scholarly journals A Novel Spirocyclic Dimer (36-286) Targeting the NF-Kappa B Pathway Displays Potent Anti-Tumor Properties in Chronic Lymphocytic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1186-1186
Author(s):  
Alexandria P Eiken ◽  
Audrey L Smith ◽  
Sarbjit Singh ◽  
Sandeep Rana ◽  
Sunandini Sharma ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Growth ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Protein Translation ◽  
Lymphocytic Leukemia ◽  
Rna Seq ◽  
Resistant Cells

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is an incurable, heterogenetic disease dependent on B cell receptor (BCR) signaling with subsequent nuclear factor-kappa B (NF-κB) activation resulting in the evasion of apoptosis and enhanced malignant B cell growth. Targeted therapies such as ibrutinib (IBR; BTK inhibitor) and venetoclax (VEN; BCL2 antagonist) have revolutionized the management of CLL, however ~20% of patients relapse, signifying the urgent need for novel therapeutics for CLL patients especially those with refractory/relapse (ref/rel) disease. Additionally, various tumor microenvironment (TME) stimuli fuel CLL growth and contribute to drug resistance through the activation of numerous signaling pathways (BCR, CD40R, TLR, BAFFR) and consequential sustained NF-κB activation. Currently, there are no FDA approved drugs that effectively target the NF-κB protein family. Herein we introduce 36-286 (N3), a novel spirocyclic dimer which displays NF-κB inhibitory activity and elicits potent anti-leukemic properties. N3 is a dimer of a spirocyclic α-methylene-γ-butyrolactone analog that covalently binds to surface exposed cysteine residues on NF-κB proteins (IKKβ and P65) (Rana S et al, 2016). Our study aims to investigate N3's mode of action (MOA) and to establish its anti-leukemic effects in CLL including drug-resistant disease, thereby introducing a novel therapeutic option for rel/ref disease. Methods: Cell growth via MTS proliferation assay was determined following treatment with N3 (0.125 - 2 μM) in a panel of malignant B cell lines [CLL (HG3, MEC1, OSUCLL), diffuse large B cell lymphoma (Pfeiffer, RC, RIVA), mantle cell lymphoma (Jeko1)], and in patient derived CLL cells stimulated with CpG ODN 2006 (CpG; 3.2 μM). Viability testing of normal B cells isolated from healthy donors was conducted following N3 treatment. Anti-tumor properties of N3 (1 - 2 μM; 4h) in the HG3 and OSUCLL cell lines were further confirmed under conditions mimicking different TME stimuli such as α-IgM (10 μg/mL), CD40L (100 ng/mL), BAFF (50 ng/mL) or CpG (3.2 μM). Protein expression of oncogenic MYC, select NF-κB pathway proteins (IKKα, IKKβ, P65, IκBα, RelB) and the anti-apoptotic protein MCL1 was determined following treatment with N3 (0.25 - 2 μM; 4h) by immunoblot (IB). Next, we induced IBR resistance in HG3 cells by prolonged exposure to increasing IBR concentrations (~10-15 fold its IC 50 in parental cells). Cell proliferation via MTS was determined following treatment with N3 on these resistant cells. To gain insight on the potential MOA of N3 in CLL, we adapted a proteomics-based approach (TMT labeled mass spectrometry) and conducted RNA-seq in OSUCLL cells treated with N3 (1 - 2 μM) for up to 24 h. Subsequent pathway analysis was performed to identify the top factors modulated by N3. Results: N3 showed remarkable efficacy (IC 50 < 0.6 μM) across all the malignant B cell lines evaluated while sparing normal B cells. In CpG stimulated primary CLL, N3 resulted in marked anti-leukemic effects (0.125 μM) comparable to IBR (1 μM). N3 induced cell apoptosis in CLL cell lines in a dose-dependent manner with marked PARP cleavage. Furthermore, our IB analyses of N3 treated CLL cell lines showed reduced levels of NF-κB pathway proteins, MYC and MCL1. Notably, N3 was effective in reducing levels of the above-mentioned proteins in the presence of the various TME stimuli. Strikingly, N3 maintained its cytotoxic effects in ibrutinib resistant HG3 cells. Studies to confirm N3's cytotoxicity in VEN resistant CLL cells are ongoing. Top ten pathways from both proteomics and RNA-seq analyses revealed an upregulation of the unfolded protein response (UPR) and inhibition of cap-dependent protein translation. IB analyses of select factors related to UPR (CHOP, XBP1, PERK, IRE1) and protein translation (eIF2α, 4E-BP1, PDCD4) in N3 treated CLL cells validated our omics' findings. Efforts to identify the proteome wide direct targets of N3 in CLL cells are currently underway. Conclusion: N3 is a novel pre-therapeutic lead that targets multiple survival and proliferation pathways through the inhibition of NF-κB activity and upregulation of UPR. We show that its highly cytotoxic in tumor B cells while sparing normal B cells. Moreover, N3 sustained its anti-tumor properties under different TME stimuli and in IBR resistant cells, indicating the potential use of this compound in rel/ref patients following evaluation in murine CLL models. Disclosures No relevant conflicts of interest to declare.

A Novel Long Non-Coding RNA, SNHG4 Complex With Eukaryotic Initiation Factor-4E and Regulate Aberrant Protein Translation In Mantle Cell Lymphoma: Implications For Novel Biomarker

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 81-81
Author(s):  
Guangzhen Hu ◽  
Thomas E Witzig ◽  
Mamta Gupta
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Rna Binding ◽  
Cell Lymphoma ◽  
Protein Translation ◽  
Rna Seq ◽  
Lymphoma Cells ◽  
Binding Motifs ◽  
Mantle Cell

Abstract Long noncoding RNAs (lncRNAs) are defined as RNA-like transcripts that are over 200 nucleotides and lack significant open reading frames. Some lncRNAs such as HOTAIR, MALAT1 and H19 have been found to be associated with clinical prognosis and are potential drivers of cancer progression in cancers of the breast, lung, and liver respectively. The role of lncRNAs in lymphoma is unknown. Dysregulation of eIF4E (a key component of the translation initiation complex eIF4F) influences global protein translation, especially the translation of “weak” mRNAs that can be malignancy-related. We and others have found that eIF4E is dysregulated in B-cell lymphoma. The aim of this study is to identify eIF4E-associated lncRNAs through next generation RNA-Sequencing (NGS RNA-Seq) and delineate their role in protein translation in lymphoma. RNA-immunoprecipitation (RNA-IP) was used to pull down eIF4E-bound lncRNA in lymphoma cells. eIF4E-bound lncRNAs were immunoprecipitated with eIF4E antibody or IgG control in Jeko, a mantle cell lymphoma (MCL) cell line and sent for microarray analysis and NGS-RNA-Seq for identification of lncRNAs. The microarray analysis showed that several lncRNAs were enriched with eIF4E antibody compared to IgG control. These included SNHG4 (13.6 fold), SNHG12 (4.8 fold), NCRNA00171 (4.8 fold) and IPW (4.6 fold), GNASAS (3.5 fold), SNHG7 (3.3 fold), NCRNA00182 (2.7 fold), NCRNA00094 (2.6 fold), NCRNA00188 (2.4 fold) and NCRNA00201 (2.1 fold). The binding of these lncRNAs to eIF4E was further confirmed by RT-PCR in Jeko, Mino and Granta MCL cell lines. Next, we looked the expression of these lncRNAs by qRT- PCR in the MCL cell lines and normal controls. We found SNHG4 and IPW to be overexpressed in all the MCL cell lines, while SNHG12 and NCRNA00201 were overexpressed in the selected cell lines. No significant difference was found for the expression of NCRNA00171 and NCRNA00182 in any of the MCL cell lines compared to controls. Overall, these data suggest that several lncRNA have altered expression in malignant B-cells. Considering that the microarray assay only covered a limited number of lncRNAs, we further confirmed eIF4E bound lncRNA by NGS RNA-Seq in Jeko MCL and normal control. The binding of 10/13 lncRNA mentioned above with eIF4E were found upregulated by NGS-RNA-Seq. In addition several novel lncRNAs such as SNHG1 (161.6), AC091814.2 (98.8) and RP11-304L19.5 (64.2) showed up in NGS-RNA-Seq data. These data suggest that lncRNAs, such as SNHG12, SNHG4, and SNHG1 bind to eIF4E with high affinity in malignant B-cells and might play a role in protein translation. We knocked down the expression of SNHG4 through siRNA and demonstrated that cell proliferation and global protein translation was inhibited in lymphoma cells. To further confirm the role of SNHG4 in translation regulation, a plasmid, which contains a renilla luciferase driven by SV40 promoter, was co-transfected with SNHG4 siRNA into Mino cells. The luciferase signal, decreased compared with the cells transfected with nontargeting siRNA. These data suggest that SNHG4 is involved in the regulation of protein translation. In order to clarify the mechanism of lncRNAs bound to eIF4E we searched for RNA binding sites or motifs in eIF4E protein using the web-based tools, BindN and PPRInt. Interestingly two RNA binding motifs, KNKRGGRWLITLNKQQRRS and SHADTATKSGSTTKNR, were found in eIF4E based on the prediction. To examine whether lncRNAs bind with eIF4E through these RNA binding motifs, an eIF4E mutant plasmid with both RNA binding motifs deleted (eIF4EDel), was constructed and transfected transiently into HEK-293T cells along with eIF4EWT plasmid. RNA-IP data showed that the lncRNAs SNHG12, SNHG4 and SNHG1 were not able to bind with eIF4E in eIF4EDel-transfected cells compared with that of eIF4EWT, suggesting that these lncRNAs complex with eIF4E through RNA-binding motifs within the eIF4E. Overall, our results show that the lncRNAs, SNHG1 and SNHG4 are able to bind with eIF4E and regulate protein translation. Since lncRNAs had been found to play roles in the regulation of gene expression, including transcription, splicing and mRNA stability, our results may broaden the view of the functional role of lncRNAs in translation in lymphoma cells and in other cancers. Furthermore, our results also suggested that SNHG4 lncRNAs might be served as potential biomarkers for MCL and other B cell lymphomas for translation therapy. Disclosures: No relevant conflicts of interest to declare.

An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2327-2335 ◽  
Author(s):  
A. Alfarano ◽  
S. Indraccolo ◽  
P. Circosta ◽  
S. Minuzzo ◽  
A. Vallario ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Rt Pcr ◽  
Alternatively Spliced

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

Proliferation of B Cells from Chronic Lymphocytic Leukemia Is Selectively Promoted by B Cell Growth Factor

1989 ◽  
Vol 81 (2) ◽  
pp. 91-97 ◽  
Author(s):  
Melchor Alvarez-Mon ◽  
Antonio de la Hera ◽  
Maria Luisa Gaspar ◽  
Alberto Orfao ◽  
Juan Casas ◽  
...  
Keyword(s):  
B Cells ◽  
Growth Factor ◽  
Cell Growth ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
B Cell Growth Factor

scholarly journals FTY720 demonstrates promising preclinical activity for chronic lymphocytic leukemia and lymphoblastic leukemia/lymphoma

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 275-284 ◽  
Author(s):  
Qing Liu ◽  
Xiaobin Zhao ◽  
Frank Frissora ◽  
Yihui Ma ◽  
Ramasamy Santhanam ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Raji Cell ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
B Cell Malignancies ◽  
Pp2a Activation

FTY720 is an immunosuppressant developed to prevent organ transplant rejection. Recent studies indicate an additional role for FTY720 in inducing cell apoptosis. We demonstrate here that FTY720 mediates toxic effects in cell lines representing different B-cell malignancies and primary B cells from patients with chronic lymphocytic leukemia (CLL). In contrast to previous reports in T-cell lines, FTY720-induced toxicity in the Raji cell line and primary CLL B cells is independent of activation of caspases or poly(ADP-ribose) polymerase processing. Further, pancaspase inhibitor Z-VAD-fmk failed to rescue these cells from apoptosis mediated by FTY720. FTY720 induced down-regulation of Mcl-1 but not Bcl-2 in CLL B cells. Overexpression of Bcl-2 failed to protect transformed B cells from FTY720-induced apoptosis, suggesting a Bcl-2–independent mechanism. Interestingly, FTY720 induced protein phosphatase 2a (PP2a) activation and downstream dephosphorylation of ERK1/2, whereas okadaic acid at concentrations that inhibited the FTY720-induced PP2a activation also resulted in inhibition of FTY720-mediated apoptosis and restoration of baseline ERK1/2 phosphorylation in primary CLL cells, indicating a role for PP2a activation in FTY720-induced cytotoxicity. Further, FTY720 treatment resulted in significant prolonged survival in a xenograft severe combined immunodeficiency (SCID) mouse model of disseminated B-cell lymphoma/leukemia. These results provide the first evidence for the potential use of FTY720 as a therapeutic agent in a variety of B-cell malignancies, including CLL.

scholarly journals Utility of Quantitative Flow Cytometry Immunophenotypic Analysis of CD5 Expression in Small B-Cell Neoplasms

2014 ◽  
Vol 138 (7) ◽  
pp. 903-909 ◽  
Author(s):  
Pramoda Challagundla ◽  
Jeffrey L. Jorgensen ◽  
Rashmi Kanagal-Shamanna ◽  
Inga Gurevich ◽  
Diane M. Pierson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Positive Events ◽  
Expression Levels ◽  
Mantle Cell ◽  
Homogeneous Pattern

Context.—The value of assessing CD5 expression in the differential diagnosis of small B-cell neoplasms is well established. Assessment is usually done qualitatively. Objectives.—To assess CD5 expression levels by quantitative flow cytometry immunophenotyping and to determine possible differences among various small B-cell neoplasms. Design.—We performed 4-color flow cytometry analysis on specimens of peripheral blood and bone marrow aspirate and quantified CD5 expression in various small B-cell lymphomas and leukemias. We also assessed CD5 levels in peripheral blood samples of healthy blood donors. Results.—Cases of chronic lymphocytic leukemia and mantle cell lymphoma had higher levels of CD5 compared with control B cells (P < .001). Cases of marginal zone lymphoma and hairy cell leukemia had CD5 levels similar to control B cells (P = .35 and P = .14, respectively), whereas cases of follicular lymphoma and lymphoplasmacytic lymphoma had significantly lower CD5 levels than control B cells (P < .001 and P = .04, respectively). In B-cell neoplasms, a high level of CD5 expression was correlated with a homogeneous pattern of positive events, whereas lower CD5 levels were correlated with heterogeneous patterns of positive events. Conclusions.—Using flow cytometric immunophenotypic analysis to quantify CD5 levels can aid in diagnosis. CD5 expression levels are higher in patients with chronic lymphocytic leukemia and mantle cell lymphoma, and expression is observed in a homogeneous pattern, as compared with other B-cell neoplasms that are either negative for CD5 or express CD5 at lower levels with a heterogeneous pattern. However, there is some overlap in CD5 expression levels between a subset of atypical chronic lymphocytic leukemia and marginal zone lymphoma cases.

Integrative Transcriptome and Quantitative Proteome Analyses Identify METTL3 As a Key Regulator for Aberrant RNA Processing in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12
Author(s):  
Yiming Wu ◽  
Meiling Jin ◽  
Kevyn Hart ◽  
Aijun Liao ◽  
Stacey M. Fernandes ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Stop Codon ◽  
Protein Translation ◽  
Rna Metabolism ◽  
Mrna Processing ◽  
Lymphocytic Leukemia ◽  
Rna Seq ◽  
Healthy Donors

Aberrant mRNA processing is known to drive the pathogenesis of chronic lymphocytic leukemia (CLL). Recurrent gene mutations in the RNA splicing factor SF3B1 and widespread RNA intronic polyadenylation impact genome-wide gene expression and inactivate tumor suppressors, respectively. Nevertheless, how mRNA processing is regulated and exerts its function in CLL remain elusive. To comprehensively characterize the role of mRNA processing in CLL, we performed RNA sequencing (RNA-seq) and Tandem Mass Tag (TMT) proteomics using normal and CLL B cells derived from healthy donors (n=5) and untreated CLL patients (n=22). We detected 328 proteins differentially expressed between normal and CLL B cells (|Log2FC|>0.58, q<0.05). Gene set enrichment analysis (GSEA) revealed that proteins involved in RNA metabolism (transcription, splicing, modification, 3'end processing, nuclear export, decay) were upregulated in CLL, while those impacting translation were downregulated. These findings were validated by immunoblotting in an independent set of samples (n=10). However, we observed no significant gene expression changes of RNA metabolism at the transcript level, indicating that regulation of these proteins occurred post-transcriptionally. Since N6-methyladenosine (m6A) is the most abundant RNA internal modification and has emerged as a key regulator for RNA metabolism, we sought to determine whether m6A is dysregulated in CLL cells. With an m6A dotblot assay and HPLC-MS, we consistently detected increased level of m6A in mRNA from CLL cells compared with normal B cells. As one of the most upregulated proteins in CLL, METTL3 writes m6A and promotes translation efficiency through its writer and reader functions, respectively. When we knocked down (KD) METTL3 in CLL cell lines (HG3, MEC1) as well as in primary CLL cells, we observed significant cell death and growth disadvantage in CLL compared to control cells, highlighting METTL3 is essential for CLL survival. We next examined whether KD of METTL3 affects m6A and RNA translation using m6A dotblot and O-propargyl-puromycin run on assays. Loss of METTL3 had subtle impact on m6A levels but it significantly decreased protein translation (t test, p<0.01) in all the cell lines tested (HG3, MEC1, JeKo-1, Mino). To define the target protein that METTL3 affects, we performed an integrated Ribosome profiling and RNA-seq analysis using HG3 and Mino cells with or without METTL3. At both transcriptome and translatome levels, loss of METTL3 significantly decreased genes enriched in the mTORC1 pathway, which has an essential role in translation (Metascape, hypergeometric test, q<0.05). Furthermore, it also decreased the translation efficiencies of genes involved in mRNA processing, DNA synthesis, and cell cycle pathways. This observation suggests that upregulation of METTL3 in CLL cells may regulate protein translation of the RNA metabolism pathway. Since m6A at the stop codon region is critical for METTL3 regulating protein translation, we performed MAZTER sequencing to determine m6A modification sites in normal and CLL B cells derived from healthy donors (n=5) and untreated CLL patients (n=11). We identified 214 genes with significant differential m6A modification at the stop codon region (delta cleavage efficiency>0.1, Wilcoxon rank-sum test, p<0.1, within DRACA motif) between normal and CLL B cells. These genes were highly enriched for mRNA processing (Metascape, q=0.017), supporting our notion that METTL3 may modulate protein expression of mRNA processing genes by recognizing m6A modification via its reader function in CLL. Consistent with its role in regulating protein expression, we detected downregulation of splicing factors (SF3A1, SF3A2, SF3B1, U2AF1) in various METTL3 KD cell lines (HG3, MEC1, JeKo-1, Mino) at only protein level but not transcription level. These data link METTL3 upregulation with RNA metabolism protein enrichment in CLL. Altogether, our integrated analysis uncovered a novel regulatory axis of METTL3 in CLL biology. We demonstrated that CLL cells have an increased m6A modification and upregulation of METTL3 at the protein level, resulting in translation of RNA metabolism related genes through its reader function by the recognition of m6A modification. Our results collectively suggest METTL3 as a central regulator for mRNA processing in CLL and provide a rationale for targeting METTL3 in this disease. Disclosures Brown: Janssen, Teva: Speakers Bureau; Gilead, Loxo, Sun, Verastem: Research Funding; Abbvie, Acerta, AstraZeneca, Beigene, Invectys, Juno/Celgene, Kite, Morphosys, Novartis, Octapharma, Pharmacyclics, Sunesis, TG Therapeutics, Verastem: Consultancy. Rosen:Seattle Genetics: Consultancy; NeoGenomics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; Pebromene: Consultancy; Celgene: Speakers Bureau; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau. Siddiqi:TG Therapeutics: Research Funding; Janssen: Speakers Bureau; Seattle Genetics: Speakers Bureau; Oncternal: Research Funding; BeiGene: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Juno: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Research Funding, Speakers Bureau.

Anti-CD20 Antibody Rituximab and Anti-CD23 Antibody IDEC-152 Induce Apoptosis of Malignant B-Cells in Combination with Chemical Antagonists of XIAP.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1401-1401 ◽  
Author(s):  
Massimo Mangiola ◽  
Kate Welsh ◽  
Shinichi Kitada ◽  
Irene M. Pedersen ◽  
Nuzhat Pathan ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Lymphoma Cell ◽  
Efficient Induction ◽  
Anti Cd20

Abstract We tested the effects of Rituximab (anti-CD20) and IDEC-152 (anti-CD23) on apoptosis of B-cell malignancies, using established non-Hodgkin’s B-Cell lymphoma cell lines and freshly isolated Chronic Lymphocytic Leukemia (CLL) B-cells. We used monolayers of stably transfected CHO-cells expressing FcRγIII-A to present antibody to B-cells and promote crosslinking. Established B-cell lymphomas (n = 3) were cultured in the presence of FcRγIIIA-expressing CHO monolayer with or without MAbs and apoptosis was measured by annexin V/propidium iodide staining at various times thereafter. Both antibodies induced time-dependent apoptosis of B-cell lymphoma cell lines. After 48 hrs of treatment with either Rituximab or IDEC-152, the majority of the malignant B-cells were apoptotic (remaining viable cells = 28.7% ± 0.2137% for Rituximab and 30.87% ± 0.7332% for IDEC-152). Rituximab and IDEC-152 also induced marked increases in caspase activity in B-cell lymphoma cell lines, with fold-increases above baseline control cells of 25 ± 0.9031 and 24 ± 0.3839, respectively. In contrast, neither Rituximab nor IDEC-152 induced striking effects on primary CLL B-cells (n = 6). We therefore tested the combination of Rituximab or IDEC-152 with other agents that target anti-apoptotic proteins, exploring whether more efficient induction of apoptosis can be achieved. We cultured lymphoma cell lines and primary CLL specimens with chemical antagonists of XIAP (Schimmer, et al. Cancer Cell5: 25, 2004), an anti-apoptotic protein that inhibits effector caspases. When used at concentrations where XIAP antagonists alone were non-apoptotic (approximately 2.5 μM), a significant increase in apoptosis was achieved in cultures of lymphoma and CLL cells treated with either Rituximab or IDEC-152. These findings suggest that Rituximab or IDEC-152 may more efficiently induce apoptosis of malignant B-cells when combined with an apoptosis-sensitizing agent. (Supported by CA-81534; CA-78040; and an unrestricted grant from Genentech, Inc.).

FTY720 (2-Amino-2-[2-(4-octylphenyl) ethyl] Propane 1, 3-diol hydrochloride), Mediates Cytotoxicity through Caspase Independent and Protein Phosphatase 2A Dependent Mechanisms in Chronic Lymphocytic Leukemia and Lymphoblastic Leukemia/Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2095-2095
Author(s):  
Qing Liu ◽  
Xiaobin Zhao ◽  
Frank Frissora ◽  
Yihui Ma ◽  
Ramasamy Santhanam ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Protein Phosphatase ◽  
Lymphocytic Leukemia ◽  
Pp2a Activity ◽  
Phosphatase 2A ◽  
Induced Apoptosis ◽  
Pp2a Activation

Abstract FTY720 (2-Amino-2-[2-(4-octylphenyl) ethyl] propane 1, 3-diol hydrochloride) is a synthetic compound produced by modification of a natural immunosuppressant, ISP-1. It is an immunosuppressive agent that is being developed to prevent organ transplant rejection. Recent studies indicate additional role for FTY720 in inducing cell apoptosis. We demonstrate here a novel mechanism by which FTY720 mediates cytotoxic effects in cell lines representing different B cell malignancies and primary B cells from chronic lymphocytic leukemia (CLL) patients. FTY720 induced apoptosis as detected by annexin V/ propidium iodide staining in representative B cell lines and CLL patient derived CD19+ B cells in time and dose dependent manner (p<0.0001, untreated vs 10mM-treated CLL cells, n=15). In contrast to previous reports in T cell lines, FTY720 induced cytotoxicity in Raji cell line and primary CLL cells is independent of activation of caspase 3, 8 and 9 or poly-ADP ribose polymerase cleavage. Further, pan-caspase inhibitor Z-VAD-fmk rescued these cells from fludarabine but not FTY720 induced apoptosis (p=0.001 fludarabine vs fludarabine+z-VAD-fmk; p=0.99 FTY720 vs FTY720+z-VAD-fmk, n=5). Over-expression of Bcl-2 failed to protect transformed B-cells from FTY720 induced apoptosis suggesting Bcl-2 independent cytotoxic effect. Interestingly, FTY720 induced consistent increase in protein phosphatase 2a (PP2a) activity and concentrations of okadaic acid that inhibited the FTY720-induced PP2A activity also resulted in inhibition of FTY720-mediated cytotoxicity in B cell lines and primary CLL cells, indicating a role for PP2A activation in FTY720 induced cytotoxicity. Consistent with its activation of PP2A, FTY720 induced dephosphorylation of of Erk1/2 in CLL B cells. Further, FTY720 treatment resulted in significant in-vivo therapeutic efficacy associated with prolonged survival in a xenograft SCID mouse model of disseminated B cell lymphoma/leukemia (median survival time for FTY720 treated mice was 47 days (95 %CI 39–53) compared to 18 days in placebo controls (95% CI 17–19) P<0.0001-FTY720 vs placebo). These results provide first evidence for a PP2A dependent and caspase independent cytotoxicity of FTY720 in B cells. The novel caspase and Bcl-2 independent mechanism of cytotoxicity concurrent with identification of PP2a activation as a surrogate marker of cell killing provide further justification for clinical development of this agent in lymphoid leukemia.

Sign in / Sign up

Export Citation Format

Share Document