Chronic Lymphocytic Leukemia Cell Lines Research Articles

Idelalisib for the treatment of indolent non-Hodgkin's lymphoma

Idelalisib is a novel, highly selective, small-molecule, tyrosine kinase inhibitor with potent activity against phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (PI3Kδ). Given the highly selective inhibition of this compound, studies have shown that idelalisib is able to achieve significantly increased apoptotic activity in B-cell acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia (CLL) cell lines, without significant increase in apoptosis among normal T and natural killer cells. Recent studies have suggested potential clinical benefit with idelalisib as either a single agent or in combination with established chemotherapeutic compounds including bendamustine and rituximab in relapsed/refractory CLL. This review will focus on the preclinical pharmacology, pharmacokinetics and clinical utility of idelalisib in the treatment of various indolent forms of non-Hodgkin's lymphoma.

Profiling the Lipid Raft Proteome from Human MEC1 Chronic Lymphocytic Leukemia Cells

Lipid rafts are specialized micro-domains located in the outer plasma membrane of cells and play important roles in various cellular functions, including cell signalling, secretory and endocytic pathways. Cell surface profiling, in particular the lipid raft proteome, has attracted interest in oncology due to the potential use of raft proteins as novel targets for diagnostics and therapeutics. Three different methods have been used to identify the lipid raft proteome from the human chronic lymphocytic leukemia (CLL) cell line MEC1. Firstly, lipid raft proteins were enriched and identified using sucrose gradient ultracentrifugation and 2D liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). To confirm protein localization to the lipid raft, proteomes were compared before and after cholesterol depletion by methyl-β-cyclodextrin (MβCD) using isobaric tags for relative and absolute quantitation (iTRAQ)-labeling coupled to 2D LC-MS/MS. Lipid raft proteins were also identified by immuno-precipitation of crosslinked CD20, a tetraspanin protein that translocates to lipid raft following treatment with the therapeutic antibody, rituximab. In total, 643 proteins were found in lipid rafts of CLL cells (580 following sucrose gradient ultracentrifugation, 181 depleted by MβCD and 199 isolated by immunoprecipitation) and 64 proteins were identified by all 3 methods). These data represents the first comprehensive profile of the lipid raft proteome in CLL cells and includes 30 proteins with no previous known association to the lipid raft. These proteins may represent novel diagnostic and therapeutic targets for CLL.

Targeting chronic lymphocytic leukemia using CIGB-300, a clinical-stage CK2-specific cell-permeable peptide inhibitor

Chronic lymphocytic leukemia (CLL) remains an incurable malignancy, urging for the identification of new molecular targets for therapeutic intervention. CLL cells rely on overexpression and hyperactivation of the ubiquitous serine/threonine protein kinase CK2 for their viability in vitro. CIGB-300 is a cell-permeable selective CK2 inhibitor peptide undergoing clinical trials for several cancers. Here, we show that CIGB-300 promotes activation of the tumor suppressor PTEN and abrogates PI3K-mediated downstream signaling in CLL cells. In accordance, CIGB-300 decreases the viability and proliferation of CLL cell lines, promotes apoptosis of primary leukemia cells and displays antitumor efficacy in a xenograft mouse model of human CLL. Our studies provide pre-clinical support for the testing and possible inclusion of CK2 inhibitors in the clinical arsenal against CLL.

Epigenetic inactivation of miR-9 family microRNAs in chronic lymphocytic leukemia - implications on constitutive activation of NFκB pathway

BackgroundThe miR-9 family microRNAs have been identified as a tumor suppressor miRNA in cancers. We postulated that miR-9-1, miR-9-2 and miR-9-3 might be inactivated by DNA hypermethylation in chronic lymphocytic leukemia (CLL).MethodsMethylation of miR-9-1, miR-9-2 and miR-9-3 was studied in eight normal controls including normal bone marrow, buffy coat, and CD19-sorted peripheral blood B-cells from healthy individuals, seven CLL cell lines, and seventy-eight diagnostic CLL samples by methylation-specific polymerase chain reaction.ResultsThe promoters of miR-9-3 and miR-9-1 were both unmethylated in normal controls, but methylated in five (71.4%) and one of seven CLL cell lines respectively. However, miR-9-2 promoter was methylated in normal controls including CD19 + ve B-cells, hence suggestive of a tissue-specific but not tumor-specific methylation, and thus not further studied. Different MSP statuses of miR-9-3, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite methylation analysis. 5-Aza-2′-deoxycytidine treatment resulted in miR-9-3 promoter demethylation and re-expression of pri-miR-9-3 in I83-E95 and WAC3CD5+ cells, which were homozygously methylated for miR-9-3. Moreover, overexpression of miR-9 led to suppressed cell proliferation and enhanced apoptosis together with downregulation of NFκB1 in I83-E95 cells, supporting a tumor suppressor role of miR-9-3 in CLL. In primary CLL samples, miR-9-3 was detected in 17% and miR-9-1 methylation in none of the patients at diagnosis. Moreover, miR-9-3 methylation was associated with advanced Rai stage (≥ stage 2) (P = 0.04).ConclusionsOf the miR-9 family, miR-9-3 is a tumor suppressor miRNA relatively frequently methylated, and hence silenced in CLL; whereas miR-9-1 methylation is rare in CLL. The role of miR-9-3 methylation in the constitutive activation of NFκB signaling pathway in CLL warrants further study.

ADARB1 Is Involved In a Reduced Maturation Of The miR15a/Mir-16-1 Family In Chronic Lymphocytic Leukemia

MiRNAs are transcribed as long primary precursors (pri-miRNAs), which are cleaved in the nucleus by DROSHA in combination with cofactors. The product of pri-miRNA cleavage is the precursor miRNA (pre-miRNA), which is exported to the cytoplasm and further cleaved by DICER1 into a functioning miRNA:miRNA* complex.The MIR15A/-16-1 tumor suppressor microRNAs (miRNAs) are localized in chromosomal band 13q14.3 that is recurrently deleted in more than 50% of patients with chronic lymphocytic leukemia (CLL). MiR-15a/16 are also less abundant in patients without genomic deletion, suggesting that their down regulation in CLL can also be caused by posttranscriptional mechanisms. Intriguingly, in human CLL cells the primary transcripts (pri-miRNAs) of miR-15a/-16/-15b are elevated and processing intermediates (pre-miRNAs) are reduced in cells from CLL patients (n=38) compared to non-malignant b-cells (n=14). 22/38 (58%) of CLL patients displayed a high pri-miR to pre-miR ratio (higher than 1SD of non-malignant cells) and were classified to harbor a putative processing defect. We recently reported epigenetic aberrations at 13q14 in 58/61 (95%) patients, suggesting 13q14 to be affected in all patients. In line with this concept defective processing of 13q14 miRNAs did not correlate with overall survival, cytogenetic aberrations, immunoglobulin mutation status or time since diagnosis.Using miR-15a/-16-1 pri-miR hairpin sequences coupled to luciferase reporters as DROSHA substrates, intracellular pri-miRNA processing could be shown to be reduced in viable primary CLL cells from patients with a processing defect compared to CLL cells from patients without processing defect and compared to non-malignant B-cells (n=5, n=3, n=4 respectively, p<0.05). This was not seen for processing of pri-miR-155 that was used as negative control. MiRNAs that are putatively affected by the processing defect in addition to the miR-15/-16 family were identified by comparison of the mature miRNome of patients with the processing defect compared to patients without (n=23). In addition to the miR-15/-16 family, in the processing defective patient cells levels of miR-886-3p, miR-7, miR-181d and miR-338-3p were significantly reduced, suggesting that these miRNAs could as well be affected by the processing defect. Intriguingly, the gene targets of these miRNAs are significantly enriched for being downstream members of the immunoglobulin receptor pathway that plays a central role in CLL pathology (KEGG pathways p=2.39 x 10^-9, “miRSystem pathways” p=8 x 10^-4).In order to characterize the molecular mechanism of the processing defect, the expression of cofactors required for DROSHA processing was quantified with Q-RT-PCR in CLL patients with and without a processing defect (n=38). DROSHA, DGCR8, p68, p72 and KHSRP were not significantly down regulated in CLL cells with a processing defect compared to cells with intact processing. For the identification of candidate genes involved in the processing defect, CLL cell lines were screened for processing activity of pri-miRs. Compared to LCL-WEI, Granta, EHEB, JVM-1, JVM-3 and MEC-1 cells, a reduced processing of pri-miR-16-1 was observed in MEC-2 and I83-E95 cells while pri-miR-34a and pri-miR-155 processing was unaffected. ADARB1 gene was significantly up regulated in the processing defective MEC-2 but not MEC-1 cells. ADARB1 belongs to the family of adenosine deaminases that posttranscriptionally introduce inosines into RNA, and its ectopic overexpression reduced processing of pri-miR-16-1 in cell lines. Intriguingly, this inhibition of processing was not reduced upon removal of the enzymatic deaminating activity of ADARB1, but was abrogated by deletion of the RNA-binding domain and the nuclear targeting of the protein as expected.Thus, we show that apart from genomic deletion or transcriptional down regulation, aberrant processing of microRNA leads to specific reduction microRNAs in leukemic cells. This represents a novel oncogenic mechanism in the pathogenesis of CLL. Disclosures:No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia-Derived Exosomes Stimulate Cells From The Microenvironment

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the blood and the primary lymphoid organs of long-lasting, mature, but non-functional B lymphocytes. Although CLL B cells can survive for long time periods in vivo, cells are undergoing apoptosis relatively quickly in vitro. This spontaneous apoptosis and their sensitivity to drugs is strongly reduced in presence of bone marrow mesenchymal stem cells (MSC) and endothelial cells (EC), which provide anti-apoptotic stimuli to CLL cells via direct contact or secretion of soluble factors. We recently reported the first profiling of circulating miRNA obtained from plasma of CLL patients (Moussay et al., PNAS, 2011). Specific miRNAs were found at higher level in the plasma of CLL patients compared to healthy donors. Exosomes, which are small extracellular vesicles of 50-150 nm originating from endosomes, are now known to efficiently transport nucleic acids and transfer mRNA, microRNA and proteins to target cells. Therefore, exosomes constitute a new component of intercellular communication and their role in CLL remains totally unknown.The specific miRNA signature from plasma of CLL patients combined with our observations that primary CLL B cells can transfer vesicles to MSC through 0.4 µm culture inserts in vitro prompted us to investigate whether CLL B cells secrete exosomes that could modify cells of the bone marrow microenvironment to produce tumor growth promoting factors locally in order to favor their own survival.We isolated, purified and characterized exosomes derived from CLL cell lines, primary cells culture supernatants and plasma from CLL patients. Proteins, mRNA and microRNAs contents were evaluated by high-throughput methods (LC-MS, microarrays) revealing in particular the presence of oncogenic molecules. In vitro, purified CLL-exosomes were found to rapidly enter target cells (already after 1h in MSC and endothelial cells) and to transfer proteins and miRNA. Flow cytometry showed that transferred proteins were expressed at cell surface. Luciferase reporter assay confirmed that miRNAs were efficient in targeting cellular mRNA. Exosomes could also be taken up ex vivo and in vivo by mouse bone marrow cells. Functionally, CLL-exosomes activated key signaling pathways (PI3K, AKT, and MAPK) Immunoblotting indicated the rapid phosphorylation of kinases after 5 min of incubation with CLL-exosomes and the subsequent activation of the canonical NF-kB pathway. We also observed that CLL-exosomes modulated gene expression in target cells among which cytokines (BAFF, IL-6, and IL-8), chemokines (CCL2/MCP-1, CCL5/RANTES, and CXCL1), and other factors involved in cell adhesion and migration (ICAM-1 and MMP-1). These factors were also secreted in the supernatants of MSC and EC as detected by antibody arrays. Exosomes were also shown to increase MSC and EC proliferation, to stimulate actin remodeling, cell migration and to enhance EC angiogenic capabilities (tube formation and aortic ring assays).In conclusion, CLL-exosomes contain pro-oncogenic molecules and strongly affect key functions of MSC and EC which are critical component of the bone marrow microenvironment. Activation of these cells by CLL-exosomes led to release of cytokines/chemokines and oncogenic factors that could promote angiogenesis and also favor leukemic cells survival and migration.Our findings may lead to applications in both diagnosis and therapy development. Molecules identified at the surface or inside CLL-exosomes may be further used as cancer biomarkers. Finally, the description of cell-to-cell communication mechanisms will generate opportunities of innovative therapeutic strategies and confirms the crucial role of exosomes in the development of CLL. Disclosures:No relevant conflicts of interest to declare.

Arsenic Trioxide Induces Apoptosis In Chronic Lymphocytic Leukemic Cells Through Down-Regulation Of Survivin Via The p53-Dependent Signaling Pathway

Arsenic trioxide induces cell apoptosis in a variety of tumor cells. However, the molecular mechanism underlying the killing effects of arsenic trioxide on tumor cells is not fully understood. Here, we demonstrate a signal pathway of arsenic trioxide treatment that leads to apoptosis in a B-cell chronic lymphocytic leukemia (WSU-CLL) cell line. Arsenic trioxide treatment significantly inhibited proliferation and induced apoptosis of WSU-CLL cells in a time- and dose-dependent manner. This anti-tumor effect of arsenic trioxide on WSU-CLL cells was mediated through the down-regulation of survivin and up-regulation of p53. Knock-down of intracellular survivin with siRNA potentiated the inhibitory effect of arsenic trioxide on proliferation in WSU-CLL cells. Also, knock-down of p53 by a specific siRNA prevented the down-regulation of survivin by arsenic trioxide and reduced the cytotoxic effect of arsenic trioxide to WSU-CLL cells. These results indicate that arsenic trioxide may be used to as novel therapeutic agent for chronic lymphocytic leukemia, which down-regulates survivin expression via a p53-dependent signaling pathway. Disclosures:No relevant conflicts of interest to declare.

P53 Pathway Alterations Influence Survival and Treatment Response In Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy, with survival rates that range from months to decades. Numerous genetic markers have been identifying that predict for poor survival outcomes. One of the most significant prognostic risk factors is loss of integrity of the p53 pathway. It is well known that loss of the short arm of chromosome 17 (17p-), containing the p53 locus, is a major predictor poor outcome in CLL, however, there is a lack of clarity how specific p53 mutations influence cancer progression. It is our contention that understanding the impact of p53 mutations in CLL progression will facilitate the development of better treatments options. We are using a mouse model of B-CLL (Eµ-TCL1) that also carry a p53 hot-spot mutation p53R172H, corresponding to R175H in humans, to study the impact of p53 mutation on treatment response and survival. Cohorts of Eµ-TCL1 and Eµ-TCL1;p53R172H/+ were treated with fludarabine at 35mg/kg per day for 5 days each month. We have observed significant changes in survival and selective pressure on the p53 locus following fludarabine treatment, leading to a refractory population of clones. Mice in the Eu-TCL1 cohort received a median of 1 treatment cycle (Range; 1-3), while the mice in the Eµ-TCL1;p53R172H/+ cohort received a median of 3 treatment cycles (Range;1-4), suggesting that the mutant cohort are likely to benefit initially with treatment only to succumb to a refractory clone of CLL. Mice in the Eµ-TCL1 cohort had an overall survival of 373 days (range; 341-464 days) in comparison to 322 days (range; 270-382days) in the Eµ-TCL1;p53R172H/+ cohort, indicating a trend toward inferior survival in the Eµ-TCL1;p53R172H/+ cohort. Interestingly, mice in the Eµ-TCL1 cohort had significant splenomegaly (40%) as compared to the Eµ-TCL1;p53R172H/+ cohort (0%). However, we observed that all Eµ-TCL1;p53R172H/+ had extensive extranodal disease with mice having abdominal and/or cervical mass, which is suggestive of Richter's transformation. Together, these data indicate that fludarabine based therapies may have a profound negative transformational impact on CLL progression when used as a treatment strategy in the context of p53 mutations. These results would suggest that less cytotoxic therapies may benefit patients harboring p53 mutations. To this end, we are evaluating the effect of Ibrutinib therapy in the context of p53 mutations. We are currently treating cohorts of Eµ-TCL1 and Eµ-TCL1;p53R172H/+ mice with Ibrutinib 25mg/kg/day. Our preliminary results suggest Ibrutinib's innovative mechanism may lessen some of these selective pressures on the p53 pathway and may indicate Ibrutinib is a superior treatment option compared to fludarabine when p53 mutations are present. In conjunction with these mouse studies, we are also determining the impact of p53 mutation in human patients by examining changes in CLL patient samples and cell lines following fludaribine and Ibrutinib treatment. This dual mouse/human strategy represents an ideal system in which to study genetic anomalies in the p53 pathway and examine the dynamic molecular pressures exerted by both fludarabine and Ibrutinib in CLL. Disclosures:No relevant conflicts of interest to declare.

Preclinical Testing Of a Novel Axl-Kinase Inhibitor In Chronic Lymphocytic Leukemia

The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their ability to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models.In vitro studies: We measured soluble Axl plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in 71 CLL patients and 24 healthy donors. Soluble Axl levels were not significantly higher in CLL patients compared to healthy donors (p=0. 11). However, in CLL patients high sAxl plasma concentrations were differentially expressed with some patients exhibiting normal and others elevated plasma concentrations. The latter showed an association with shorter time to first treatment (p=0.0005) and several poor prognostic markers (e.g. CD38, FISH cytogentics, Binet stage).Freshly isolated PBMC (>90% CD5+CD19+) from CLL patients were incubated in serum free medium for 48h containing concentrations series of 2 different Axl inhibitors: BMS777607, a previously published inhibitor of the MET kinase family, and LDC2636, a novel inhibitor of the TAM receptor tyrosine kinase (RTK) family with high affinity to Axl. Viability of CLL cells was assessed by trypan blue staining and flow cytometry employing annexin V staining. Cellular polarization was analyzed by time-lapse microscopy. We detected cytotoxic effects in a patient-dependent manner that were more prevalent in LDC2636 as compared to BMS777607 treated cells (IC50= 0.21 µM vs. 2.88 µM, p<0.05, n=5). The cellular polarization of the remaining viable cells was significantly reduced in a dose dependent fashion in comparison to vehicle only controls (LDC2636 IC50 = 7.2 µM, p<0.00001; BMS777607: IC50=6.2µM; p=0.0004). Of note, both Axl inhibitors exhibited significantly weaker effects on both, the viability and polarization of normal PBMC over the whole concentration range tested (p<0.05, n=5).In vivo studies To verify our hypothesis that reduced cell polarization results in decreased homing of leukemic cells in vivo we employed a recently developed adoptive transfer model of CLL. In this model NOD/SCID/IL2Rgcnull(NSG) mice were pre-treated with a single intraperitoneal (i.p.) bolus of LDC2636 or BMS777607 (20 mg/kg) and subsequently transplanted with primary CLL cells. Both Axl inhibitors significantly reduced the homing capacity of CLL cells to the BM of NSG mice by 46% and 59%, respectively, compared to vehicle treated controls (LDC2636: p=0.0063, BMS777607 p=0.0007; n=4).To evaluate if LDC2636 also exhibits effects in a disease-relevant CLL model, we applied a MEC-1 xenograft model which causes a lethal leukemia in NSG mice. We pretreated the mice with 40mg/kg Axl inhibitor or vehicle-only control i.p. and subsequently transplanted the CLL cell line MEC-1 intravenously. The following four days the mice were injected again with 40mg/kg LDC2636 or vehicle-only i.p. We evaluated the survival time and found that mice treated with LDC2636 lived significantly longer than vehicle-only controls (24 vs. 18 days median survival, p=0.0016, n=15). Mice that received only LDC2636 and no Mec-1 cells did not show any effect.These data demonstrate that Axl inhibitors exert potent in vitro and in vivo activity against human CLL cells, which is caused at least in part by the suppression of CLL homing to their supportive stromal niches. Disclosures:Schultz-Fademrecht:Lead Discovery Center GmbH: Employment. Unger:Lead Discovery Center GmbH: Employment. Klebl:Lead Discovery Center GmbH: Employment. Choidas:Lead Discovery Center GmbH: Employment.

Low-Dose Lenalidomide Improves CAR-Based Immunotherapy In CLL By Reverting T-Cell Defects In Vivo

Chronic Lymphocytic Leukemia (CLL) is a chronic lymphoid malignancy characterized by immune suppression that is responsible for an increase in infection susceptibility but also concurs to a reduced ability of the immune system to promote an effective response against the leukemic cells. Tumor-immunosuppressive mechanisms are essentially due to the capacity of CLL cells of modifying the surrounding microenvironment including immune effectors likely contributing to disease progression but also to limited effectiveness of current immunotherapy approaches.Lenalidomide is an immunomodulatory agent (IMID) able to induce significant long-lasting responses in CLL patients. The exact mechanism of anti-tumor activity of lenalidomide remains undefined, but it also implies the modulation of tumor microenvironment through down-regulation of critical cytokines and activation of immune effector cells. In addition, lenalidomide was shown to reverse, in vitro, defects in immunological synapse formation between T cells and CLL cells, by interfering with several cytoskeletal molecules.Chimeric antigen receptors (CARs) molecules are emerging as a powerful tool to redirect T-cell specificity against leukemia. CARs are artificial molecules constituted by an extracellular-antigen-binding domain consisting of the variable chains of a monoclonal antibody, linked together as a single chain Fv (scFV), and an intracellular signaling region, usually the zeta chain of the TCR/CD3 complex, that is immediately triggered after antigen recognition. Therefore, CARs take advantage of both the antigen binding non MHC-restricted-properties of monoclonal antibodies and of the typical T-cell mediated effector functions. Given the characteristic T cell defects occurring in vivo in CLL patients, it becomes very intriguing to explore the possibility of a novel CLL therapy combining a CAR-based immunotherapy with low doses of lenalidomide, in order to maximize the effect of the immune attack by reverting in vivo the acquired T cell defects.We studied the in vivo cytotoxic effects on the tumor microenvironment upon lenalidomide treatment utilizing the Rag2-/-γc-/--xenograft model of human CLL based on transplantation of the CLL cell line MEC1 into Rag2-/-γc-/--mice. Utilizing the CAR.CD23 tool as previously published by our group, we also performed experiments where MEC-1-trasplanted-Rag2-/-γc-/- mice were injected with CAR.CD23 T cells from CLL patients together with lenalidomide at low concentrations, uneffective in monotherapy. In these animals, a decrease of the percentage of CD19+leukemic cells was observed in all lymphoid and non-lymphoid tissues after 20 days of treatment, as compared to controls treated with CAR.CD23 T cells or lenalidomide alone. This combination resulted also in improved survival of the treated cohort (NT+lenalidomide vs CAR+lenalidomide: p<0.03, n=7). The effect of the combination with low dose lenalidomide was more effective also when compared to the addition of human recombinant IL-2 as in traditional immunotherapeutic settings.In accordance to the in vivo efficacy, CAR T cells were observed in all leukemic sites suggesting an ability to migrate and home in vivo. In addition, when purified from the bone marrow CD23.CAR+T cells were still able to mount a tumor-specific cytotoxic response in vitro, reaching more than 50% of tumor lysis in both the conditions with lenalidomide and IL-2, compared to 20% of tumor lysis exerted by unmanipulated T cells. Indeed, ex vivo T cells were for the majority effector memory cells and the CD23.CAR was still expressed on their surface.These results conceivably support the use in the CLL therapeutical setting of low doses lenalidomide to improve CARs cytotoxic response and avoid the potential impairment of an effective immune response. Disclosures:No relevant conflicts of interest to declare.

Synthetic Lethality In CLL With DNA Damage Response Defect By Targeting ATR Pathway

▪Ataxia Telangiectasia Mutated (ATM) protein coordinates responses to DNA double strand breaks (DSBs) and the ATM-null status caused by biallelic ATM gene inactivation in chronic lymphocytic leukemia (CLL) results in resistance to p53-dependent apoptosis. Accordingly, alternative strategies to target ATM-null CLL are needed.ATM is a serine/threonine protein kinase that synchronises rapid DNA damage response (DDR) to DNA double strand breaks (DSBs) with activation of cell cycle checkpoints, DNA repair and apoptosis via p53 activation. ATM-null cells are defective in a type of DSB repair that involves homologous recombination and rely on co-operating and compensatory DNA repair pathways for their survival. Therefore, inhibition of DNA repair pathways to which CLL cells with loss of ATM signalling become addicted could provide ‘synthetic lethality' and induce tumour specific killing. Indeed, we have recently shown that inhibition of a single strand break protein PARP induces differential killing of ATM-null CLL tumours. Here we expand the concept of synthetic lethality in ATM-null CLL and address the question of whether ATM-null deficient CLL cells can be targeted by inhibition of the ATR protein that governs responses to post-replicative damage and co-operates with ATM.First, we addressed the status of the ATR pathway in primary CLL cells and consistent with previous findings we observed that initiation of cell cycling is required for both ATR upregulation and activation of ATR target Chk1 in response to replicating stress inducing agent hydroxyurea.We then proceeded with testing viability of the isogenic CLL cell line CII, with and without stable ATM knock down, in the presence or absence of increasing doses of ATR inhibitor AZD6738. We observed a uniform loss of cellular viability in the presence of 1 or 3 μM of inhibitor in ATM-null cells but not in the ATM-wt counterpart. Similar observation was made in primary CLL cells initiated to cycle in the presence of stimulatory oligonucleotide-ODN2006/IL2 support.To confirm the cytotoxic effect of AZD6738 in vivo we used an ATM null primary CLL xenograft model. Representative primary CLL tumour cells with 15% bialleic ATM inactivation, as assessed by percentage of 11q deletion and allelic frequency of ATM mutation 4220T>C, was engrafted in the presence of activated autologous T lymphocytes into 10 NOG mice. Upon detection of engraftment in peripheral blood, animals were treated by oral administration of either AZD6738 (50mg/kg) or vehicle alone over a 2 week period, and tumour load measured by FACS analysis of CD45+ CD19+ human cells in infiltrated spleens. We observed a reduction in tumour cell numbers in AZD6738-treated compared to vehicle-treated spleens and current investigations are underway to determine whether this difference can be attributed to the selective disappearance of CLL population with biallelic ATM loss.We suggest that targeting ATR pathway provides an attractive approach for selective killing of ATM-null CLL cells and that this approach should be considered as a future therapeutic strategy for this CLL subtype. Disclosures:Off Label Use: ATR inhibitor AZD6738 targets ATM-null phenotype inducing synthetic lethality. Jeff:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.

Transcriptional Mistargeting Of NFAT2 In CLL

Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy of mature B cells and constitutes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. The clinical course of CLL can be predicted by serveral prognostic markers like CD38, ZAP70 and cytogenetic abnormalities. While the treatment of CLL has significantly improved during recent years, it remains an essentially incurable disease and the molecular events that lead to its development are still largely elusive. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated by a network of several kinases. Several recent studies have demonstrated that Ca2+/NFAT signaling is involved in the pathogenesis of a wide array of different tumor types including pancreatic adenocarcinoma, breast cancer and Non Hodgkin´s lymphoma. In this study we investigated the significance of the Ca2+/NFAT signaling pathway in B-CLL.For this purpose, we analyzed CLL cell lines (MEC-1, JVM-3) as well as primary blood samples from patients with CLL (n=30). The analyzed patient population exhibited a representative distribution of age, sex, Binet stage, WBC count, cytogenetics and IGVH mutational status. We detected a profound overexpression of NFAT2 mRNA as well as NFAT2 protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited an at least three fold overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors. In one case, NFAT2 expression in CLL cells was 200 times higher than in the corresponding controls. This profound overexpression of NFAT2 in CLL cells could be confirmed on the protein level using Western Blotting and Immunocytochemistry. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in healthy B cells. NFAT2 nuclear translocation could be inhibited using pretreatment with Cyclosporin A demonstrating that this process was still calcineurin-dependent in CLL cells. We could further show that nuclear NFAT2 in CLL cells was able to bind DNA using electrophoretic mobility shift assays (EMSA). To assess the transcriptional activity of NFAT2 in human CLL we determined the expression of the apoptosis regulators OX40L, osteopontin and PD-L2, which we previously identified as NFAT2 target genes in a gene expression analysis with CD19+CD5+ CLL cells from TCL1 transgenic mice with intact NFAT2 and NFAT2 deletion, respectively. Interestingly, qRT-PCR revealed a tremendous reduction of all three target genes in the analyzed CLL samples as compared to control B cells from healthy donors. This is particularly remarkable, since in the TCL1 mouse model we observed a similar reduction of the expression of these genes in CLL cells with NFAT2 ablation.In summary, these results provide strong evidence that the Ca2+/NFAT signaling axis is constitutively activated in CD19+CD5+ CLL cells. Our data suggest that the profound overexpression of NFAT2 in CLL cells leads to its targeting to aberrant genetic loci different from its phsiological target genes resulting in a consecutive knock out phenotype with respect to the expression of the apoptosis regulators OX40, osteopontin and PD-L2 in CLL. Further investigation is therefore warranted to decipher the therapeutic potential of modulating the Ca2+/Calcineurin/NFAT signaling pathway in this disease. Disclosures:No relevant conflicts of interest to declare.

Realgar induces apoptosis in the chronic lymphocytic leukemia cell line MEC-1

The aim of the present study was to investigate the effect of realgar on the viability, proliferation and apoptosis in the human chronic lymphocytic leukemia (CLL) cell line, MEC‑1. Potential mechanisms mediating the effect were also explored in the experiment. Cultured MEC‑1 cells were incubated with various concentrations of realgar for 24, 48 and 72 h. A WST‑8 assay was employed to evaluate the effect on cell viability. Inhibitory effects on cell proliferation were determined using a 5‑bromodeoxyuridine cell proliferation ELISA. The apoptotic effect on MEC‑1 cells was evaluated by annexin V‑fluorescein isothiocyanate/propidium iodide dual staining, followed by flow cytometry. Quantitative polymerase chain reaction was performed to determine the mRNA expression levels of BCL2‑associated X protein (BAX), BCL2‑like 1 (Bcl-xL), v‑myc myelocytomatosis viral oncogene homolog (avian; c‑Myc) and cyclin‑dependent kinase inhibitor 1A (p21). It was found that viability and proliferation were significantly reduced while apoptotic rates increased in MEC‑1 cells following exposure to realgar. Furthermore, mRNA expression of BAX and c‑Myc was upregulated and downregulated, respectively, in realgar‑treated MEC‑1 cells. In conclusion, the results showed that realgar inhibits viability and prolife-ration and induces apoptosis of MEC‑1 cells in a dose‑ and time‑dependent manner. The effect may depend on the mitochondrial apoptosis pathway. The results of the present study may be beneficial in the identification of a new target therapy for CLL.

Characterization of a New Chronic Lymphocytic Leukemia Cell Line for Mechanistic In Vitro and In Vivo Studies Relevant to Disease

Studies of chronic lymphocytic leukemia (CLL) have yielded substantial progress, however a lack of immortalized cell lines representative of the primary disease has hampered a full understanding of disease pathogenesis and development of new treatments. Here we describe a novel CLL cell line (OSU-CLL) generated by EBV transformation, which displays a similar cytogenetic and immunophenotype observed in the patient’s CLL (CD5 positive with trisomy 12 and 19). A companion cell line was also generated from the same patient (OSU-NB). This cell line lacked typical CLL characteristics, and is likely derived from the patient’s normal B cells. In vitro migration assays demonstrated that OSU-CLL exhibits migratory properties similar to primary CLL cells whereas OSU-NB has significantly reduced ability to migrate spontaneously or towards chemokine. Microarray analysis demonstrated distinct gene expression patterns in the two cell lines, including genes on chromosomes 12 and 19, which is consistent with the cytogenetic profile in this cell line. Finally, OSU-CLL was readily transplantable into NOG mice, producing uniform engraftment by three weeks with leukemic cells detectable in the peripheral blood spleen and bone marrow. These studies describe a new CLL cell line that extends currently available models to study gene function in this disease.

Pharmacological targeting of eIF4E in primary CLL lymphocytes

Pharmacological targeting of eIF4E in primary CLL lymphocytes

Determination of 10B in lymphoma human cells after boron carrier treatment: comparison of 10BPA and immuno-nanoparticles

AbstractCurrent cancer treatments lead to insufficient distribution of therapeutic agents in tumor cells due to their lack of selectivity often causing adverse effects in the normal cell uptake of the drug. The challenge is to design therapeutic drugs able to maximize the treatment of malignant cancers while minimizing their adverse effects. In this study, 10B incorporation in the B chronic lymphocytic leukemia cell line MEC-1 was investigated. The cells were treated with l-p-10borophenylalanine (10BPA) and 10boron immuno-nanoparticles (BNPs) loaded with 10BPA with or without the anti-CD20 monoclonal antibody for different contact times. The uptake of 10B by the cells was determined by the inductively coupled plasma mass spectrometry (ICP-MS) after acid mineralization. To obtain accurate and consistent data, the analytical procedure was optimized using factorial experimental design. It was observed that BNP loaded with 10BPA and anti-CD20 represents the best carrier system for 10B in B cells for long time (&gt;10 h) whereas 10BPA seems to be the top drug for short time (&lt; 4 h) procedures.

A Proline/Arginine-Rich End Leucine-Rich Repeat Protein (PRELP) Variant Is Uniquely Expressed in Chronic Lymphocytic Leukemia Cells

Proline/arginine-rich end leucine-rich repeat protein (PRELP) belongs to the small leucine-rich proteoglycan (SLRP) family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1). As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR) in all CLL patients (30/30), as well as in some patients with mantle cell lymphoma (3/5), but not in healthy donor leukocytes (0/20) or tumor samples from other hematological malignancies (0/35). PRELP was also detected in CLL cell-lines (4/4) but not in cell-lines from other hematological tumors (0/9). PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies.

Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells.

e22198 Background: The receptor tyrosine kinase ROR1 has important functions during normal embryogenesis. It is overexpressed and of importance for the survival of different malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). ROR1 inhibition in CLL cells and cell lines with high expression of ROR1 induced specific apoptosis of the cells. There is however little information on the expression and function of ROR1 in melanoma. Here, we investigated ROR1 expression, phosphorylation and the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on seven melanoma cell lines. Methods: ROR1 expression and phosphorylation was analysed in a series of malignant melanoma cell lines using RT-PCR, immunocytochemistry, flow cytometry and western blot. Annexin V/PI staining, MTT assay, PARP and caspase 8 and 9 cleavage as well as MCL-1 protein were used for detection of apoptosis induced by anti-ROR1 mAbs. The cytotoxic effects of anti-ROR1 mAbs were evaluated in the absence or presence of complement (CDC) or immune effector cells (ADCC). Transfection of melanoma cell lines using ROR1 siRNA was used for gene silencing. Results: ROR1 was shown to be overexpressed to a varying degree at protein level. ROR1 was phosphorylated at tyrosine and serine residues. Three out of four anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in the presence of complement or immune effector cells. ESTDAB081 and 094 cell lines were resistant to direct apoptosis of the four anti-ROR1 mAbs alone compared to the other melanoma cell lines, but not to complement dependent cytotoxicity (CDC) and antibody dependent cell mediated cytotoxicity (ADCC). ROR1 siRNA transfection induced down-regulation of the ROR1 expression both at the mRNA and protein levels proceeded by a significant apoptosis of the melanoma cells. Conclusions: The results indicate that ROR1 may play an important role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable targeted for mAb therapy.

Hsp90 Inhibitor SNX-7081 Dysregulates Proteins Involved with DNA Repair and Replication and the Cell Cycle in Human Chronic Lymphocytic Leukemia (CLL) Cells

The proteomic effects of the Hsp90 inhibitor, SNX-7081, have been determined on the p53-mutated B-cell chronic lymphocytic leukemia (CLL) cell line, MEC1. Following SNX-7081 treatment (500 nM, 24 h), 51 proteins changed abundance by more than 2-fold (p < 0.05); 7 proteins increased while 44 proteins decreased. Proteins identified as differentially abundant by LC-MS/MS were validated by Western blotting (DDB1, PCNA, MCM2, Hsp90, Hsp70, GRP78, PDIA6, HLA-DR). RT-PCR showed that SNX-7081 unexpectedly modulates a number of these proteins in MEC1 cells at the mRNA level (PCNA, MCM2, Nup155, Hsp70, GRP78, PDIA6, and HLA-DR). Pathway analysis determined that 3 of the differentially abundant proteins (cyclin D1, c-Myc and pRb) were functionally related. p53 levels did not change upon SNX-7081 treatment of p53 wild-type Raji cells or p53-mutated MEC1 and U266 cells, indicating that SNX-7081 has a p53-independent mechanism. The decreases in DDB1, MCM2, c-Myc, and PCNA and increases of pRb and cyclin D1 were confirmed in MEC1, U266, Raji, and p53 null HL60 cells by Western blotting. These data suggest that SNX-7081 arrests the cell cycle and inhibits DNA replication and r epair and provides evidence for the mechanism of the observed synergy between Hsp90 inhibitors and drugs that induce DNA strand breaks.

Molecular characterization of neoplastic and normal “sister” lymphoblastoid B-cell lines from chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) B-cells resemble self-renewing CD5 + B-cells carrying auto/xeno-antigen-reactive B-cell receptors (BCRs) and multiple innate pattern-recognition receptors, such as Toll-like receptors and scavenger receptors. Integration of signals from BCRs with multiple surface membrane receptors determines whether the cells will be proliferating, anergic or apoptotic. To better understand the role of antigen in leukemogenesis, CLL cell lines producing monoclonal antibodies (mAbs) will facilitate structural analysis of antigens and supply DNA for genetic studies. We present here a comprehensive genotypic and phenotypic characterization of available CLL and normal B-cell-derived lymphoblastoid cell lines (LCLs) from the same individuals (n = 17). Authenticity and verification studies of CLL-patient origin were done by IGHV sequencing, fluorescence in situ hybridization (FISH) and DNA/short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA.