in inducing apoptosis in lymphoma cells from MCL patients. We conclude that inhibition of the proteasome might be a promising therapeutic approach for this incurable disease.

Objective Although peripheral blood B-CLL cells are arrested in G0 phase of the cell cycle, a proliferating pool of cells in proliferation centers might be involved in disease progression. We have previously described an in vitro model of this proliferating pool of cells using B-CLL cells stimulated with immunostimulatory oligonucleotides (CpG-ODN) and interleukin-2. Lactacystin is a specific inhibitor of the proteasome and is a potent apoptosis inductor in resting peripheral B-CLL cells. In the present study, we investigated the effect of proteasome inhibition in proliferating B-CLL cells. Methods The effect of proteasome inhibition was analyzed using thymidine incorporation, annexin V assays, and TUNEL staining. Immunoblots were performed to evaluate expression of proteins involved in cell cycle and apoptosis regulation. Results Lactacystin blocked cell cycle progression in activated B-CLL cells and inhibited degradation of p27. Upregulation of cyclin D2 and D3 in activated B-CLL cells was inhibited while the expression of cdk2, cdk4, and cyclin E remained unchanged. Activated B-CLL cells were more susceptible to apoptosis induction as compared to resting B-CLL cells. Apoptosis induction was accompanied by cleavage of Bax, procaspase 8, procaspase 9, and procaspase 3. However, a broad-spectrum caspase inhibitor (z-VAD.fmk) only partially inhibited cell death although DNA degradation was completely inhibited. Conclusion Proteasome inhibition is highly effective in proliferating B-CLL cells and induces apoptosis using a caspase-dependent and -independent pathway.

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.

ZAP-70 is a key signaling molecule in T cells. It couples the antigen-activated T-cell receptor to downstream signaling pathways. Its expression in leukemic B-cells derived from a subgroup of patients with chronic lymphocytic leukemia (CLL) is associated with an aggressive course of the disease. However, its implication for the pathogenesis of aggressive CLL is still unclear. In this study, we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor, recruiting protein kinase C-betaII (PKC-betaII) into lipid raft domains. Subsequently, PKC-betaII is activated and shuttles from the plasma membrane to the mitochondria. We unravel that the antiapoptotic protein Bcl-2 and its antagonistic BH3-protein Bim(EL) are putative substrates for PKC-betaII. PKC-betaII-mediated phosphorylation of Bcl-2 augments its antiapoptotic function by increasing its ability to sequester more pro-apoptotic Bim(EL.) In addition, the phosphorylation of Bim(EL) by PKC-betaII leads to its proteasomal degradation. These changes confer leukemic cells to a more antiapoptotic state with aggressiveness of the disease. Most importantly, these molecular changes can be therapeutically targeted with the small molecule inhibitor Enzastaurin. We provide evidence that this compound is highly active in leukemic cells and augments the cytotoxic effects of standard chemotherapeutic drugs.

Diffuse large B-cell lymphoma (DLBCL) is a common lymphoma entity. Although a significant amount of DLBCL patients can be cured with modern chemotherapeutic regimens, a substantial proportion of patients die because of progressive disease. Therefore, new therapeutic strategies are clearly needed. Inhibitors of mTOR [mammalian target of rapamycin (Rap)] represent a new class of antiproliferative drugs with applications as immunosuppressive and anticancer agents. Extensive safety data exist on the mTOR inhibitor RAD001, which is already approved as an immunosuppressant in organ transplant recipients. Rap and RAD001 inhibited cell cycle progression in DLBCL cells by inducing a G1 arrest without inducing apoptosis. Phosphorylation of the main targets of mTOR, p70 s6 kinase and 4-EBP-1 was reduced in cells cultured in the presence of RAD001. Cell cycle arrest was accompanied by reduced phosphorylation of the retinoblastoma protein (RB) as well as reduced expression of cyclin D3 and A in all cell lines. Although the effect of the chemotherapeutic agent vincristine (vin) was not enhanced by RAD001, rituximab-induced cytotoxicity was augmented in the rituximab-sensitive cell lines. mTOR inhibition is a promising therapeutic strategy in DLBCL by inducing a G1 arrest and augments rituximab-induced cytotoxicity. Therefore, combination of these drugs might be an interesting new therapeutic approach in DLBCL patients.

The clinical course of chronic lymphocytic leukemia is variable. While some patients have indolent disease, others require aggressive treatment within a short time after diagnosis. Differences in the expression of proteins regulating cell cycle and apoptosis may be responsible for the heterogeneous course of the disease. Recently, protein ZAP 70 [zeta-chain (T-cell receptor) associated protein kinase 70 kDa] has been found to be differentially expressed within two biologic subgroups, characterized by the presence or absence of somatic mutations in specific immunoglobulin heavy-chain variable region genes. In the present work, we analyzed highly purified B-CLL cells from 60 patients for ZAP 70 expression and the expression of cyclin E, bcl-2, bax, and mcl-1 as well as the ratios of bcl-2/bax and mcl-1/bax. The results indicate that cyclin E is expressed significantly higher in ZAP 70-positive as in ZAP 70-negative samples. We did not observe significant differences within the expression of Bcl-2 family member proteins. We conclude that higher cyclin E expression in samples of ZAP 70-positive patients may reflect a larger proliferating compartment in vivo compared to ZAP 70-negative patients and that cyclin E may add prognostic information in this context for patients with B-CLL.

Chronic lymphocytic B-cell leukemia (B-CLL) is an incurable disease characterized by the accumulation of monoclonal mature B cells, although disease progression relies upon cycling B-CLL cells in proliferation centers in central lymph organs. Rapamycin and its analogs are immunosuppressant drugs that exert their activity by specific inhibition of the mammalian target of rapamycin (mTOR). mTOR inhibition induces cell cycle arrest not only in normal lymphocytes but also in malignant cells. Therefore, rapamycins have recently entered the field of cancer treatment. In the present review we discuss how progression through the cell cycle is regulated in B-CLL cells and how rapamycin and its analogs can be used as target therapies against proliferating B-CLL cells. We also focus on additional effects of rapamycin, such as targeting the interaction between malignant B cells and the microenvironment.

In B-cell chronic lymphocytic leukemia (B-CLL), malignant cells seem to be arrested in the G0/early G1phase of the cell cycle, and defective apoptosis might be involved in disease progression. However, increasing evidence exists that B-CLL is more than a disease consisting of slowly accumulating resting B cells: a proliferating pool of cells has been described in lymph nodes and bone marrow and might feed the accumulating pool in the blood. Rapamycin has been reported to inhibit cell cycle progression in a variety of cell types, including human B cells, and has shown activity against a broad range of human tumor cell lines. Therefore, we investigated the ability of rapamycin to block cell cycle progression in proliferating B-CLL cells. We have recently demonstrated that stimulation with CpG-oligonucleotides and interleukin-2 provides a valuable model for studying cell cycle regulation in malignant B cells. In our present study, we demonstrated that rapamycin induced cell cycle arrest in proliferating B-CLL cells and inhibited phosphorylation of p70s6 kinase (p70s6k). In contrast to previous reports on nonmalignant B cells, the expression of the cell cycle inhibitor p27 was not changed in rapamycin-treated leukemic cells. Treatment with rapamycin prevented retinoblastoma protein (RB) phosphorylation in B-CLL cells without affecting the expression of cyclin D2, but cyclin D3 was no longer detectable in rapamycin-treated B-CLL cells. In addition, rapamycin treatment inhibited cyclin-dependent kinase 2 activity by preventing up-regulation of cyclin E and cyclin A. Interestingly, survivin, which is expressed in the proliferation centers of B-CLL patients in vivo, is not up-regulated in rapamycin-treated cells. Therefore, rapamycin interferes with the expression of many critical molecules for cell cycle regulation in cycling B-CLL cells. We conclude from our study that rapamycin might be an attractive substance for therapy for B-CLL patients by inducing a G1 arrest in proliferating tumor cells.

In the present study we analyzed the role of phophatidylinositol-3 kinase (PI-3K) in B chronic lymphocytic leukemia (B-CLL) cells. PI-3K is activated by many stimuli and is linked to several different signaling pathways. We demonstrated that inhibition of PI-3K by a specific inhibitor, LY294002, induced apoptosis in B-CLL cells in vitro. This effect was specific for the inhibition of PI-3K because inhibition of other signaling pathways such as extracellular signaling-regulated kinase (ERK), p38, or p70S6 kinase did not affect spontaneous apoptosis. Furthermore, PI-3K was constitutively activated in freshly isolated B-CLL cells. Corresponding to enhanced apoptosis, LY294002 down-regulated expression of the antiapoptotic proteins X-linked inhibitor of apoptosis protein (XIAP) and Mcl-1. Next, we investigated which factors downstream of PI-3K were activated in B-CLL cells. We demonstrated that protein kinase B/Akt is expressed in all tested CLL samples but no activation of Akt was detected. In contrast, we observed a constitutive activation of protein kinase Cdelta (PKCdelta) in freshly isolated B-CLL cells. PKCdelta is linked to PI-3K and is phosphorylated at Thr505 in response to PI-3K activation. We further demonstrated that tyrosine phosphorylation and activity of PKCdelta were dependent on PI-3K activity in B-CLL cells. Inhibition of PKCdelta by the specific inhibitor Rottlerin strikingly enhanced apoptosis. In contrast, peripheral blood B cells of healthy donors were resistant to inhibition of PI-3K or PKCdelta. We conclude that activated PI-3K might be important in the pathogenesis of B-CLL, and survival signals might be mediated via PKCdelta. Therefore, inhibition of PI-3K or PKCdelta may be an innovative approach to treat B-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 314 Defects in the apoptosis program are a hallmark of chronic lymphocytic leukemia (CLL), characterized by high expression levels of bcl2 and Mcl1. Notably, this de-regulation of anti-apoptotic proteins is not sufficient to maintain long-term survival of CLL cells, which remain highly dependent on pro-survival factors provided by the leukemia-microenvironment. Bone marrow stromal cells (BMSCs) play an important role for microenvironment mediated survival of CLL cells, based on the provision of soluble and membrane-bound factors. The stroma-CLL interactions not only protect CLL cells from spontaneous, but also from drug-induced apoptosis, clinically recognized as minimal-residual disease. Therefore, understanding the molecular mechanisms of CLL-stroma interactions may offer new therapeutic options and help in eradicating CLL cells from the bone-marrow niche. Here we describe that monoclonal B-cells from CLL patients impose morphological and genetic changes in stromal cells, which become reminiscent of cancer-associated fibroblast (CAF). Comparative gene expression profiles indicate that contact with primary CLL cells induce the expression of pro-inflammatory genes in stromal cells. Further characterization of the underlying signaling pathways activated in stromal cells revealed that CLL cells induce the expression of protein-kinase C-β in BMSCs. Blocking the up-regulation of PKC-β by siRNA abrogated the pro-survival effects of stromal cells on CLL cells. Furthermore, following induction of PKC-β, BMSCs activate NF-kappaB through a Bcl10-independent, but NEMO/IKKgamma-dependent pathway. Gene expression profiling of NEMO-proficient and deficient BMSCs indicated that NF-kappaB regulates the expression of pro-inflammatory cytokines and adhesion molecules by stromal cells, required to promote survival of CLL. Interference with the NF-kappaB activation in BMSCs abrogated the pro-survival effects of stromal cells on CLL, similar to PKC-β deficient stromal cells. To demonstrate that this pathway is also important in vivo, Tcl1-CLL was transplanted into syngeneic PKC-β knock-out and wild-type mice. Notably, all PKC-β wild-type mice died of a CLL-like disease, whereas PKC-β kock-out animals were entirely resistant to CLL transplants. Immunofluorescence staining of PKC-β in bone marrow trephine biopsies indicated that this pathway is also activated in mesenchymal stromal cells of CLL patients. Importantly, our data provide further evidence that the PKC-β – NF-kappaB pathway is also activated in stomal cells by monoclonal B-cells from ALL and mantle-cell lymphoma (MCL) patients. Conclusively, we describe a novel survival signaling pathway activated by monoclonal B-cells in BMSCs. Interference with the PKC-β-NF-kappaB pathway activated in the leukemia/lymphoma microenvironment may offer new therapeutic options to fully eradicate malignant B-cells from bone-marrow niches. Disclosures: No relevant conflicts of interest to declare.

Abstract 2735 Poster Board II-711 Patients with hematologic malignancies have a high risk of developing invasive fungal infections. The higher risk is attributed to host defense impairment due to intensive cytotoxic chemotherapies, hematopoetic stem cell transplantation, and immunosuppressive agents. As early treatment initiation in patients with invasive fungal infections has a profound impact on mortality rates, different antifungal treatment strategies like prophylaxis, empirical, pre-emptive and targeted treatment after proven fungal infection have been developed. Azoles are the most broadly used antifungal drugs inhibiting CYP51. They play a pivotal role in the treatment and prophylaxis of systemic and dermal mycoses. Azoles inhibit the sterol 14a-demethylase activity and block sterol biosynthesis, which is lethal in unicellular organisms. However, in mammalian cells, it has been shown that they lower endogenous cholesterol production. As lipid rafts consist of sphingolipids and cholesterol and lipid rafts have been demonstrated to be crucial for rituximab induced cell death, we asked whether rituximab exhibits its full anti-lymphoma effect in the presence of azoles. We now demonstrate that antifungal treatment with itraconazole impairs rituximab's anti-lymphoma effect both in vitro and in vivo. Using a mouse xenograft model, no tumor growth was observed in mice treated with rituximab. In contrast, in the presence of itraconazole there was tumor growth, indicating that the concentration was sufficient to antagonize rituximab′s anti-lymphoma effect. On the molecular level, CD20 raft recruitment was inhibited in the presence of itraconazole. Furthermore, the translocation of CD20 into lipid rafts has been shown to be crucial for calcium influx and apoptosis. Therefore, we tested the rituximab induced calcium flux in the absence and presence of itraconazole. Indeed, calcium flux was almost completely abolished in the presence of itraconazole. The fact that antibody treatment failure in the presence of itraconazole only affects targeted therapy against raft associated molecules i.e. CD20 and CD52 but not the non raft associated molecule CD33 further supports our proposed mechanism of action. Together, our data suggest that concomitant treatment of itraconazole and rituximab attenuates rituximab's anti-lymphoma effect. As chemotherapy by itself has high response rates in lymphoma, the loss of rituximab mediated cell death in the presence of itraconacole may be missed in our daily routine. 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.

Purpose: We have previously shown that PKC delta is constitutively activated in B-CLL cells. In the present study, we have analyzed the mechanism of apotosis induction as well as the effets of PKC delta in the presence of survival signal and chemotherapeutic agents. Methods: The mitochondrial membrane potential, presence of active caspase 3, conformational status of Bax and apoptosis assays (Annexin V stain, Tunel assay) were performed using flow cytometric analysis. Expression of BCL-2 family proteins and XIAP as well as processing of caspase 8 and 9 was revealed in western blot experiments. Results: Rottlerin at 5μM (which is not toxic for normal lymphocytes) induced apoptosis in a large amount of B-CLL samples. Rottlerin was equally effective in zap70 positive- and negative samples. Rottlerin induced apoptosis was in part caspase dependent and involved processing of caspase 3, 8 and 9. Expression of antiapoptotic proteins MCL-1 and XIAP was reduced in Rottlerin treated cells and BAX expression increased and Bax conformation changed to its proapoptotic form.IN contrast, expression of bcl-2 was not changed. B-CL cells are likely to receive survival signals from the microenvironment and might be rescued from cell death induced by chemotherapeutic agents. Rottlerin was very effective in inhibiting survival signals like IL-4 or stromal cell contact. In addition, treatment with Rottlerin enhanced the cytotoxicity of the chemotherapeutic agents Fludarabine, Vncristine and Daunorubicine. Conclusion: Inhibition of PKC delta might be a powerful treatment option for B-CLL given its potential to induce apoptosis, inhibit antiapoptotic survival signals and augment the cytotoxicity of chemotherapeutic agents.