scholarly journals The Novel Berbamine Derivative, Cbbm, Downregulates c-Myc to Block Diffuse Large B-Cell Lymphoma Cell Maintenance

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 212-212
Author(s):  
Lei Zhang ◽  
Rongzhen Xu ◽  
Ling Li ◽  
Xiaoying Zhao ◽  
Jinwen Huang
Keyword(s):  
Cell Lines ◽  
Cell Lymphoma ◽  
Hematopoietic Cells ◽  
B Cell Lymphoma ◽  
Vehicle Control ◽  
Dependent Manner ◽  
Protein Levels ◽  
Large B Cell Lymphoma ◽  
Induced Apoptosis ◽  
Dose Dependent

Abstract Diffuse large B-cell lymphoma (DLBCL), is one of the most common lymphoma subtypes worldwide, and represents 30% of non-Hodgkin lymphoma (NHL). The current DLBCL chemotherapy R-CHOP regimen is limited by lack of specificity and toxicity to normal hematopoietic cells. Identification of a targeted therapeutic approach is an unmet need for the disease. Through translocations, amplifications or overexpression, c-Myc is dysregulated in many human cancers, including DLBCL. Aberrant overexpression of c-Myc promotes tumorigenesis by activating target genes critical for cell proliferation/survival. In our preliminary study, c-Myc protein levels in 119 cases of newly diagnosed DLBCL were evaluated by immunohistochemistry. We found that c-Myc levels were evident (>30% positive cells) in 40.52% (47/116) and high (>80% positive cells) in 6.90% (8/116) of patients. We also observed that relative to c-Myc negative cases, c-Myc positive cases (>30% positive cells) were associated with adverse prognostic indicators including high international prognostic index (IPI) score (≥60 years old group) (p=0.033), high Ki-67 index (p=0.002), high central nervous system involvement (p=0.003) and low complete response rate (p=0.028), confirming that c-Myc plays a critical role in DLBCL pathogenesis. Currently, c-Myc is considered to be an "undrugable" oncoprotein. Chemotherapeutic agents do not affect c-Myc activity. Our previous work in T-cell lymphoma (Cancer Cell, 32:115-128) demonstrated that berbamine, a natural bis-benzylisoquinoline alkaloid, effectively reduced N-Methyl-N-Nitrosourea (MNU)-induced lymphoma burden by disrupting the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII)γ/c-Myc axis. We also showed that CaMKIIγ stabilizes c-Myc by phosphorylating it at the serine 62 residue (Ser62). We created the berbamine derivative 4-chlorobenzoyl berbamine (CBBM) (Fig.1), and evaluated its efficacy in vitro using DLBCL cell lines (n=5) which expressed high levels of c-Myc. The IC50 of CBBM was an order of magnitude lower than that of berbamine across a panel of DLBCL cell lines: the IC50 (μmol/L) of CBBM vs. berbamine in these cells were OCI-Ly3 (1.39±0.04 vs 12.20±0.71), OCI-Ly10 (2.56±0.04 vs 23.66±0.53), U2932 (3.89±0.07 vs 17.78±0.29), SU-DHL 16 (1.82±0.05 vs 17.56±0.71) and Pfeiffer (3.73±0.06 vs 26.16±0.56). CBBM also induced apoptosis in a dose-dependent manner in these cell lines while sparing normal hematopoietic cells from healthy donors (n=2). As an example, CBBM treatment dose-dependently induced apoptosis on OCI-Ly3 cells (vehicle control was 4.43±0.06%, 4 μmol/L CBBM was 24.43±0.72% and 6 μmol/L CBBM was 75.43±0.25%, vehicle control vs CBBM at 4 μmol/L, p<0.001). Conversely, more than 85% of normal hematopoietic cells from mobilized healthy donor peripheral blood were still viable after exposure of CBBM at 10 μmol/L, which is a lethal dose for DLBCL lines. Moreover, CBBM treatment significantly reduced cycling of DLBCL cells (SG2M fractions: vehicle control 68.11±0.40%, CBBM 4 μmol/L 38.36±0.34%, p<0.001). Mechanistically, exposure of DLBCL lines (n=5) to CBBM decreased c-Myc protein levels in a dose-dependent manner. We also validated the reduction of PD-L1 expression, a reported c-Myc target through immunoblots. The proteasome inhibitor MG132 prevented CBBM-induced c-Myc protein degradation, indicating that reduced c-Myc after CBBM treatment is related to downregulation of protein stability. Importantly, we also explored the effect of CBBM on the CaMKIIγ/c-Myc axis. CBBM treatment reduced phospho-c-Myc (Ser62) levels in all the lines. We observed CBBM-mediated inhibitory effects (IC50 of five lines) was correlated with the basal level of CaMKIIγ (R2 = -0.8269, p=0.0323) (Fig.2). Beyond targeting c-Myc, CBBM also exhibited immune modulator potency including downregulation of autocrine interleukin 10 (IL10) level assayed by ELISA, leading to reduction of the downstream JAK2/STAT3 levels. Currently, we are in the process of testing CBBM in vivo effects using an OCI-Ly3 xenograft model. Although the overall survival result is pending, following 4 weeks of CBBM treatment (1 g/kg, i.g.), tumors from animals receiving drug treatment show decreased c-Myc expression levels relative to that of vehicle treated animals. Collectively, our results support further evaluation of CBBM as a promising compound to treat c-Myc associated DLBCL. Disclosures No relevant conflicts of interest to declare.

scholarly journals MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Danxia Zhu ◽  
Cheng Fang ◽  
Wenting He ◽  
Chen Wu ◽  
Xiaodong Li ◽  
...  
Keyword(s):  
B Cell ◽  
Binding Site ◽  
Cell Lines ◽  
Expression Vector ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Protein Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.

HDAC Class I Inhibition Acetylates a Non-Histone Protein STAT3 by Modulating p300-STAT3-HDAC1 Interaction In Activated B- Cell Like (ABC) Diffuse Large B Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 115-115
Author(s):  
Mamta Gupta ◽  
Jing Jing Han ◽  
Mary Stenson ◽  
Linda Wellik ◽  
Thomas E. Witzig
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Dependent Manner ◽  
Hdac Inhibition ◽  
Over Expression ◽  
Stat3 Signaling ◽  
Large B Cell Lymphoma ◽  
Dose Dependent

Abstract Abstract 115 Patients with diffuse large B- cell lymphoma (DLBCL) tumors that have an activated B-cell like (ABC) gene expression profile have a poorer prognosis. Understanding the mechanism(s) used by ABC tumor cells to resist the effects of common chemotherapy agents may lead to alternative approaches for the treatment of these tumors. ABC cell lines have been shown to have high levels of phosphorylated STAT3 (pSTAT3); however, the mechanisms that regulate STAT3 signaling in ABC DLBCL remain unclear. Histone deacetylases (HDACs) are enzymes that can deacetylate both non-histone and histone substrates. In this study we tested the hypothesis that HDACs in the tumor cells target a non-histone protein STAT3 in ABC DLBCL. In studies of HDAC expression in DLBCL tumors, we found over-expression of the type 1 HDACs, specifically HDAC1and HDAC3, in the pSTAT3- positive ABC tumors as compared to germinal centre B like (GCB) tumors. We then performed a co-immunoprecipitation (Co-IP) assay to learn the functional interaction between STAT3 and HDAC1. We found that STAT3 formed complexes with HDAC1 or HDAC3. Further Co-IP studies demonstrated that p300, a histone acetyltransferase (HAT), STAT3, and HDAC1 are all in the same complex. To determine whether p300 acetylates STAT3 in ABC cells, we immuno-precipitated endogenous p300 and blotted with acetylated STAT3 and showed that p300 acetylates STAT3 at lysine 685. We next tested whether HDAC inhibition could affect p300 mediated STAT3 acetylation in ABC cells. Inhibition of HDAC activity through the HDAC inhibitor LBH589 (LBH, Novartis Pharmaceuticals) increased STAT3 acetylation in a dose- dependent manner. Similar results were obtained when we used antiacetyl- lysine antibody. Furthermore HDAC1 over-expression inhibits STAT3 acetylation at lysine 685. This data implies a tight regulation of STAT3 acetylation and deacetylases in vivo in ABC lymphoma. In addition to acetylation, STAT3 can be modified by phosphorylation, thus the effect of HDAC inhibition on pSTAT3 both at serine and tyrosine residues was studied. We observed a dose-dependent decrease in pSTAT3 with some inhibitory effect on total STAT3. LBH was found to mediate STAT3 dephosphorylation by inhibiting the tyrosine phosphorylation of JAK2 and TYK2, the STAT3 upstream activators, in a dose- dependent manner. Since ABC lymphoma has higher levels of HDAC1 or HDAC3 and pSTAT3/STAT3 than GCB, we hypothesized that ABC cells will be more sensitive to HDAC inhibition than GCB. In fact, when ABC and GCB DLBCL cells were treated with LBH we observed that LBH was more cytotoxic to ABC than GCB as evidenced by annexin/PI staining and PARP cleavage. LD90 was 25 nM for ABC cells, however GCB cells required 5 times more LBH to kill 90% cells. STAT3 activation regulates genes involved in cell survival, including Bcl-2, Mcl-1, Bcl-XL, and c-Myc. LBH treatment resulted in down-regulation of Mcl-1 and c-Myc in ABC cells but has no effect in GCB cells; however, Bcl-2 and Bcl-XL levels were not decreased in both the subtype. Having established that HDAC1 physically associated with STAT3 and that LBH treatment elevated STAT3 acetylation in ABC cells, we proceeded to deplete endogenous HDAC1 with siRNA in Ly3 cells and found that HDAC1 knockdown up-regulated STAT3 acetylation indicating that HDAC1 negatively regulates the acetylation in vivo. HDAC1 inhibition also prevented phopshorylation of STAT3 and induces aopotosis in ABC cells. In summary, we have demonstrated that a key consequence of HATs and HDACs expression and activity is modulation of the STAT3 pathway in ABC lymphoma. Inhibition of this pathway with the HDAC inhibitor LBH inhibits constitutive STAT3 signaling and induces Mcl-1 mediated apoptosis. These studies provide the rationale for targeting the poorly responsive ABC-type DLBCL by inhibiting HDAC activity with epigenetic inhibitors such as LBH. We are currently testing LBH589 in relapsed DLBCL in a phase I clinical trial. Disclosures: No relevant conflicts of interest to declare.

Heat Shock Protein 90 (HSP90) Is a Rational Therapeutic Target in Diffuse Large B-Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 829-829
Author(s):  
Jeremy S. Abramson ◽  
Wen Chen ◽  
Hidenobu Takahashi ◽  
Przemyslaw Juszczynski ◽  
Jeffery L. Kutok ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Binding Domain ◽  
Atp Binding ◽  
Pi3k Inhibitors ◽  
Large B Cell Lymphoma ◽  
Dose Dependent ◽  
Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease that is currently treated with a homogeneous approach based on empiric combination chemotherapy. To elucidate molecular differences in DLBCL subsets and identify more rational treatment targets, coordinate transcriptional profiling was used to define three groups of DLBCLs: “Oxidative Phosphorylation” (OxPhos), “B-cell Receptor/Proliferation” (BCR), and “Host Response” (HR). Of interest, the HSP90 α and β isoforms were differentially expressed in the DLBCL subsets; the stress-induced α isoform was more abundant in OxPhos tumors (p &lt; .0001) whereas the constitutively expressed β isoform was increased in BCR DLBCLs (p &lt; .0001). HSP90 α and β differ in the length of their N-termini but share a common ATP binding domain, the target site for recently developed HSP90 inhibitors, such as IPI-504 (Infinity Pharmaceuticals, Cambridge, MA). Although differential HSP90 isoform expression likely reflects underlying biological differences in the DLBCL subsets, the shared ATP binding domain suggests that both isoforms, and the majority of DLBCLs, may be effectively targeted with HSP90 inhibitors. For these reasons, we determined the IC50 and cytotoxic potential of IPI-504 in an extensive series of DLBCL cell lines. IPI-504 inhibited the proliferation of DLBCL cell lines at IC50s ranging from 0.04 μM in the most sensitive line to 12.67 μM in the most resistant line. Annexin V/PI staining revealed that IPI-504 was cytotoxic to most DLBCL cell lines at low micromolar doses; even cell lines that were relatively resistant to IPI-504 at lower doses (2 μM) underwent apoptosis with prolonged exposure to increased IPI-504 doses (72 hrs. and 8 μM). Cell lines that were most sensitive to IPI-504 expressed high levels of a major HSP90 client protein, pAKT, and exhibited a dose-dependent decrease in pAKT levels following IPI-504 treatment. For these reasons, we assessed the efficacy of IPI-504 in association with a PI3K/AKT pathway inhibitor, LY24009, in the DLBCL panel. Combined treatment with the HSP90 and PI3K inhibitors was highly synergistic (Method of Chou and Talalay; Calcusyn software, Biosoft, Ferguson, MO), inhibiting cellular proliferation by over 95% in evaluated DLBCL cell lines. In contrast, the combination of concurrent IPI-504 and an empiric chemotherapeutic agent, doxorubicin, was antagonistic, underscoring the importance of rational combination therapy. We conclude that: 1) HSP90 isoforms are differentially expressed in primary DLBCL subsets; and 2) inhibition of HSP90, via the conserved ATP binding domain, leads to dose-dependent apoptosis that is associated with depletion of pAKT in DLBCL cell lines. The HSP90 inhibitor IPI-504 possesses both single-agent efficacy as well as synergy when combined with PI3K inhibitors, suggesting that this rational targeted approach warrants further clinical investigation.

Role of phosphatidylinositol 3′-kinase/AKT pathway in diffuse large B-cell lymphoma survival

Blood ◽  
2006 ◽  
Vol 108 (13) ◽  
pp. 4178-4186 ◽  
Author(s):  
Shahab Uddin ◽  
Azhar R. Hussain ◽  
Abdul K. Siraj ◽  
Pulicat S. Manogaran ◽  
Naif A. Al-Jomah ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Akt Pathway ◽  
Phosphatidylinositol 3 Kinase ◽  
Large B Cell Lymphoma ◽  
Induced Apoptosis ◽  
Large B Cell

Abstract Phosphatidylinositol 3′-kinase (PI3K) is a key player in cell-growth signaling in a number of lymphoid malignancies, but its role in diffuse large B-cell lymphoma (DLBCL) has not been fully elucidated. Therefore, we investigated the role of the PI3K/AKT pathway in a panel of 5 DLBCL cell lines and 100 clinical samples. Inhibition of PI3K by a specific inhibitor, LY294002, induced apoptosis in SUDHL4, SUDHL5, and SUDHL10 (LY-sensitive) cells, whereas SUDHL8 and OCI-LY19 (LY-resistant) cells were refractory to LY294002-induced apoptosis. AKT was phosphorylated in 5 of 5 DLBCL cell lines and inhibition of PI3K caused dephosphorylation/inactivation of constitutively active AKT, FOXO transcription factor, and GSK3 in LY-sensitive cell lines. In addition, there was a decrease in the expression level of inhibitory apoptotic protein, XIAP, in the DLBCL cell lines sensitive to LY294002 after treatment. However, no effect was observed in XIAP protein levels in the resistant DLBCL cell lines following LY294002 treatment. Finally, using immunohistochemistry, p-AKT was detected in 52% of DLBCL tumors tested. Furthermore, in univariate analysis, high p-AKT expression was associated with short survival. In multivariate analysis, this correlation was no longer significant. Altogether, these results suggest that the PI3K/AKT pathway may be a potential target for therapeutic intervention in DLBCL.

scholarly journals Endogenous Neurotrophins and Trk Signaling in Diffuse Large B Cell Lymphoma Cell Lines Are Involved in Sensitivity to Rituximab-Induced Apoptosis

PLoS ONE ◽  
2011 ◽  
Vol 6 (11) ◽  
pp. e27213 ◽  
Author(s):  
Cynthia Bellanger ◽  
Lydie Dubanet ◽  
Marie-Claude Lise ◽  
Anne-Laure Fauchais ◽  
Dominique Bordessoule ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Large B Cell Lymphoma ◽  
Induced Apoptosis ◽  
Large B Cell

Histone Deacetylase Inhibition with LBH589 Inhibits the Rapamycin Insensitive Rictor-mTOR (mTORC2) Complex and Translation Initiation Factor eIF4E Activation in Diffuse Large B-Cell Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 603-603
Author(s):  
Mamta Gupta ◽  
Stephen M. Ansell ◽  
Anne J. Novak ◽  
Thomas E. Witzig
Keyword(s):  
B Cell ◽  
Histone Deacetylase ◽  
Hdac Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mtor Pathway ◽  
Dependent Manner ◽  
Large B Cell Lymphoma ◽  
Dose Dependent ◽  
Large B Cell

Abstract Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma and the treatment of these patients has improved with rituximab-based chemo-immunotherapy. However, approximately 40% of patients die of their disease and new agents with novel mechanisms of action are needed for this disease. The mammalian target of rapamycin (mTOR) has emerged as an important therapeutic target in cancer cells and rapamycin and its derivatives, which specifically inhibit mTOR, are now being actively evaluated. Recent clinical trials of mTOR inhibitors have demonstrated that a fraction of patients (35%) with relapsed DLBCL respond to single-agent temsirolimus (Smith S et al J Clin Oncol 26; May 20 Supplement abstract 8514) or everolimus (Reeder et al 2007 ASH Annual Meeting Abstracts 110 (11) abstract 121) in relapsed DLBCL, however most patients are resistant to this therapy. The mechanism of this resistance remains a subject of major therapeutic significance. Herein, we report that cells from DLBCL lines (DHL-6, Ly7 and Ly3) and primary tissues from DLBCL patients (n=10) display persistent activation of the mTOR pathway as determined by phosphorylation of mTOR targets S6 ribosomal protein (S6rp) and 4E-binding protein 1 (4E-BP1). Treatment of human DLBCL cells with various doses of rapamycin predictably demonstrated a decrease in proliferation but less than 10% reduction in overall cell survival. However, rapamycin suppressed the phosphorylation of S6rp and 4E-BP1, indicating an inhibition of raptor-mTOR (mTORC1) signaling. Paradoxically, rapamycin also concurrently increased, through a negative feedback mechanism, the phosphorylation of Akt that may contribute to drug resistance. Interestingly we found that rapamycin treatment also increased the phosphorylation of eIF4E, a survival protein downstream of mTOR, which may also be responsible for resistance of rapamycin along with Akt. To determine whether the observations found in vitro are clinically relevant, we obtained peripheral blood samples from patients with aggressive lymphoma treated with the rapamycin derivative everolimus. The levels of phosphorylated Akt and eIF4E were increased in 3 of 3 patient samples at 48 hrs and after 1 cycle compared to untreated control. Our observations therefore suggest that rapamycin derivatives potently activate Akt and eIF4E activity via activation of the mTORC2 assembly, in addition to its well-characterized ability to suppress the mTORC1 pathway. Treatment of DLBCL cells with histone deacetylase (HDAC) inhibitor LBH589 induced growth inhibition of DLBCL cells at nanomolar concentrations in a dose dependent manner in association with hyperacetylation of histones H3 and H4. LBH alone was able to inhibit the phosphorylation of S6rp and 4E-BP1, while combined treatment with rapamycin inhibited the phosphorylation of S6rp and 4EBP1 to a greater extent than either agent alone. Surprisingly LBH inhibited constitutive as well as rapamycin-induced activation of Akt and eIF4E in a time and dose dependent manner. Our co-immunoprecipitation data suggest that LBH alone was able to alter the level of intact mTORC2 by reducing the amounts of rictor bound to mTOR, which is further decreased when combined with rapamycin. In support of this model, rapamycin combined with LBH exhibited enhanced synergistic inhibitory effects on survival and proliferation of DLBCL cells. The mTOR pathway is also considered to control the translation of specific mRNA species, some of which are involved in cell cycle control and angiogenesis (e.g. cyclin D1, c-Myc and HIF-1a). LBH alone down-regulated expression of c-Myc and HIF-1a, while have no effect on cyclin D1 expression. Combination of LBH with rapamycin further decreased expression of c-Myc and HIF-1a. Overall these results indicate that pharmacological inhibition of the mTOR pathway by rapamycin and LBH interferes with essential survival and proliferating pathways in DLBCL cells. We propose that the rapamycin-induced functional blockade of Akt and eIF4E is inhibited by LBH, and the combination increased the anti tumor activity of rapamycin. In summary, our data provide a mechanistic basis for enhancing mTORtargeted cancer therapy by combining an mTOR inhibitor with a HDAC inhibitor such as LBH. A phase I/II trial of this combination is planned.

PX-478, a Novel Small Molecule Inhibitor of Hypoxia Inducible Factor-1 (HIF-1) Downregulates HIF and Induces Cytotoxicity in Diffuse Large B Cell Lymphoma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2713-2713 ◽  
Author(s):  
Savita Bhalla ◽  
Leo I. Gordon ◽  
Amareshwar Singh ◽  
Sairah Ahmed ◽  
Danijela Dokic ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Small Molecule ◽  
B Cell Lymphoma ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Large B Cell Lymphoma ◽  
Molecule Inhibitor ◽  
Dependent Cell ◽  
Dose Dependent

Abstract Abstract 2713 Poster Board II-689 Introduction: HIF-1 is a transcription factor that serves as a master regulator of cellular responses to hypoxia and regulates genes required for adaptation to hypoxia. Although the expression of HIF-1α subunit is constitutive, HIF-1α protein levels are regulated in response to oxygen tension. Under normoxic conditions, HIF-1α is degraded by the proteasome, and HIF-1 remains inactive. In hypoxia, HIF-1α is stabilized and forms a complex with HIF-1β that allows HIF-1 to function as a transcription factor. Thus, HIF-1α is activated only during hypoxia under normal physiologic conditions. By contrast, HIF-1α is frequently activated in cancer cells, including under normoxic conditions by oncogene products or impaired activity of tumor suppressor genes. We previously reported that there is constitutive stabilization of HIF-1α in many non-Hodgkin lymphoma (NHL) cell lines as well as among a significant fraction of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma patients (Evens et al, BJH, 2008), implicating a potential role of dysregulated HIF activation in NHL. Constitutive expression of HIF-1α enhances vascularization, increases glucose metabolism, and induces the expression of anti-apoptotic proteins in cancer tissues. HIF-1α is thought to be one of the most important molecular targets in the treatment of cancer. PX-478 is a novel small molecule inhibitor of HIF-1α being developed for the treatment of cancer. Methods: We treated several DLBCL cell lines (SUDHL4, SUDHL6, and SUDHL10) with increasing concentrations (2–25μM) of PX-478 for 4 hours followed by 20-hour incubation under normoxic (5% oxygen) or hypoxic (1.5% oxygen) conditions. Cell viability was assessed by MTT assay. Expression of HIF-1α and HIF-2α were measured by Western blotting after pre-incubation of cells with 5–25μM PX-478 followed by 20-hour incubation under normoxia or hypoxia. Results: Under hypoxic conditions, dose-dependent downregulation of HIF-1α protein levels were noted with complete absence of HIF-1α by 20μM. Interestingly, lower concentrations of PX-478 were needed for effective HIF-1α downregulation in normoxia. Of note, there was no change in HIF-2α protein levels observed in either normoxic or hypoxic conditions. In cell viability studies, time- and dose-dependent cell death of PX-478 was documented in all cell lines with an associated IC50 of 15–20μM (figure below). Conclusion: Our observations suggest that the novel small molecule HIF-1α inhibitor, PX-478, effectively downregulates HIF-1α protein at low concentrations and induces cell death in DLBCL cells. Further studies to elucidate the mechanisms of HIF-1α dependent cell death in lymphoma and the associated novel therapeutics to target this pathway are warranted. Disclosures: Gordon: Cure Tech: Membership on an entity's Board of Directors or advisory committees.

Anti-Apoptotic Bcl2 and Bcl-Xl Proteins Involved in Stroma-Induced Drug Tolerance to Hsp70 and Hsp90 Inhibitors in Leukemia and B-Cell Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4900-4900
Author(s):  
Eloisi Caldas Lopes ◽  
Fabian M Correa ◽  
Ling-Bo Shen ◽  
Jae-Hung Shieh ◽  
Tony Taldone ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Hsp90 Inhibitor ◽  
Large B Cell Lymphoma ◽  
Induced Apoptosis ◽  
Large B Cell

Abstract Abstract 4900 Background: Multiple studies have demonstrated that the bone marrow stromal microenvironment contributes to the survival of hematologic malignant cells, eventually leading to relapse. However, molecular mechanisms associated with this stromal niche remain unclear. The human bone marrow stromal cell lines, HS-5 and HS-27, provide physical contact with hematologic cells, while HS-5 cells secrete more growth factors and cytokines than HS-27 stromal cells. Our objective is to dissect the mechanisms underlying stromal-mediated drug tolerance in leukemia and lymphoma cells, which could potentially lead to novel therapies for various leukemia. Methods and Results: A panel of leukemia and B-cell lymphoma cell lines were used in this project, including Kasumi1 (AML: Acute Myeloid Leukemia) and OCILy1 (DLBCL: Diffuse Large B-Cell Lymphoma) cells and their respective sub-lines resistant to heat shock protein-70 and −90 (HSP70/90) inhibitors. To determine the ability of stromal cell lines to confer tolerance to HSP-inhibitors, Kasumi1 and OCILy1 (sensitive and resistant) cells were cultured alone or in the presence of the HS-27 or HS-5 cells with HSP70 inhibitor or HSP90 inhibitor for 48h. The resulting cultures were then harvested and analyzed for apoptosis and by western blot. Both HS-5 and HS-27 stromal cells markedly protected OCILy1 and Kasumi1 cells from HSP70 inhibitor induced apoptosis. At a dose of 0.5 μM, % apoptotic cells were 74.0±1.6% for OCILy1 alone, 38.3±2.1% for OCILy1 with HS-5 and 42.2±1.8% for OCILY1 with HS-27. At a dose of 1 μM of HSP90 inhibitor, apoptosis rate are 61.9±1.5% for OCILy1 alone, 28.2±2.2% for OCILy1 with HS-5 and 36.4±1.9% for OCILy1 with HS-27. A similar HSP inhibitor induced apoptosis was also observed in Kasumi1 cells. In contrast, both Kasumi1 and OCILy1 HSP70/90 inhibitor resistant sub-lines in the presence or absence of the stromal cells did not respond to treatment with respective inhibitors. Further study reveals the stromal cells up-modulated the expression of the anti-apoptotic proteins Bcl2 and Bcl-xL in both Kasumi and OCILY1 cells. Conclusions: Our results demonstrate that the stromal niche is able to mediate tolerance to HSP70 and HSP90 inhibitors in Leukemia and B-cell lymphoma via up-regulation of antiapoptotic proteins such as Bcl2 and Bcl-xL. The Bcl2 protein is deregulated and plays a crucial role in diffuse large B-cell lymphoma (DLBCL) with the t(14;18) translocation. Our finding elucidates one of the drug-specific mechanisms that suggest a promising combination therapy targeting both HSP70 and HSP90 to reduce antineoplastic resistance and relapse, and thereby improve survival for patients with leukemia and lymphoma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals BTK and PI3K Inhibitors Reveal Synergistic Inhibitory Anti-Tumoral Effects in Canine Diffuse Large B-Cell Lymphoma Cells

2021 ◽  
Vol 22 (23) ◽  
pp. 12673
Author(s):  
Weibo Kong ◽  
Sina Sender ◽  
Leila Taher ◽  
Simon Villa-Perez ◽  
Yixuan Ma ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Morphological Changes ◽  
B Cell Lymphoma ◽  
Dependent Manner ◽  
Combined Application ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Bruton’s tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3β, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL.

scholarly journals ASB2 is a direct target of FLI1 that sustains NF-κB pathway activation in germinal center-derived diffuse large B-cell lymphoma

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Giulio Sartori ◽  
Sara Napoli ◽  
Luciano Cascione ◽  
Elaine Yee Lin Chung ◽  
Valdemar Priebe ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Transcriptional Level ◽  
Rna Seq ◽  
Protein Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background Diffuse large B-cell lymphoma (DLBCL) comprises at least two main biologically distinct entities: germinal center B-cell (GCB) and activated B-cell (ABC) subtype. Albeit sharing common lesions, GCB and ABC DLBCL present subtype-specific oncogenic pathway perturbations. ABC DLBCL is typically characterized by a constitutively active NF-kB. However, the latter is seen in also 30% of GCB DLBCL. Another recurrent lesion in DLBCL is an 11q24.3 gain, associated with the overexpression of two ETS transcription factors, ETS1 and FLI1. Here, we showed that FLI1 is more expressed in GCB than ABC DLBCL and we characterized its transcriptional network. Methods Gene expression data were obtained from public datasets GSE98588, phs001444.v2.p1, GSE95013 and GSE10846. ChIP-Seq for FLI1 paired with transcriptome analysis (RNA-Seq) after FLI1 silencing (siRNAs) was performed. Sequencing was carried out using the NextSeq 500 (Illumina). Detection of peaks was done using HOMER (v2.6); differential expressed genes were identified using moderated t-test (limma R-package) and functionally annotated with g:Profiler. ChIP-Seq and RNA-Seq data from GCB DLBCL cell lines after FLI1 downregulation were integrated to identify putative direct targets of FLI1. Results Analysis of clinical DLBCL specimens showed that FLI1 gene was more frequently expressed at higher levels in GCB than in ABC DLBCL and its  protein levels were higher in GCB than in ABC DLBCL cell lines. Genes negatively regulated by FLI1 included tumor suppressor genes involved in negative regulation of cell cycle and hypoxia. Among positively regulated targets of FLI1, we found genes annotated for immune response, MYC targets, NF-κB and BCR signaling and NOTCH pathway genes. Of note, direct targets of FLI1 overlapped with genes regulated by ETS1, the other transcription factor gained at the 11q24.3 locus in DLBCL, suggesting a functional convergence within the ETS family. Positive targets of FLI1 included the NF-κB-associated ASB2 a putative essential gene for DLBCL cell survival. ASB2 gene downregulation was toxic in GCB DLBCL cell lines and induced NF-κB inhibition via downregulation of RelB and increased IκBα. Additionally, downregulation of FLI1, but not ASB2, caused reduction of NF-κB1 and RelA protein levels. Conclusions We conclude that FLI1 directly regulates a network of biologically crucial genes and processes in GCB DLBCL. FLI1 regulates both the classical NF-κB pathway at the transcriptional level, and the alternative NF-κB pathway, via ASB2. FLI1 and ASB2 inhibition represents a potential novel therapeutic approach for GCB DLBCL.

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