Elevated Serum IL-10 Levels in Patients with Diffuse Large B Cell Lymphoma: A Mechanism of Aberrant JAK2 Kinase Activation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 960-960
Author(s):  
Mamta Gupta ◽  
Mary Stenson ◽  
Jing Jing Han ◽  
Matthew J Maurer ◽  
Linda Wellik ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Elevated Serum ◽  
B Cell Lymphoma ◽  
Cytokine Signaling ◽  
Dependent Manner ◽  
Kinase Activation ◽  
Stat3 Phosphorylation ◽  
Dose Dependent

Abstract Abstract 960 Diffuse large B cell lymphoma (DLBCL) is an aggressive lymphoma whose survival depends on various signaling pathways one of which is Signal transducer and activator of transcription 3 (STAT3)/Janus kinase 2 (JAK2). We have demonstrated that JAK2 is constitutively activated at the auto-phosphorylation site in many cases of DLBCL. The most common mechanism causing abnormal JAK2 activation is through dysregulated cytokine signaling. Cytokines are deregulated in several hematological malignancies and may play a role in tumor cell growth through receptor-mediated and ligand dependent activation of the JAK2 kinase. In this study we investigated the role of cytokine signaling in JAK2 activation in DLBCL and use of a novel JAK2 inhibitor (JAK2i) to inhibit IL-10 induced JAK/STAT signaling. We studied serum cytokines from 70 patients with new untreated DLBCL who participated in a clinical trial in the North Central Cancer Treatment Group (Micallef, IN et al Blood 2011). Compared to control sera, patients with DLBCL had higher levels of several JAK2 kinase related cytokines (IL-2, IL-6, IL-10, and EGF). IL-10 and IL-6 were significantly higher in DLBCL patients vs controls with p values >0.03 and >0.001, respectively. We next examined whether DLBCL cell lines produced IL-10. The phospho-JAK2 (pJAK2) positive DLBCL cell lines Ly10, DHL2 and HBL1 produced more IL-10 (40–80 pg/ml) than the pJAK2 negative cell lines Ly1, Ly19 and DHL6 (0–5 pg/ml). Analysis of the surface expression of IL-10 receptors (IL-10R) determined that most p-JAK2 positive DLBCL cells express either IL-10Ra or IL-10Rb or both. IL-10 had no effect on DLBCL cell survival in vitro; however, it did promote their proliferation. Only IL-10 (but not the other elevated cytokines) was specifically able to activate JAK2 and STAT3 phosphorylation in a subgroup of DLBCL cell lines. IL-10 was not able to activate JAK1, STAT1 and STAT5, which suggests a specific role of IL-10 in the JAK2 pathway. Moreover, neutralizing antibody to IL-10 inhibited IL-10-induced JAK2 and STAT3 tyrosine phosphorylation. We studied the effect of SAR302503 (SAR503, Sanofi, Cambridge, MA), a selective JAK2i currently in clinical trial for myelofibrosis on constitutive JAK2 signaling in DLBCL cells. SAR503 was able to inhibit JAK2 and STAT3 phosphorylation in a dose and time dependent manner in a variety of DLBCL cell lines and patient samples. JAK2 inhibition with SAR2503 caused a dose-dependent inhibitory effect on survival of pJAK2 positive DLBCL cells not observed in pJAK2 negative cells. Activation of STAT3 signaling up-regulates the expression of various genes involved in cell survival and proliferation such as bcl-xl, bcl-2, mcl-1 and c-myc. We observed a dose-dependent decrease in c-myc protein and mRNA levels in Ly3 and DHL2 cells with SAR503 treatment; however, SAR503 did not effect expression of bcl2, mcl-1 and bcl-xl proteins in DLBCL cells. Interestingly, JAK2 inhibition with SAR503 inhibited the autocrine secretion of IL-10 in a dose-dependent manner. Next, we determined if higher pre-treatment serum IL-10 correlates with the overall survival of 81 DLBCL patients. IL-10 by ELISA demonstrated that IL-10 levels were high in 51% (41/81) of patients (median, 57.7pg/ml; range, 26.1–503.7), and low in 49% (40/81) (median, 15.9 pg/ml; range, 0–25.9). Using a cut-off value of 26.1pg/ml, patients with a high serum IL-10 level had a significantly worse event-free survival (p=0.01). Clinical correlation of serum IL-10 with disease parameters showed that the IL-10 level was correlated with elevated serum LDH (p= 0.0085) and IPI score (p=0.01); there was no correlation with the number of extranodal sites, age, or B symptoms. DLBCL tumors from 40 patients were classified into germinal center B-cell type (GCB) and non-GCB type using the Hans algorithm. There was a clear trend towards a higher serum IL-10 in non-GCB DLBCL patients (p <0.06). In summary, our data identifies mechanisms of JAK2 kinase activation in DLBCL and supports the hypothesis that the IL-10-JAK2-cmyc pathway is activated in patients with DLBCL. Finally our data provide a mechanistic basis of selecting and targeting DLBCL tumor cells with high IL-10 levels and/or constitutive JAK2 activity with potent and novel JAK2 inhibitors such as SAR503. Disclosures: No relevant conflicts of interest to declare.

The xCT Antiporter Is a Therapeutic Target in the ABC Subtype of DLBCL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3996-3996
Author(s):  
Xiaolei Wei ◽  
Yun Mai ◽  
Ru Feng ◽  
B. Hilda Ye
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
Dependent Manner ◽  
Ros Accumulation

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in the adult population and can be subdivided into two main subtypes, i.e. GCB-DLBCL and ABC-DLBCL. While both subtypes are derived from normal germinal center (GC) B cells, they differ in B cell maturation stage, transformation pathway, and clinical behavior. When treated with either the combination chemotherapy CHOP or the immuno-chemotherapy R-CHOP, the survival outcome of ABC-DLBCL patients is typically much worse than that of GCB-DLBCL patients. Although the molecular mechanisms underlying this survival disparity remain poorly understood, an attractive hypothesis is that there exist subtype-specific resistance mechanisms directed against the chemo-therapy drugs in the original CHOP formulation. In support of this notion, our previous study has revealed that Doxorubicin (Dox), the main cytotoxic ingredient in CHOP, has subtype-specific mechanisms of cytotoxicity in DLBCLs due to differences in its subcellular distribution pattern. In particular, Dox-induced cytotoxicity in ABC-DLBCLs is largely dependent on oxidative stress rather than DNA damage response. Based on these findings, we hypothesize that agents capable of disturbing the redox balance in ABC-DLBCL cells could potentiate the therapeutic activity of first line lymphoma therapy. As the major route of cystine uptake from extracellular space, the xCT cystine/glutamate antiporter controls the rate-limiting step for glutathione (GSH) synthesis in several types of cancer cells, including CLL. We focused the current study on xCT because its protein stability is known to be positively regulated by a splicing variant of CD44 and we have recently published that expression of CD44 and CD44V6 are poor prognosticators for DLBCL. Indeed, we found that surface CD44 is exclusively expressed in ABC-DLBCL (6/6) but not GCB-DLBCL (0/5) cell lines. In addition, the xCT proteins in two ABC-DLBCL cell lines, Riva and SuDHL2, are extraordinarily stable, with half-lives exceeding 24 hours. As such, transient transfection using siRNA oligos was ineffective in reducing the endogenous xCT protein in ABC-DLBCL cell lines. To circumvent this issue, we turned to a clinically approved anti-inflammatory drug, sulfasalazine (SASP), which is a validated xCT inhibitor in its intact form. When Riva and SuDHL2 cells were treated overnight with the IC50 dose of SASP, the endogenous GSH pool was drastically reduced, leading to significant increase in intracellular ROS, p38 and JNK activation, and progressive apoptosis. Unexpectedly, we found that Dox-treated cells had significantly elevated GSH levels, possibly the result of an antioxidant response to Dox-triggered ROS accumulation. This increase in GSH was completely suppressed when the IC25 dosage of SASP was included in the Dox treatment. As expected, SASP/Dox combination significantly enhanced Dox-triggered ROS accumulation and synergistically promoted cell death in Riva and SuDHL2 cells. Mechanistically, p38 activation and cell death induced by SASP/Dox combination could be markedly attenuated by pretreatment with glutathione monoethyl ester, demonstrating the critical role of oxidative stress. Furthermore, cytotoxicity triggered by SASP/Dox could also be suppressed by the p38 inhibitor, SB203580. We have developed stable cell lines expressing xCT shRNA to confirm the results obtained with SASP. In vivo interactions between SASP and Dox are also being evaluated in xenograft-based ABC-DLBCL models. In summary, we report here for the first time a critical role of xCT in sustaining in vivo GSH production in ABC-DLBCL cells. More importantly, pharmacologic inhibition of xCT function in ABC-DLBCL cells not only prevented Dox-induced endogenous GSH increase, but also potentiated Dox-induced ROS accumulation and cytotoxicity in a p38-dependent manner. With additional evidence from ongoing experiments, our study aims to provide a mechanistic basis for development of novel therapies that target either xCT or redox homeostasis to improve treatment outcomes for ABC-DLBCLs. Disclosures No relevant conflicts of interest to declare.

Activation Of TAK1 By MYD88 L265P Drives Malignant B Cell Growth In Non-Hodgkin Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 245-245
Author(s):  
Stephen M Ansell ◽  
Lucy S. Hodge ◽  
Frank Secreto ◽  
Michelle Manske ◽  
Esteban Braggio ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Wild Type ◽  
Dose Dependent

Abstract Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHL) including the rare lymphoplasmacytic lymphoma, Waldenström’s macroglobulinemia (WM). Using whole exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). MYD88L265P was detected at lower frequencies in other indolent lymphomas including LPL (0%), MALT (4%), nodal MZL (5%) and splenic MZL (8%); all but one MYD88L265P was heterozygous. Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B cell survival, therapeutic targeting of MYD88 signaling pathways may be useful clinically. However, while the effects of MYD88L265P on the activity of IRAK1/4 and NF-κB are have been studied previously, we are lacking a thorough characterization of the role of intermediary signaling proteins such as TRAF6 and TAK1 on the biology of MYD88L265P-expressing B cells. A better understanding of the proteins involved in MYD88L265P signaling may lead to the development of more targeted and effective therapeutic approaches. In an attempt to identify MYD88L265P –specific therapeutic targets we first wanted to characterize the role of intermediary signaling proteins that facilitate the downstream activation of NF-κB. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodimer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-κB. We monitored the formation of a complex comprised of MYD88, IRAK1, IRAK4 and TRAF6 and immunoprecipitation of either endogenous IRAK4 or IRAK1 revealed constitutive association of IRAK with TRAF6 and MYD88L265P. To assess if the formation of a MYD88L265P/IRAK/TRAF6 complex results in downstream activation of TAK1, constitutive TAK1 phosphorylation was measured and detected in all three cell lines that express MYD88L265P. An association between TAK1 and TRAF6, another measure of TAK1 activation, was also detectable. When a similar analysis of TAK1 was performed in DLBCL cells expressing wild-type MYD88, no phosphorylation of TAK1 was detected, nor was TAK1 associated with TRAF6. IRAK1, IRAK4, TAK1, TRAF6, and MYD88 were expressed at similar levels in all cell lines studied and therefore did not contribute the differences in MYD88 complex formation observed between cell lines. These studies were further confirmed using HEK 293T cells that were transduced with either a vector control plasmid or HA-tagged MYD88WT or MYD88L265P expression plasmids. Together, these studies suggest that MYD88L265P forms a complex with IRAK and TRAF6 resulting in constitutive activation of TAK1 and NF-κB. To confirm the significance of TAK1-mediated MYD88L265P signaling on lymphoma cell growth, the effect of the selective TAK1 inhibitor, (5Z)-7-Oxozeaenol, on cell proliferation was tested. All MYD88L265P-expressing cell lines were sensitive to TAK1 inhibition in a dose-dependent manner (0-10 μM). In contrast, NHL cells expressing MYD88WT were found to be insensitive to TAK1 inhibition. We next tested the impact of the TAK1 inhibitor on a MYD88L265P positive WM patient sample. Similar to what was seen in the WM cell lines, the TAK1 inhibitor inhibited WM cell growth and survival in a dose dependent manner. Additionally, the TAK1 inhibitor significantly reduced the level of IL-10 secreted by each of the cell lines. Together, these data suggest that MYD88L265P drives cell proliferation and cytokine secretion through a TAK1-dependent mechanism. In conclusion, we are the first to validate by NGS in a large patient cohort the high prevalence and specificity of MYD88L265P in WM. Cells harboring the L265P mutation but not wild-type MYD88 exhibit constitutive signaling leading to the hyperactivation of NF-κB. We have established the role of TAK1 as an integral component of MYD88L265P signaling in both WM and DLBCL cell. Our data suggest that targeting TAK1 clinically may be an effective strategy for the treatment of WM and other lymphomas driven by MYD88L265P signaling. Disclosures: Fonseca: millennium: Consultancy; amgen: Consultancy; Binding site: Consultancy; onyx: Consultancy; medtronic: Consultancy; Genzyme: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; lilly: Consultancy; Onyx: Research Funding; cylene: Research Funding.

scholarly journals B-myb antisense oligonucleotides inhibit proliferation of human hematopoietic cell lines

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2708-2716 ◽  
Author(s):  
M Arsura ◽  
M Introna ◽  
F Passerini ◽  
A Mantovani ◽  
J Golay
Keyword(s):  
Cell Lines ◽  
Antisense Oligonucleotides ◽  
Hematopoietic Cell ◽  
Dependent Manner ◽  
Myb Gene ◽  
Myb Genes ◽  
Dose Dependent ◽  
Lymphoid Cell Lines ◽  
Hematopoietic Cell Lines

Abstract The B-myb gene is highly homologous to the c-myb protooncogene in several domains and also shares some of the functions of c-myb in that it can act as a transcriptional activator. In addition, the expression of both the B-myb and c-myb genes correlates with proliferation of normal hematopoietic cells. We investigated more directly the role of B- myb in proliferation of hematopoietic cell lines using B-myb-specific antisense oligonucleotides. We showed that several anti-B-myb oligonucleotides, complementary to distinct regions of the gene, inhibit significantly and in a dose-dependent manner the proliferation of all myeloid or lymphoid cell lines tested. This block in proliferation was not accompanied by detectable differentiation of U937 or HL60 cells to macrophages or granulocytes either spontaneously or after exposure to chemical agents. These data suggest that the B-myb gene, like c-myb, is necessary for hematopoietic cell proliferation.

Mutations of the Tumor Suppressor Gene SOCS-1 in Classical Hodgkin Lymphoma Are Frequent and Associated with Nuclear Phospho-STAT5 Accumulation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 19-19 ◽  
Author(s):  
Marc A. Weniger ◽  
Ingo Melzner ◽  
Christiane K. Menz ◽  
Silke Wegener ◽  
Alexandra J. Bucur ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Tumor Suppressor Gene ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Suppressor Gene ◽  
Cytokine Signaling ◽  
Sequencing Analysis ◽  
Classical Hodgkin Lymphoma

Abstract The suppressors of cytokine signaling (SOCS) are critically involved in the regulation of cellular proliferation, survival, and apoptosis via cytokine-induced JAK/STAT signaling. SOCS-1 silencing by aberrant DNA methylation contributes to oncogenesis in various B-cell neoplasias and carcinomas. Recently, we showed an alternative loss of SOCS-1 function due to deleterious SOCS-1 mutations in a major subset of primary mediastinal B-cell lymphoma (PMBL) and in the PMBL line MedB-1, and a biallelic SOCS-1 deletion in PMBL line Karpas1106P (BLOOD, 105, 2535–42, 2005). For both cell lines our previous data demonstrated retarded JAK2 degradation and sustained phospho-JAK2 action leading to enhanced DNA binding of phospho-STAT5. Here we analysed SOCS-1 in laser-microdissected Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). We detected SOCS-1 mutations in HRS cells of eight of 19 cHL samples and in three of five Hodgkin lymphoma (HL)-derived cell lines by sequencing analysis. Moreover, we found a significant association between mutated SOCS-1 of isolated HRS cells and nuclear phospho-STAT5 accumulation in HRS cells of cHL tumor tissue (p<0.01). Collectively, these findings support the concept that PMBL and cHL share many overlapping features, and that defective tumor suppressor gene SOCS-1 triggers an oncogenic pathway operative in both lymphomas.

Janus Kinase (JAK)-2 Does Not Play a Role in Bcr-Abl Signaling in Philadelphia Chromosome (Ph)-Positive (p190) Acute Lymphoblastic Leukemia (ALL) Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4241-4241
Author(s):  
Stefan H. Faderl ◽  
Quin Van ◽  
Patricia E. Koch ◽  
David M. Harris ◽  
Inbal Hallevi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Janus Kinase ◽  
Myelogenous Leukemia ◽  
Dependent Manner ◽  
Western Immunoblotting ◽  
Gm Csf ◽  
Dose Dependent

Abstract Novel immunochemotherapy regimens combined with imatinib mesylate (IA) have significantly improved treatment outcome of Ph+ ALL. Nevertheless, most adult patients with Ph+ ALL relapse and succumb to their disease. Recent reports suggested that Jak-2 is engaged in the signaling of Bcr-Abl in chronic myelogenous leukemia (CML) cells. Because Jak-2 inhibitory agents are currently investigated in clinical trials, we sought to explore the role of Jak-2 in the signaling of Bcr-Abl in Ph+ ALL assuming that inhibition of Jak-2 might be beneficial in the treatment of Ph+ ALL. To do this, we used our Ph+ (p190) ALL cell lines Z-119 and Z-181 (Estrov et al. J Cell Physiol166: 618, 1996). We chose these cells because in both lines Jak-2 can be activated. Both Z-119 and Z-181 cells express granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors and GM-CSF activates Jak-2 and stimulates the proliferation of both cell lines. Using a clonogenic assay, we found that IA inhibited the proliferation of these cells at concentrations ranging from 50 to 500 nM. Because Bcr-Abl was found to activate the signal transducer and activator of transcription (STAT)-5 in CML cells, we used Western immunoblotting and found that IA inhibited the phosphorylation (p) of STAT5 in a dose-dependent manner in Ph+ ALL cells. To test whether JAk-2 plays a role in Bcr-Abl (p190) signaling we incubated Z-181 cells for 4 hours with or without 50, 100, 250, and 500 nM IA, extracted cellular protein and immunoprecipitated total STAT5 protein. Then, using Western immunoblotting we detected the Bcr-Abl p190 protein in all STAT5 immunoprecipitates and by using specific pSTAT5 antibodies, we demonstrated that IA induced a dose-dependent reduction in the levels of pSTAT5, but not of p190 protein, suggesting that the p190 Bcr-Abl kinase binds to and activates STAT5. Remarkably, neither Jak-2 nor pJak-2 was detected in either immunoprecipitate. To further delineate the role of Jak-2 in Bcr-Abl signaling we extracted protein from Z-181 cells and immunoprecipitated Jak-2. Neither Bcr-Abl nor STAT5 was detected in these immunoprecipitates, confirming that Jak-2 does not bind Bcr-Abl p190 protein and does not participate in the activation of STAT5. Taken together, our data suggest that Bcr-Abl (p190) binds and phosphorylates STAT5 whereas, Jak-2 is not engaged in Bcr-Abl (p190) signaling in Ph+ ALL cells.

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.

Disruption Of Super Enhancer-Driven Cancer Dependencies In Diffuse Large B-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3021-3021 ◽  
Author(s):  
Bjoern Chapuy ◽  
McKeown Michael ◽  
Charles Y. Lin ◽  
Stefano Monti ◽  
Margaretha GM Roemer ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Dana Farber Cancer Institute ◽  
Super Enhancer ◽  
Equity Ownership

Abstract Diffuse large B-cell lymphoma (DLBCL) exhibits significant biological and transcriptional heterogeneity which is conferred, in part, by pathologic modulation of lineage-specific and growth-associated master regulatory transcription factors (TF). Chromatin associated with TF binding sites is markedly enriched in histone proteins that are post-translationally modified by lysine side-chain acetylation. This mark facilitates the opening of chromatin and recruits a class of co-activators which recognize ε-acetyl lysine through a bromodomain. The sub-family of bromodomain and extra-terminal domain (BET) co-activators (BRD2, BRD3 and BRD4) are appealing, in part, because transgenic expression of BRD2 caused a DLBCL-like neoplasm in mice. We recently developed the first BET inhibitor, JQ1, and now explore the role of BET bromodomains in oncogenic transcription and assess BET family members as therapeutic targets in DLBCL. Nanomolar doses of JQ1 and 3 structurally dissimilar BET bromodomain inhibitors decreased the cellular proliferation of a broad panel of DLBCL cell lines of all transcriptionally defined types whereas the inactive enantiomer, JQ1R, had no effect. BRD2 and BRD4 depletion similarly decreased the proliferation of multiple DLBCL cell lines. We next explored the therapeutic potential of BET inhibition in two independent DLBCL xenotransplantation models, Ly1 and Toledo. In the first xenograft model, JQ1-treated mice had a prolongation of overall survival (p = 0.003). In the second model, JQ1-treated animals had significantly delayed tumor progression and decreased lymphomatous infiltration of spleen and bone marrow. To define the transcriptional pathways regulated by BET bromodomain proteins, we performed transcriptional profiling of multiple vehicle and JQ1-treated DLBCL cell lines. Following JQ1 treatment, we observed downregulation of multiple MYD88/TLR and BCR signaling pathway components and functionally validated MYC and E2F target gene sets. BET inhibition decreased MYC transcripts and protein in the DLBCL cell line panel suggesting that BET bromodomains directly modulate MYC transcription. In contrast, JQ1 treatment did not measurably alter E2F1 transcript or protein abundance suggesting a co-activator role of the BET bromodomains for E2F1. To explore the role of BET bromodomains in oncogenic E2F1 transcriptional signaling, we performed ChIPSeq experiments in Ly1 cells, using a chemical genetic approach. Rank-ordering of all transcriptionally active promoters based on H3K4me3 enrichment and RNA Pol II occupancy identifies pervasive binding and spatial colocalization of BRD4 and E2F1 to active promoter elements. We identified a JQ1-mediated transcriptional elongation defect across E2F1-bound promoters, responsible for the downregulation of E2F1 targets. As oncogenic TFs may signal to RNA Pol II through distal enhancer elements, we also characterized the genome-wide localization of BRD4 to enhancers in the Ly1 DLBCL cell line. Rank-ordering of enhancer regions by H3K27ac enrichment reveals that BRD4 binds to the vast majority of active enhancers in the Ly1 genome. Strikingly, the BRD4 load is asymmetrically distributed throughout the genome at enhancer sites with only a small subset of BRD-loaded “super enhancers (SE)”, 285/18330 (1.6%), accounting for 32% of all BRD4 enhancer binding in the cell. The POU2AF1 locus emerged as the most BRD4-overloaded enhancer in Ly1. BET inhibition reduced RNA Pol II elongation of POU2AF1, with a concomitant increase in promoter-paused RNA Pol II near the transcriptional start site. Accordingly, JQ1 treatment decreased POU2AF1 transcript abundance and protein expression and reduced the expression of a POU2AF1 target gene set. POU2AF1 depletion with independent shRNAs significantly decreased the proliferation of Ly1 and enforced POU2AF1 expression decreased the sensitivity of Ly1 cells to JQ1 treatment. Additional super enhancer-driven genes that were sensitive to JQ1 treatment include ones which promote and maintain the B-cell gene expression program and limit plasma cell differentiation. Our data suggest that BET inhibition limits the growth of DLBCLs by at least two complementary activities: a specific effect on genes that define a given cell type by high BRD4 loading at enhancers and the selective suppression of transcription at E2F- and MYC- driven target genes. + Contributed equally Disclosures: Qi: Patent for JQ1: holds patent for JQ1, holds patent for JQ1 Patents & Royalties. Young:Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Enzon Pharmaceuticals: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Bradner:Tensha Therapeutics: Equity Ownership, Scientific founder of Tensha which is translating drug-like derivatives of the JQ1 chemical probe of BET bromodomains used in this study, as cancer therpeutics. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity. Other; Syros Pharmaceuticals: Equity Ownership, Scientific founder of Syros which is discovering Super Enhancers as a new class of gene control elements. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity., Scientific founder of Syros which is discovering Super Enhancers as a new class of gene control elements. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity. Other.

scholarly journals Role of the microRNA-7 (miR-7) in the Inhibition of Chemoresistance and Migration of Lymphoma Non-Hodgkin's through Regulation of YY1 and KLF4

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5220-5220 ◽  
Author(s):  
Mario Morales-Martinez ◽  
Gabriel G Vega ◽  
Natividad Neri Munoz ◽  
M. J Nambo ◽  
Isabel Alvarado ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
B Cell Lymphoma ◽  
Negative Regulation ◽  
Negative Regulator ◽  
Induced Expression ◽  
Klf4 Expression ◽  
And Migration

Since the discovery and description of microRNAs (miRs), these molecules have become very important, specifically due to their participation in the regulation of proteins and transcription factors involved in the development of cancer. The microRNA-7 (miR-7) has been described as a negative regulator of several proteins involved in cancer such as YY1 and KLF4 in various cancer subtypes. We have recently reported that YY1 and KLF4 play a role in Non-Hodgkin Lymphoma (NHL) and that the expression of KLF4 is regulated by YY1 (Valencia-Hipolito 2014, Morales-Martinez 2019). Therefore, in this study we analyzed the role of miR-7 in NHL through its effect on the negative regulation of YY1 and KLF4 in vitro, and also assessed its expression in clinical specimens from lymphoma patients. Expression of KLF4, YY1 and miR-7 was analyzed used real-time PCR in NHL-B cell lines (Ramos, Raji, DHL4 and 2F7). The expression of KLF4 and YY1 was inverse correlated with miR-7 expression, where Raji has higher miR-7 expression and DHL4 lower, and this correlated with inverse expression of YY1 and KLF4 in both cell lines, respectively. The possible regulation of YY1 and KLF4 by miR-7 was analyzed by using the inducible expression or inhibition of miR-7, (transfection with pre-miR-7 or mimetic miR-7 respectively) as well using reporter-system plasmids containing the 3 'UTR region of YY1 or KLF4. The role of miR-7 in NHL, through the negative regulation of YY1 and KLF4 was determined by chemoresistance/proliferation, and migration assays. The clinical implications of miR-7 in the negative regulation of YY1 and KLF4, were analyzed by ISH and IHC in a TMA with 43 samples of NHL subtypes: DLBCL and Follicular Lymphoma (FL). Our results showed an inverse correlation of miR-7 expression with KLF4 and YY1 expression in B-NHL cell lines, miR-7 is able to regulate the expression of YY1 and KLF4 by direct binding in the 3 'UTR region. Thus, the induced expression of miR-7 inhibited the constitutive expression of YY1 / KLF4, whereas the inhibition of miR-7 correlated with an increase in the expression of YY1 / KLF4. Likewise, the induced expression of miR-7 reverses the chemo resistance and inhibits the migration capacity of NHL-B cell lines. Also, all tumor tissues expressing miR-7 demonstrated a negative correlation with YY1 and KLF4 expression, which was more significant in the FL. Additionally the expression of miR-7 in FL was associated with clinical outcome. Our results show for the first time that miR-7 has a role in NHL through the negative regulation of YY1 and KLF4. These results confirm YY1 and KLF4 as possible therapeutic targets through the regulation of miR-7. References: -Valencia-Hipόlito A, et al Expression of KLF4 is a predictive marker for survival in pediatric Burkitt lymphoma. Leuk Lymphoma. 2014;55(8):1806-14 -Morales-Martinez M, Valencia-Hipolito A, Vega GG, Neri N, Nambo MJ, Alvarado I, Cuadra I, Duran-Padilla MA, Martinez-Maza O, Huerta-Yepez S, Vega MI. Regulation of Krüppel-Like Factor 4 (KLF4) expression through the transcription factor Yin-Yang 1 (YY1) in non-Hodgkin B-cell lymphoma. Oncotarget. 2019;10(22):2173-2188. Disclosures No relevant conflicts of interest to declare.

scholarly journals Klotho/IGF-1R Regulates Tumor Growth and Predicts Prognosis in Diffuse Large B-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2924-2924
Author(s):  
Xiangxiang Zhou ◽  
Ying Li ◽  
Xinyu Li ◽  
Lingyun Geng ◽  
Ya Zhang ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Cell Lines ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
Control Group ◽  
Klotho Protein ◽  
Xenograft Models

Abstract Introduction: Klotho is an anti-aging gene with an extracellular domain. Mice with Klotho knockout exhibited obvious impairment in B-cell development. Evolving evidence indicates that Klotho modulates the proliferation and survival via targeting insulin-like growth factor-1 receptor (IGF-1R) in several cancers. However, the expression and biological role of Klotho in B-cell non-Hodgkin lymphoma (B-NHL) has not been elucidated to date. We hypothesized that Klotho could modulate the tumor growth and predicts prognosis in diffuse large B-cell lymphoma (DLBCL) through inhibiting IGF-1R activation. The aim of this study is to characterize the functional significance of Klotho and the therapeutic potential of its secreted form in DLBCL. Methods: Lymph nodes samples from 50 de novo DLBCL and 20 reactive hyperplasia cases were collected with informed consents. Klotho expression were assessed by Immunohistochemistry. CD19+ B-cells and peripheral blood mononuclear cells were isolated with informed consents from healthy donors. Expression levels of Klotho mRNA and protein in DLBCL cells were determined by quantitative RT-PCR and western blotting. Lentivirus vectors either encoding Klotho (LV-KL) or empty lentiviral vector (LV-Con) were stably transfected into DLBCL cells. Cell viability and apoptosis were analyzed by cell counting kit-8 and Annexin V-PE/7AAD staining. Animal experiments were performed in accordance with the principles of the Institutional Animal Care. SCID-Beige mice were subcutaneously injected with DLBCL cells to establish xenograft model. Results: We observed markedly decreased level of Klotho protein in DLBCL lymph nodes (Fig. 1A). Expression of Klotho protein exhibited significantly negative correlation with Ann Arbor stage of DLBCL patients (p=0.002). Level of Klotho protein was negatively correlates with the media overall survival (OS), suggesting lower Klotho expression is associated with poor OS in DLBCL ((Fig. 1B, p=0.045). Reduction of Klotho was also confirmed in DLBCL cell lines at mRNA and protein level (Fig. 1C). We next functionally interrogated the role of Klotho in DLBCL cell lines and xenograft models. Stably expression of LV-KL in DLBCL cell lines resulted in dramatically decreased cell proliferation and incremental apoptotic rates when compared to LV-Con (Fig. 2A and B). We validated the changed expression of critical targets known to govern apoptosis in DLBCL cells transfected with LV-KL. Xenograft models with Klotho overexpression revealed significantly abrogated tumor growth compared to control group (Fig. 2C). Interestingly, lower levels of Ki67 were observed in mice treated with LV-KL (Fig. 2D). These results highlighted the proliferation-inhibitory and apoptosis-inductive activities of Klotho in DLBCL cells. The underlying mechanism driving the tumor suppressive potential of Klotho was investigated. Surprisingly, we observed that the Klotho-induced inhibition of cell viability was only fewer restored by IGF-1 in DLBCL cells transfected with LV-KL (Fig. 3A). Reductive phosphorylation of IGF-1R and its downstream targets (AKT and ERK1/2) were observed in DLBCL cells with Klotho overexpression (Fig. 3B). In addition, we evaluated the regulation of Klotho on IGF-1R signaling in vivo. Decreased phosphrolation of IGF-1R as well as its downstream targets were observed in mice treated with LV-KL compared to the control group (Fig. 3C). Lastly, we explored the activity of secreted Klotho protein (rhKL). The rhKL was found to be active in vitro and significantly reduced the viabilities of DLBCL cells (Fig. 3D). Moreover, combination with rhKL increased the sensitivity of DLBCL cells to adriamycin. The in vivo activity of rhKL in DLBCL xenograft model was also detected. Significantly decreased tumor volumes were noted in mice treated with rhKL compared with those treated with vehicle control (Fig. 3E). Moreover, reductive expression level of Ki67 was observed in rhKL-treated group (Fig. 3F). Conclusions: Our observations identified for the first time that loss of Klotho expression contributed to the development and poor prognosis via activating IGF-1R in DLBCL. Given the in vivo tumor suppressive activity of secreted Klotho protein, it may serve as a potential strategy for the development of novel therapeutic interventions for DLBCL. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Unveiling the Role of MicroRNA155 in Diffuse Large B Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2024-2024
Author(s):  
Deepak Rai ◽  
Shailaja Karanti ◽  
Patricia Dahia ◽  
Ricardo C.T. Aguiar
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
B Cell ◽  
Cell Lines ◽  
Mhc Class Ii ◽  
B Cell Lymphoma ◽  
Tissue Cell ◽  
Transcription Analysis ◽  
Mrna Cleavage

Abstract MicroRNAs (miR) are non-coding RNAs that regulate gene expression by pairing to 3UTRs of target genes inducing translational repression or mRNA cleavage. New evidence suggests that the latter mechanism markedly contributes to miRNA effects. Hence, global gene expression analyses may help elucidate the functional role of miRNA by recognizing pathways modified by their abnormal expression and identifying direct targets. MiR155, the product of the non coding gene BIC, is overexpressed in lymphomas and its role in tumorigenesis is supported by the development of B-cell malignancies in miR155 transgenic mice. However, the functional consequences of miR155 overexpression in tumor development remain unclear. To address this issue, we developed a semi-quantitative RT-PCR assay that specifically amplifies either the nuclear unspliced BIC mRNA (target of the RNase III Drosha) or the spliced BIC mRNA. We found a marked correlation between the expression levels of these two mRNAs, which in turn agreed with the levels of mature miR155 detected in northern blots. Of the 22 DLBCL cell lines studied, only 5 (DHL6, Ly3, Ly10, Farage, RCK-8) expressed significantly high levels of BIC and miR155. To isolate the effects of miR155 in DLBCL we genetically modified its expression and performed global transcription analysis on microarray. In brief, we cloned the BIC transcript in a MSCV-GFP bicistronic retrovirus and confirmed in transduced HeLa cells that the mature miR155 was expressed when this transcript was driven by an LTR promoter. Next, we used two DLBCL cell lines with low levels of miR155 (Ly8 and Ly19) to generate unique populations expressing miR155 or MSCV alone. RNA was isolated from GFP-sorted cells, hybridized to the Affymetrix U133Plus2.0 chip and the data analyzed with dChip. Remarkably, and in agreement with the role of miRNAs, supervised analysis (fold diff >1.7) revealed a vast predominance (>90%) of downregulated genes when comparing miR155 expressing cells to MSCV only. These gene groups included predicted miR155 targets and were significantly enriched for molecules involved in the immune response (p<.001), including MHC class II, chemokine receptors, TDT, NFAT and CD24, a particularly relevant target for miR155 inhibition since its activation induces apoptosis in lymphomas. To validate and extend our findings, we queried public expression datasets of primary DLBCL. First, we used our cell lines expression data to confirm that the BIC probe in the Affymetrix chip reliably reflected the expression of miR155. Since DLBCL entails at least two groups of tumors reflecting distinct normal B-cells (GC and ABC) and the expression of miRNA and its targets are highly tissue/cell specific, we compartmentalized these analyses within the groups of ABC and GC tumors. We selected the tumors within each group with the highest and lowest levels of BIC (20% percentile) and performed unsupervised hierarchical clustering analysis (filtering parameters 0.5<SD/mean<10). In agreement with our data in the GC-type miR155-expressing cell lines, we found that in GC-, but not in ABC tumors, the expression of BIC inversely correlated with that of the genes related to the immune response. Notably, the pro-apoptotic molecule CD24 was significantly downregulated (p<0.02) in BIC overexpressing primary DLBCL, underscoring the need for further characterization of the signals relayed by this surface molecule and its potential as a rational drug target. Our data start to delineate the effects of miR155 in DLBCL and show the potential of expression arrays to identify miRNA targets modified by mRNA cleavage.

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