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.

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.

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.

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 &lt; 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.

Sustained Signaling through the B-Cell Receptor Induces Mcl-1 and Promotes Survival of Chronic Lymphocytic Leukemia B-Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 178-178
Author(s):  
Dimitar G. Efremov ◽  
Aleksandar Petlickovski ◽  
Luca Laurenti ◽  
Xiaoping Li ◽  
Sara Marietti ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Spontaneous Apoptosis ◽  
B Cell Survival ◽  
Bcr Signaling ◽  
Induced Apoptosis

Abstract The clinical course of chronic lymphocytic leukemia (CLL) differs significantly between patients with mutated (M-CLL) and unmutated (U-CLL) immunoglobulin V genes, implying a role for B-cell receptor (BCR) signaling in the pathogenesis of this disease. BCR stimulation in normal B-cells triggers several crucial signaling pathways, including PI3K/Akt, IKK/NF- κB and the mitogen-activated protein kinases Erk, JNK and p38 MAPK, which can induce proliferation, survival, differentiation or apoptosis, depending on the nature and context of the antigenic stimulation. We have now investigated activation of these downstream signaling pathways, as well as induction of anti-apoptotic proteins and survival of CLL B-cells stimulated with soluble (sol-IgM) and immobilized (imm-IgM) anti-IgM antibodies, which were used to mimic stimulation with soluble and particulate/membrane-bound antigen, respectively. Stimulation with sol-IgM revealed similar activation patterns in the 10 U-CLL and 12 M-CLL cases that partially resembled the pattern described for tolerant B-cells. The response in the U-CLL cases was characterized by transient (<45 minutes) phosphorylation of Akt and Erk, no activation of JNK and p38 MAPK, and activation of IKKβ in 50% of the cases. Most M-CLL cases showed similar activation of Akt and Erk, but lacked activation of IKKβ, whereas three M-CLL cases were completely non-responsive. To investigate the effects on CLL B-cell survival, 14 U-CLL and 19 M-CLL cases were analyzed by Annexin V/PI staining after 48 hours stimulation with sol-IgM. A 10–40% increase in apoptotic cells was observed in the majority of cases from both CLL subsets (p<0.001 with respect to spontaneous apoptosis). Induction of apoptosis was confirmed by analyzing cleavage of the Caspase 3 substrate PARP, and was accompanied by an approximately 50% reduction in the levels of Mcl-1, an antiapoptotic protein implicated in CLL B-cell survival and resistance to chemotherapy. A markedly different response was induced by imm-IgM, which was characterized by activation of IKKβ in all cases and sustained Akt and Erk phosphorylation that persisted over 24 hours. This response resulted in a 2.5 fold mean increase in the levels of Mcl-1, whereas no changes were observed in the levels of Bcl-2 and Bcl-xL. Imm-IgM slightly reduced the percentage of cells undergoing spontaneous apoptosis after 48 hours, but significantly protected from fludarabine- and methylprednisolone-induced apoptosis. To investigate which of the three imm-IgM activated pathways is responsible for induction of Mcl-1 and protection from chemotherapy-induced apoptosis, we incubated CLL B-cells with LY294002, U0126 and BAY-11 (inhibitors of PI3K, ERK and NF- κB, respectively) prior to stimulation with imm-IgM and addition of fludarabine. Induction of Mcl-1 and inhibition of fludarabine-induced PARP cleavage were significantly abrogated only by LY294002, indicating that the PI3K/Akt pathway is the major link between the BCR and apoptosis resistance of CLL B-cells. In conclusion, this study shows that the response of CLL B-cells to BCR stimulation primarily depends on the nature of the antigenic stimulus. Moreover, it shows that only sustained BCR signaling can promote survival of CLL B-cells, and raises the possibility that the distinct clinical and biological behavior of U-CLL and M-CLL is determined by the availability of such stimulation.

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

Abstract 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.

Mediation of apoptosis by and antitumor activity of lumiliximab in chronic lymphocytic leukemia cells and CD23+ lymphoma cell lines

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1594-1602 ◽  
Author(s):  
Nuzhat I. Pathan ◽  
Peter Chu ◽  
Kandasamy Hariharan ◽  
Carolyn Cheney ◽  
Arturo Molina ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Antitumor Activity ◽  
B Cell ◽  
Cell Lines ◽  
Human Monoclonal Antibody ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Synergistic Cytotoxicity ◽  
Induced Apoptosis

AbstractLumiliximab is a chimeric macaque-human monoclonal antibody to CD23, a protein expressed on virtually all chronic lymphocytic leukemia (CLL) cells. We examined the ability of lumiliximab to mediate apoptosis, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity against primary CLL cells and CD23-expressing B-cell lines. Our data suggest that lumiliximab kills CLL cells and CD23-expressing B cells predominantly by apoptosis, which occurs through the intrinsic pathway. Lumiliximab-induced apoptosis was accompanied by the down-regulation of antiapoptotic proteins Bcl-2, Bcl-XL, and XIAP, activation of Bax, and release of cytochrome c from the mitochondria. We also found that the addition of lumiliximab to rituximab or fludarabine results in synergistic cytotoxicity of primary CLL cells and CD23-expressing B-cell lines. We investigated the in vivo activity of lumiliximab in a human disseminated CD23+ B-cell lymphoma SCID mouse model and found greater antitumor activity with it than with control antibody. We also found that paralysis-free survival was greater with lumiliximab plus rituximab or fludarabine than with any of those agents alone. These results suggest that lumiliximab may be an effective treatment alone or in combination with rituximab or chemotherapy agents in CLL or other CD23-overexpressing B-cell malignancies.

DNA Hypomethylation Leads to Aberrant Expression of PD-1 in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3504-3504 ◽  
Author(s):  
Eun Joon Lee ◽  
Jimei Liu ◽  
Ethan Speir ◽  
James Wilson ◽  
Hena Joshi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphocytic Leukemia ◽  
Healthy Controls ◽  
Dna Hypomethylation ◽  
Bisulfite Pyrosequencing ◽  
Enhancer Region

Abstract Abstract 3504 Programmed Death-1 (PD-1) is an inhibitory cell surface receptor of the immunoglobulin superfamily expressed on activated lymphocytes, monocytes and dendritic cells. Although PD-1 function is best characterized in T-cells, it is known that PD-1 also suppresses the immune response of B lymphocytes through protein phosphatase recruitment and dephosphorylation of signaling molecules downstream of the B-cell receptor (BCR). Recent studies have found that PD-1 expression is elevated at the mRNA as well as the protein levels in B cells obtained from chronic lymphocytic leukemia (CLL) patients compared to those from healthy controls. Using genome-wide DNA methylation sequencing, we identified PD-1 as one of the significantly hypomethylated genes in CLL compared to normal B-cell samples. Three differentially methylated regions (DMRs) were discovered in the first intron, proximal promoter and up-stream enhancer regions. We validated these DMRs in 43 CLL and 7 normal control samples using bisulfite pyrosequencing. The pyrosequencing analysis further confirmed that all three regions were significantly hypomethylated in CLL patient samples (p<0.001). These epigenetic changes resulted in the overexpression of PD-1 in primary CLL B cells, which was confirmed by real-time quantitative PCR. In B cells isolated from healthy controls, flow cytometry analysis showed that only approximately 1% expressed PD-1, whereas PD-1 positive B cells in CLL patients ranged from 5% to 64%. No correlation between PD-1 status and IGHV mutations or CD38 expression was observed. To elucidate the mechanisms of epigenetic regulation of PD-1 expression, we studied five non-Hodgkin's lymphoma cell lines including Mec-1, Granta 519, RL, Raji and DB. Bisulfite pyrosequencing results showed that only the up-stream enhancer is differentially methylated in these cell lines. Treatment of lymphoma cell lines with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors can up-regulate PD-1 expression in RL, Raji and DB cell lines in which the up-stream enhancer region is hypermethylated; however, the same treatments decreased the PD-1 expression in Mec-1 cells, which are demethylated in the PD-1 enhancer region and express PD-1 on the cell surface. Chromatin immunoprecipitation (ChIP) analysis revealed that H3K4me3 and H3K4me1 modifications were significantly enriched in the promoter and enhancer regions in PD-1 positive Mec-1 cells, respectively. However, H3K27me3 modification was enriched in both promoter and enhancer regions in PD-1 negative RL cells, while enrichment of H3K3me3 and H3K4me1 modification was significantly decreased. These results suggest that coordinated regulation of enhancer activity by DNA methylation and histone modification is crucial for PD-1 expression in CLL B cells. Furthermore, we mapped the nucleosome occupancy in the enhancer regions using a high-resolution, single-molecule approach. The nucleosome mapping results revealed nucleosome-depleted regions in Mec-1 cells, but not in RL cells. This novel finding demonstrates the complexity of epigenetic regulation of PD-1 expression. To determine the function of PD-1 in CLL, we co-cultured the Mec-1 cell line and primary CLL B-cells with a hepatocyte cell line Huh7.5 that overexpresses exogenous PD-1 ligand, PD-L1. Surprisingly, unlike PD-1 positive normal B cells, we did not observe increased apoptosis in the co-cultured CLL B cells, suggesting that PD-1 has a different functional role in CLL compared to normal B cells. In summary, we present here the novel finding that DNA hypomethylation in the enhancer region of PD-1 leads to aberrant overexpression of PD-1 on CLL B-cell surfaces and that in CLL PD-1 may have a different function than in normal B cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells

2021 ◽  
Author(s):  
Sarah Wilmore ◽  
Karly-Rai Rogers-Broadway ◽  
Joe Taylor ◽  
Elizabeth Lemm ◽  
Rachel Fell ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mrna Translation ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Mrna Stabilization

AbstractSignaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

scholarly journals Emergence of a B-cell lymphoblastic lymphoma in a patient with B-cell chronic lymphocytic leukemia: evidence for the single-cell origin of the two tumors

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 797-804
Author(s):  
V Pistoia ◽  
S Roncella ◽  
PF Di Celle ◽  
M Sessarego ◽  
G Cutrona ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Chromosomal Abnormalities ◽  
Cell Clone ◽  
Lymphoblastic Lymphoma ◽  
Lymphocytic Leukemia ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Chain Gene

A patient is described who presented with a chronic lymphocytic leukemia (CLL) and later developed a lymphoblastic lymphoma. The cells from the CLL were typical mature B lymphocytes as could be assessed by morphologic, cytochemical, and surface marker analyses. The cells from the lymphoblastic lymphoma were immature B cells that expressed CD10, CD20, and HLA-DR markers, but not surface Ig or cytoplasmic mu chains, and were negative for terminal deoxynucleotidyl transferase (TdT). The cells of two continuous cell lines, obtained from the bone marrow and the peripheral blood of the patient, had the same phenotype as the lymphoblastic lymphoma cells, did not contain the Epstein-Barr virus genome, and displayed malignant features in vitro, including the capacity to form colonies in agar. The two cell lines also shared identical chromosomal abnormalities, a finding which suggests that they derived from the same malignant cell already present in vivo. Such chromosomal abnormalities were not seen in the karyotype of the peripheral blood cells at the onset of the disease. Analysis of the Ig heavy chain genes using a DJ-specific probe showed the very same monoclonal rearrangement in the cells from the B-CLL, the lymphoblastic lymphoma and the two cell lines, thus demonstrating their common clonal origin. By contrast, a monoclonal rearrangement of the lambda chain gene locus was found in the B-CLL cells only, a finding consistent with their exclusive capacity to express surface IgM lambda. This patient represents a rare case in whom a chronic lymphoproliferative disorder with mature malignant cells transforms into a lymphoblastic lymphoma characterized by cells frozen at a very early maturational stage. The possible mechanisms leading to such transformation within the same cell clone are discussed.

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