MEC-2 Cells Research Articles

An Antibody-Dependent Cellular Cytotoxicity Assay for Detecting Ocrelizumab Neutralizing Antibody.

Ocrelizumab (OCREVUS®) is a humanized anti-CD20 monoclonal antibody approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). Here, we discuss the strategic and technical considerations needed to develop a robust antibody-dependent cellular cytotoxicity (ADCC)-based neutralizing antibody (NAb) assay to detect anti-ocrelizumab NAb in patients enrolled in the ocrelizumab registered clinical trials. The NAb detection assay consisted of a two-tier assay that included a screening assay and a confirmation assay. In the screening assay, patient samples were analyzed in the presence of ocrelizumab. Samples that tested positive in the screening assay were subsequently analyzed in the confirmatory assay where another anti-CD20 mAb, obinutuzumab, was replaced by ocrelizumab, to verify NAb specificity. Both assays utilized MEC-2 cells, a chronic B cell leukemia cell line, pre-labeled with calcein AM as the target cells, and natural killer (NK) cells engineered to stably express Fc gamma receptor IIIa_ F158 as effector cells. Both cell lines were prepared to be thaw-and-use cells. The NAb assay measures fluorescence from the calcein AM released into the assay media upon the lysis of target cells by ADCC in the presence of ocrelizumab or obinutuzumab. Our validated NAb assay showed a relative sensitivity of 743ng/mL and can detect 1500ng/mL of a surrogate positive control antibody in the presence of 1500ng/mL ocrelizumab. This ADCC assay is the first reported NAb assay that directly measures target cell lysis by using thaw-and-use target and effector cells simultaneously.

Abstract 5432: Targeted alpha therapy with 212Pb-NNV003 in treatment of NHL

Abstract Introduction: CD37 has shown promise as a new therapeutic target for B-cell malignancies. CD37 is highly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies. Alpha-emitting radionuclides have demonstrated potential for cancer targeted therapies because of efficient energy deposition along the short alpha track (50-100 µm) causing localized and irreparable DNA damage while sparing surrounding healthy tissues. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the alpha-particle-generating radioisotope 212Pb for the treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Approximately 90,000 cases of CLL and NHL are expected annually in the US. Standard of care includes chemotherapy combined with anti-CD20 antibodies (obinutuzumab and rituximab) and Bruton’s tyrosine kinase inhibitor ibrutinib. While these therapies are initially effective, most patients inevitably relapse. Materials and methods: The efficacy and tolerability of a single dose 212Pb-NNV003 treatment were evaluated in disseminated models using CD37-expressing human CLL (MEC-2) and Burkitt’s lymphoma (Daudi) cells. Comparison to therapeutic doses of ibrutinib was investigated in the MEC-2 model. R2G2 or CB17-SCID mice were injected intravenously with 2.5×106 MEC-2 or 10×106 Daudi cells on day 0, followed by intravenous injection of 212Pb-NNV003 (2.5, 5 or 7.5 µCi in Daudi model and 5, 10, 15 or 20 µCi in MEC-2 model) or daily oral administration of ibrutinib (12.5 or 25 mg/kg) starting on day 2 (n=10-12 per group). 212Pb-cetuximab (unspecific isotype control), unlabelled NNV003 and NaCl were used as controls. Results: In vitro testing of the antibody showed that MEC-2 cells were 5 times more resistant to ibrutinib than Daudi cells. Treatment of mice injected intravenously with Daudi cells with 2.5 μCi 212Pb-NNV003 resulted in survival of 100% of the mice 100 days after start of treatment, while 100 days after start of treatment with 100 μg obinutuzumab only 40% of the mice were still alive. Control mice had a median survival of 7-8 weeks. Treatment of mice injected intravenously with MEC-2 cells with 15 μCi 212Pb-NNV003 resulted in survival of 90-100% for more than 25 weeks in two independent experiments. Ibrutinib treated mice with intravenous MEC-2 cells presented a median survival of only 4.3-4.4 weeks, comparable to the unlabelled NNV003 and saline groups (4.1 and 4.4 weeks). Mice receiving non-specific 212Pb-cetuximab presented a median survival of 7-9 weeks. Conclusion: The study shows that a single injection 212Pb-NNV003 is safe and effective for the treatment of CD37 positive CLL and NHL in preclinical models, with promising efficacy in both ibrutinib-resistant and sensitive CLL models. Treatment with 212Pb-NNV003 was superior to both ibrutinib and obinutuzumab. Further clinical testing is warranted. Citation Format: Jostein Dahle, Amal Saidi, Tania Stallons, Helen Heyerdahl, Ada Repetto-Llamazares, Julien Torgue. Targeted alpha therapy with 212Pb-NNV003 in treatment of NHL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5432.

CDK7 Inhibitor THZ1 Inhibits Glycolytic Metabolism By Suppressing HIF1α Activity in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL), the most common type of leukemia in Western adults, is characterized by the progressive accumulation of CD19+ malignant B cells in blood, secondary lymphoid tissues and bone marrow. Currently, CLL remains incurable in spite of the explosive development of novel therapeutics in the past decade. Cyclin-dependent kinases (CDKs) have been suggested as potential therapeutic targets for CLL. Flavopiridol, a pan-CDK inhibitor, has demonstrated safety and efficacy in patients with relapsed CLL, including patients with high-risk cytogenetic features. In this study, we assessed the therapeutic potential of a covalent CDK7 inhibitor, THZ1, in preclinical CLL models. We observed that THZ1 had a significantly lower IC50 in CLL cell lines MEC1 and MEC2 (7.23nM and 7.35nM, respectively), which was 10-fold more potent than flavopiridol and CR8, two other CDK inhibitors that were previously studied in CLL. THZ1 inhibited cell growth and induced apoptosis in MEC1, MEC2 and primary CLL cells in both a dose-dependent and time-dependent manner. The induction of apoptosis by THZ1 in MEC1, MEC2 and primary CLL cells was associated with PARP1 cleavage and down-regulation of anti-apoptotic protein MCL-1. Particularly, THZ1 was able to overcome protection signals provided by the tumor microenvironments and induce apoptosis in primary CLL cells in stromal-coculture conditions. To elucidate the mechanism of action of THZ1, we performed a time course RNA-seq expression analysis in both MEC1 and MEC2 treated with 50nM THZ1. Based on the incremental reduction of transcript levels over a 12-hour period, we identified 728 and 647 THZ1-sensitive transcripts (~5.2% and 4.5% of all active transcripts) in MEC1 and MEC2 cell lines, respectively. Pathway analysis demonstrated that THZ1 down-regulated transcripts are significantly enriched in regulation of cellular metabolism including hypoxia, mTORC1 signaling, and glycolysis pathways. In particular, when analyzing the entire THZ1 down-regulated expression signature for both MEC1 and MEC2 at 8 hours using Gene Set Enrichment Analysis (GSEA), two of the most significantly enriched gene sets contain gene expression signatures from hypoxia induction and HIF1α target genes. Several of the metabolic transcripts sensitive to THZ1 treatment including SLC2A1 , SLC2A3 , HK2 , ENO2 , and PKFKB4 were validated using quantitative RT-PCR. Immunoblot analysis demonstrated that mTORC1 substrates 4E-BP1 and p70S6K were hypophosphorylated in MEC1 and MEC2 when treated with increasing concentrations of THZ1 over time, indicating the inhibition of mTORC1 activity by THZ1. We also observed that the metabolically sensitive AKT Ser473 phosphorylation had essentially been fully repressed by THZ1 treatment in both MEC1 and MEC2. Furthermore, THZ1 was able to mitigate glucose uptake and reduce lactate production in MEC1 and MEC2 cells. THZ1 also prevented the induction of glycolytic transcripts by hypoxia and hypoxia-mimetic CoCl2 (200μM) treatment in MEC1, MEC2 and primary CLL cells. Analysis of CDK7 ChIP-seq data in Jurkat cells revealed that CDK7 directly binds to the promoter and -24kb, -35kb distal enhancers of SLC2A3 that are known to directly interact with HIF1α, suggesting that CDK7 is likely involved in transcriptional regulation of HIF1α target genes via direct enhancer binding. Experiments are currently underway to determine if CDK7 physically interacts with HIF1α under hypoxia. Overall, we observed that THZ1 could effectively disrupt CLL cell viability and overcome the protective signals present in the leukemia microenvironment by inhibiting the transcriptional activity of HIF1α, which is a key regulator of the interaction of CLL cells with the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of the CDK7 inhibitor as novel therapies for CLL. DisclosuresNo relevant conflicts of interest to declare.

High IGF1R Expression Is Associated with Worse Prognosis in CLL and Impacts Response to PI3K-δ Inhibitor Treatment

High IGF1R Expression Is Associated with Worse Prognosis in CLL and Impacts Response to PI3K-δ Inhibitor Treatment

212Pb-NNV003 as a Novel Targeted Alpha Therapy for CD37 Positive B-cell Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL)

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in western countries, accounting for approximately one quarter of all leukemias and Non-Hodgkin lymphoma (NHL) caused an estimated 200,000 cancer deaths worldwide in 2014. More than 90,000 cases of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) are expected in the US each year. Immuno-chemotherapy using anti-CD20 monoclonal antibodies (mAb) in combination with DNA alkylating agents is the front-line therapy of CLL and NHL. Despite promising initial results, relapses after repeated administration of immuno-chemotherapy are frequent and relapsed/refractory patients show poor prognosis. As CD37 is strongly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies, the development of new therapies targeting CD37 expressing cells may prove useful for relapsed or refractory patients as an alternative to CD20 targeting agents. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the in-vivo alpha-particle-generator, 212Pb. When treated with alpha radiation, targeted cancer cells are exposed to high linear energy transfer (LET). LET acts over a short range (50–100 μm), causing double strand breaks in the DNA of targeted cells while sparing adjacent normal tissues. The efficacy of a single escalating 212Pb-NNV003 administration was evaluated on disseminated models of human Burkitt’s lymphoma (Daudi) and CLL (MEC-2). 10 million Daudi cells or 2.5 million MEC-2 cells were intravenously injected in CB17-SCID or R2G2 mice and 212Pb-NNV003 was given two days later. Unspecific, 212Pb-labeled antibody was used as control. Dose range finding and tolerability studies were performed in CB17-SCID and R2G2 tumor-free mice to define the maximum tolerated dose prior to the efficacy studies. 212Pb-NNV003 displays a favorable toxicity profile after a single intravenous injection in tumor-free mice. No acute hematological toxicity was observed, and animals presented only a slight initial reduction in their platelets (PLT) counts which was fully recovered 4-weeks after injection. A single intravenous dose of 10, 15 or 20 μCi of 212Pb-NNV003 led to 70%, 90% and 100% of mice injected with MEC-2 cells being tumor free 20 weeks post cell injection. Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 4.9, 5.4 and 9.3 weeks, respectively. A single intravenous dose of 2.5, 5 and 7.5 μCi 212Pb-NNV003 led to over 80% tumor-free mice injected with Daudi cells 15 weeks post cell injection. Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 7, 7.8 and 7.7 weeks, respectively. The results of preclinical studies suggest that TAT using 212Pb-NNV003 may have positive clinical implication for the treatment of CD37 positive CLL and NHL.

Targeted Alpha Therapy with 212Pb-NNV003 for the Treatment of CD37 Positive B-Cell Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL)

BackgroundEach year more than 90,000 cases of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) are expected in the US. The standard of care is immuno-chemotherapy with alkylating agents in combination with an anti-CD20 monoclonal antibody (mAb). While immuno-chemotherapy regimens are initially effective in inducing responses, most patients inevitably relapse and the same therapies show decreasing efficacy with repeated administration.In recent years, CD37 has been considered as a target for B-cell malignancies. CD37 is strongly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies. Therapies targeting CD37 expressing cells may become a useful alternative to CD20 targeting agents. Alpha-emitting radionuclides have demonstrated good potential for cancer targeted therapies because of efficient energy deposition along the short alpha track (50-100 µm). The absorbed energy cause irreparable DNA double-strand breaks and localized cytotoxicity while sparing surrounding healthy tissues. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the alpha-particle-emitting radioisotope 212Pb.Materials and MethodsDose-dependent efficacy and tolerability of a single-dose 212Pb-NNV003 treatment was evaluated using escalating activity levels in human disseminated models of Burkitt's lymphoma (Daudi) and CLL (MEC-2). 10 million Daudi cells or 2.5 million MEC-2 cells were intravenously injected in CB17-SCID or R2G2 mice and 212Pb-NNV003 was given two days later. Unspecific, 212Pb-labeled antibody was used as control. A study of the tolerability of 212Pb-NNV003 was performed in R2G2 mice without tumor prior to the efficacy studies.Results212Pb-NNV003 displays a favorable toxicity profile in tumor-free mice single intravenous dose injections up to 15 µCi showing 100 % survival 4 weeks post-injection. No acute hematological toxicity was observed and animals who received 5, 10 or 15 μCi doses of 212Pb-NNV003 presented only a slight initial reduction in their platelets (PLT) counts which was fully recovered by 20 days after injection.A single intravenous dose of 10, 15 or 20 µCi of 212Pb-NNV003 lead to 70 %, 90 % and 100 % of mice injected with MEC-2 cells being tumor free 20 weeks post cell injection (Figure 1). Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 4.9, 5.4 and 9.3 weeks, respectively.A single intravenous dose of 2.5, 5 and 7.5 µCi 212Pb-NNV003 lead to over 80% tumor-free mice injected with Daudi cells 15 weeks post cell injection (Figure 2). Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 7, 7.8 and 7.7 weeks, respectively.ConclusionThe results of preclinical studies suggest that TAT using 212Pb-NNV003 is a safe and effective method for the treatment of CD37 positive CLL and NHL. [Display omitted] DisclosuresSaidi:Orano Med: Employment. Maaland:Nordic Nanovector: Employment. Torgue:Orano Med: Employment, Membership on an entity's Board of Directors or advisory committees. Heyerdahl:Nordic Nanovector: Employment, Equity Ownership. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties.

Fludarabine-resistance associates with ceramide metabolism and leukemia stem cell development in chronic lymphocytic leukemia.

Fludarabine (flu) -containing regimens such as flu, cyclophosphamide and rituximab have been established as one of the standard first line therapy in medically-fit chronic lymphocytic leukemia (CLL) patients. Therefore, flu-refractory (primary flu-insensitivity or flu-caused relapse) remains a major problem causing treatment failure for CLL patients. We isolated the peripheral blood mononuclear cells (PBMCs) from CLL patients and treated with flu to find flu-refractory cases, and established flu-resistant clonal cells to study molecular mechanism of flu-resistance. By comparing parental MEC-2 cells, a human CLL cell line, we found that flu-resistant clonal cells were significantly increased lethal dose 50 of flu concentration, and up-regulated expression of P-glycoprotein, a drug-resistant marker, glucosylceramide synthase (GCS), an enzyme that can convert ceramide to glucosylceramide, and CD34, a leukemia stem cell marker. Overexpression of GCS leads to promptly elimination of cellular ceramide levels and accumulation of glucosylceramide, which reduces apoptosis and promotes survival and proliferation of flu-resistant clonal cells. Furthermore, we demonstrated that the accumulation of glucosylceramide can be blocked by PDMP to restore flu-sensitivity in flu-resistant clonal cells. We also found that elevating glucosylceramide levels in flu-resistant clonal cells was associated with up-regulation of GCS and CD34 expression. Importantly, overexpression of GCS or CD34 was also determined in flu-refractory PBMCs. Our results show that flu-resistance is associated with the alteration of ceramide metabolism and the development of leukemia stem cell-like cells. The flu-resistance can be reversed by GCS inhibition as a novel strategy for overcoming drug resistance.

Abstract 848: In vitro and in vivo evaluation of the beta-emitting lutetium-177 labeled anti-CD37 antibody radionuclide conjugate 177Lu-NNV003 in DLBCL, CLL and MCL models

Abstract The aim of this study was to evaluate the cytotoxic and anti-tumor efficacy of the beta-emitting lutetium labelled NNV003 antibody (177Lu-NNV003) in non-Hodgkin lymphoma (NHL) and Chronic Lymphocytic Leukemia (CLL) models. Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) were measured in REC-1 (MCL), MEC-2 (CLL) and DOHH-2 (DLBCL) cells using FcRγIIIa immortalized NK92 cells as effector cells. Antibody-Dependent Cellular Phagocytosis (ADCP) was measured using a commercially available FcRγIIa-H reporter kit. Binding and internalization of 177Lu-NNV003 after up to 18 hours incubation were measured by a stripping buffer method. Cytotoxicity effects were evaluated by incubating cells with increasing concentrations of 177Lu-NNV003 and measuring cell proliferation. Biodistribution of 177Lu-NNV003 was measured in CB17 SCID and NSG mice with DLBCL and CLL xenografts, respectively. Therapeutic and toxic effects of 177Lu-NNV003 were measured in different strains of immunocompromised mice bearing systemic DLBCL (in RAG-2), CLL (in RAG-1) or MCL (in NOD SCID) xenografts, and compared with NaCl and unspecific 177Lu-labelled isotype control groups. NNV003 elicited ADCC lysis in all cell lines and induced ADCP in MEC-2 and DOHH-2. Minimal CDC activation was observed. 177Lu-NNV003 showed activity dependent antiproliferative effect in all cell lines. Maximum tumor uptake in vivo was 45% IA/g in MEC-2 tumors and 15% IA/g in DOHH-2 tumors. Survival was significantly extended in DOHH-2 xenograft-bearing mice treated with NNV003 (2-30 mg/kg) and 177Lu-NNV003 (200-400 MBq/kg) compared to control groups (p<0.0001). In the MEC-2 model, 200 MBq/kg 177Lu-NNV003 extended survival compared to the NNV003 and the 177Lu-isotype treatment (p<0.025). In mice bearing REC-1 xenografts, both 50 and 100 MBq/kg 177Lu-NNV003 improved survival compared to control groups (p<0.02). Transient hematological toxicity was observed in all groups treated with radioactivity, and one mouse treated with 200 MBq/kg in the MEC-2 study died of radiation toxicity. 177Lu-NNV003 showed best anti-tumor effect in the two NHL models, and less effect in the CLL model despite the high tumor uptake, which might be explained by the very aggressive growth of the MEC-2 cells in vivo. In conclusion, in vitro studies have shown that the unlabelled NNV003 antibody have an immunological cytotoxicity effect on tumor cells, mainly through ADCC, and that 177Lu-NNV003 inhibits cell growth. 177Lu-NNV003 has shown significant tumor uptake and therapeutic effect in vivo in all three models tested. The results warrant clinical testing in patients suffering from CD37-expressing B-cell malignancies. Citation Format: Astri F. Maaland, Helen Heyerdahl, Adam O'Shea, Bergthora Eiriksdottir, Jostein Dahle. In vitro and in vivo evaluation of the beta-emitting lutetium-177 labeled anti-CD37 antibody radionuclide conjugate 177Lu-NNV003 in DLBCL, CLL and MCL models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 848.

Abstract 268: PARP3 inhibitors in cancer therapy

Abstract Poly-ADP-ribose-polymerase (PARP1) is an important regulator of DNA damage response (DDR). After induction of certain types of DNA damage, including nicks and double strand breaks (DSB)s, PARP1 is rapidly recruited to altered DNA sites were its catalytic activity produces protein-conjugated long-branched-poly-ADP-ribose (PAR) chains. These protein modifications result in the recruitment and activation of multiple proteins involved in DNA repair. Although there is considerable research describing the characteristics and activity of PARP1/2, there are 15 other PARP family members that are less well characterized. Of these PARP3 has been shown to have a distinct role in DNA repair. This protein has been shown to have a critical role in DSB resolution and to interact with partner proteins know to function in classical nonhomologous end joining (NHEJ) such as DNA-PKcs, Ku70, and Ku80. In order to examine the role of PARP3 vis-à-vis chemotherapy, we utilized the specific PARP3 inhibitor, ME-0328 in combination with activated cyclophosphamide (4HC) in the chronic lymphocytic leukaemia (CLL) cell lines, MEC1 and MEC2. Results from MTT cytotoxicity assays showed that the 50% inhibitory concentration (IC50) (50% of control) of 4HC in MEC-1 cells was 12.9μM. When used in combination with a nontoxic concentration (2μM) of the specific PARP3 inhibitor ME-0328 the IC50 of 4HC was reduced to 3.3μM. In MEC-2 cells the IC50 of 4HC was 10.8μM and was reduced to 2.1μM in the presence of ME-0328 at 2μM. ABT-888, a specific PARP1/2 inhibitor in clinical trials had a minimal effect on 4HC cytotoxicity in these cell lines. 4HC results in interstrand crosslinks (ICLs) which are believed to be repaired by homologous recombination. Intriguingly, PARP3 negatively regulates class switch recombination via activation-induced cytidine deaminase in B-lymphocytes. This may be involved in ME-0328 sensitization of 4HC in B-lymphocytic malignancies. Given these preliminary results we hypothesize that the PARP3 inhibitor, ME-0328 will sensitize B-lymphocytic cancers to chemotherapies. To explore this hypothesis this project will pursue the following objectives: 1. Characterize the selective sensitization of ME-0328 with 4HC and/or bendamustine (an ICL-inducing drug utilized in the treatment of B-lymphocyte malignancies) in B-lymphoma and CLL cell lines plus CLL clinical samples. 2. Determine the mechanisms by which ME-0328 sensitizes CLL cells to 4HC and/or bendamustine (i.e. reduction of PARP1 or 3 activity and/or altered activity of known DNA repair proteins (Rad51, DNA-PK, H2AX) and/or altered activation-induced cytidine deaminase). 3. Determine the in-vivo activity of 4HC and/or bendamustine with or without ME-0328 in a Rag2 /-γcc-/- xenograft model using the MEC1-CLL cell line. The inhibition of PARP3 may increase the sensitivity of tumor cells to DNA damaging chemotherapies. Our results should stimulate development of specific PARP3 inhibitors for clinical use. Citation Format: Bahram Sharif-Askari, David Davidson, Lilian Amrein, Lawrence Panasci. PARP3 inhibitors in cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 268.

Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells

Cholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but doesn’t interfere with CD-20 in chemoimmunotherapy of chronic lymphocytic leukemia (CLL). CLL cell killing by rituximab requires cross linking of CD-20, which induces redistribution of CD-20 to lipid rafts. Lipid rafts are specialized microdomains of the plasma membrane that are enriched in sphingolipid and cholesterol, and play an important role in the initiation of many anticancer drug-induced signaling pathways and toxicological effects. Anticancer drugs are able to suppress cell growth and induce apoptosis of tumor cells through disrupting lipid raft integrity [32,47]. The redis-tribution of CD-20 may result in lipid raft disruption, activate or deactivate raft-associated proteins, such as death receptors and protein kinases in apoptotic pathway which are correlated with efficiency of complement-dependent cytotoxicity and antibody-complement-dependent cell-mediated cytotoxicity. Rituximab and other anti-CD-20 antibodies are currently key drugs in CLL chemoimmuno-therapy. Although statins have antitumor activity, they are clearly unsuitable for enhancement of chemoimmuno-sensitivity of lymphomas and leukemias.We used MEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients, treated them with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations. We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients. Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmunotherapy resistant MEC-2 cells sensitive to chemoimmunotherapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients. Conclusion:Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment. Chronic lymphocytic leukemia (CLL) is the most preva-lent hematologic malignancy affecting Caucasian adults in Western countries. In the United States, about 15,000 new cases are diagnosed every year. CLL is characterized by the accumulation of mature CD5, CD19, and CD23 B-lymphocytes in peripheral blood, bone marrow, lymph nodes and spleen. These results provide a novel strategy which could be applied to CLL treatment.

Quaking and miR-155 interactions in inflammation and leukemogenesis.

Quaking (QKI) is a tumor-suppressor gene encoding a conserved RNA-binding protein, whose expression is downregulated in several solid tumors. Here we report that QKI plays an important role in the immune response and suppression of leukemogenesis. We show that the expression of Qki is reduced in lipopolysaccharide (LPS)-challenged macrophages, suggesting that Qki is a key regulator of LPS signaling pathway. Furthermore, LPS-induced downregulation of Qki expression is miR-155-dependent. Qki overexpression impairs LPS-induced phosphorylation of JNK and particularly p38 MAPKs, in addition to increasing the production of anti-inflammatory cytokine IL-10. In contrast, Qki ablation decreases Fas expression and the rate of Caspase3/7 activity, while increasing the levels of IL-1α, IL-1β and IL-6, and p38 phosphorylation. Similarly, the p38 pathway is also a target of QKI activity in chronic lymphocytic leukemia (CLL)-derived MEC2 cells. Finally, B-CLL patients show lower levels of QKI expression compared with B cells from healthy donor, and Qki is similarily downregulated with the progression of leukemia in Eµ-miR-155 transgenic mice. Altogether, these data implicate QKI in the pathophysiology of inflammation and oncogenesis where miR-155 is involved.

Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells.

BackgroundCholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but do not interfere with CD-20 in chemo immunotherapy of chronic lymphocytic leukemia (CLL).MethodsMEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients were treated with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations.ResultsWe found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients.ConclusionOur data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment.

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.

Mevalonate pathway inhibitors in the treatment of B-cell chronic lymphocytic leukemia.

6561 Background: B-cell chronic lymphocytic leukemia (CLL) cells are arrested in G0/G1 phase of the cell cycle and are resistant to programmed cell death, hypothesized to contribute to the resistance of CLL cells to standard chemotherapy with curative intent. Methods: Mec-2 cells and Wac-3 cells are CLL cells that have been shown to be resistant to fludarabine and rituximab. We tested a novel enzyme inhibitor’s ability to render CLL cells sensitive to fludarabine and rituximab. Results: BIBB515, a lanosterol synthase inhibitor, at a concentration of 10μM, was able to reduce the cell viability from 82% in controls to 65% after 72 hours. Fludarabine 10μM alone did not reduce the cell viability, 82 % in controls compared to 80%. BIBB515+ fludarabine treatment for 72 hours, at the dose of 10μM each decreased the cell viability to 37%. Cell proliferation by MTT assay was 0.66±0.010 in control compared to 0.37±0.01 in BIBB515+fludarbine and 0.21±0.01 in BIBB515+ fludarabine+ rituximab. There is a 68% down-regulation of cell proliferation using this treatment. There was a two fold induction of CD 20 with combination treatment, and BIBB515 treatment. The mechanism of cell death in the combination treatment of BIBB515 and fludarabine may be due to the up regulation of cell surface marker CD-20. WAC-3 is another CLL cell line that is sensitive to fludarabine, and resistant to rituximab. BIBB515 sensitizes WAC-3 cells to CD 20 antibody rituximab. There is a 68.7% decrease in cell proliferation with combination treatment of BIBB515 and rituximab. Proliferation of Mec-2 cells were inhibited by 60µM and 30µM terbinafine. Ro-48-8071, showed dose-dependent activity, alone or in combination to fludarabine was seen to induce cell death in Mec-2 cells. Fludarabine alone did not have any effect on these cells. Conclusions: Inhibitors of the mevalonate pathway make resistant CLL cells sensitive to current chemotherapeutic agents. Exploiting this mechanism could alter the current treatment regimens, leading to control of the disease in advanced stages by either inducing the leukemic cells to be static or to regress. This strategy may also limit the toxicities involved with chemotherapy.

Cladribine and Fludarabine Nucleoside Change the Levels of CD Antigens on B-Lymphoproliferative Disorders

The purine analogs, fludarabine nucleoside (FdA), and cladribine (CdA) (1 μM, 24 hours), significantly changed the levels of some surface antigens on the human B-cell lines MEC2 and Raji. Changes in the surface proteins were identified using a Cluster of Differentiation (CD) antibody microarray that captures live cells and confirmed by flow cytometry. For Raji cells, CdA up-regulated CD10, CD54, CD80, and CD86, with repression of CD22, while FdA up-regulated CD20, CD54, CD80, CD86 and CD95. For MEC2 cells, CdA up-regulated CD11a, CD20, CD43, CD45, CD52, CD54, CD62L, CD80, CD86, and CD95, but FdA had no effect. Up-regulation of particular CD antigens induced on a B-cell lymphoproliferative disorder by a purine analog could provide targets for therapeutic antibodies with synergistic cell killing.

MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation

We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light ( κ) and heavy chains ( μ, δ) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19,CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bc1-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.