SIRT6 As a Potential Therapeutic Target in Diffuse Large B-Cell Lymphoma

Abstract Introduction Sirt6 is a member of the mammalian sirtuins (SIRTs) family, involved in diverse biological processes. It is dysregulated in various solid tumors and multiple myeloma. Sirt6 can be considered a double-edged sword in cancer, as its dual role of both an oncogene and a tumor suppressor. However, no literature has been reported regarding the functional significance of Sirt6 in diffuse large B-cell lymphoma (DLBCL) to date. Thus we evaluated the expression and function of Sirt6 in both DLBCL tissues and cell lines in this present study, as well as assessed the proliferation of DLBCL cells when treated with a pan-sirtuin inhibitor Nicotinamide (NAM) and a selected Sirt6 inhibitor, OSS_128167. Methods Microarray datasets GSE83632 and GSE32918 were obtained from Gene Expression Omnibus. Differential expression analysis in gene expression profiles GSE83632 were performed through classical Bayesian method provided by the Limma package and then extracted the SIRT6 gene expression value. GSE32918 was annotated with the R-package illumineHumanWGDASLv3.db data probe, converted into gene symbol, and extracted expression data of Sirt6 for survival analysis. Immunohistochemistry (IHC) was conducted to assess the expression of Sirt6 on araffin-embedded tissues from 60 de novo DLBCL patients (study group) and 30 reactive hyperplasia patients (control group) with informed contents. Then further analysis was performed to find the correlations between Sirt6 expression and the clinical characteristics of DLBCL patients. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers with informed consents. Expression levels of Sirt6 mRNA and protein in DLBCL cell lines (LY1, LY8, LY3 and Val) and PBMCs (control) were detected by quantitative RT-PCR and western blotting. Lentivirus vectors transfected DLBCL cells to stably knockdown or overexpress Sirt6. Apoptosis was assessed by PE/7AAD. DLBCL cell lines were treated with NAM and OSS_128167, cell proliferation was analyzed by cell counting kit (CCK-8). Results Bioinformatics analysis showed that SIRT6 was significantly up-regulated in DLBCL than in normal samples. High Sirt6 expression in DLBCL was turned up to be correlate with shorter overall survival according to GSE32918 (Figure 1A-B). These data indicated a relevant role for Sirt6 in the biology of DLBCL. We observed markedly increased level of Sirt6 in DLBCL lymph nodes by IHC. Expression of Sirt6 was positively correlated with Ann Arbor stage and international prognostic index (IPI) score of DLBCL patients. Aberrantly overexpression of Sirt6 was also confirmed in DLBCL cell lines at mRNA and protein level (Figure 2A-C). Our findings on Sirt6 were consistent with those from publicly available data sets. Sirt6 knockdown caused increased apoptosis rate, and on the contrary, Sirt6 over-expression had brought about decreased apoptosis (Figure 3A-C). We then investigated the function of Sirt inhibitors on the proliferation of DLBCL cells. LY1 and Val cells were treated with different doses of NAM at 24-72 hours. Cell proliferation was inhibited in a dose-dependent manner. LY1 and LY8 cells were treated with different doses of OSS_128167 at 24-72 hours and triggered similar effect, cell proliferation was inhibited by 60% at the concentration of 100µM for 48 hours (Figure 4A-B). Conclusion Our investigations identified for the first time that expression levels of Sirt6 were up-regulated in DLBCL and associated with adverse outcome by bioinformatics analysis and in vitro experiments. Knockdown of Sirt6 lead to increased apoptosis and over-expression of Sirt6 had the opposite effect. DLBCL cells treated with pan-Sirt inhibitor and selective Sirt6 inhibitor, revealed reduced proliferation in a dose-dependent manner. This study suggests that Sirt6 could be a potential molecular target for the treatment of DLBCL. Further research on it is under way. Disclosures No relevant conflicts of interest to declare.

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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 ◽

10.1182/blood.v116.21.115.115 ◽

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.

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

Blood ◽

10.1182/blood-2018-99-112006 ◽

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.

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Targeting Glycosylated Ptgds Displays Anti-Tumor Activities in Diffuse Large B-Cell Lymphoma through Down-Regulation of Wnt Pathway

Blood ◽

10.1182/blood-2020-139027 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 13-15

Author(s):

Shunfeng Hu ◽

Xiangxiang Zhou ◽

Juan Yang ◽

Shuai Ren ◽

Yiqing Cai ◽

...

Keyword(s):

Cell Proliferation ◽

Western Blotting ◽

Cell Apoptosis ◽

Wnt Pathway ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Dependent Manner ◽

Large B Cell Lymphoma ◽

Dose Dependent ◽

Large B Cell

Introduction: Prostaglandin D2 Synthase (PTGDS), a member of lipocalin superfamily, plays a dual role in catalyzing the conversion of PGH2 to PGD2 and transporting lipophilic substances. PTGDS protein is with different degrees of glycosylation. AT56 is a selective, competitive, and highly bioavailable inhibitor of PTGDS. However, the function and mechanism of PTGDS in diffuse large B-cell lymphoma (DLBCL) remains ill defined. Herein, we aimed to investigate the functional significance of PTGDS and proposed a novel therapeutic strategy for DLBCL. Methods: Lymph node biopsies from 53 de novo DLBCL patients and 28 reactive hyperplasia cases, and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were collected with informed consents. CD19+ B cells were purified by CD19+ magnetic microbeads. The expression levels of PTGDS in DLBCL cell lines and serum were detected by western blotting and ELISA, respectively. Lentivirus vectors were transfected to stably knockdown or overexpress PTGDS. After the treatment of Tunicamycin (Tun), an N-glycosylation inhibitor, western blotting and immunofluorescence were performed to validate the molecular weight and location of PTGDS protein. Results: We first evaluated the expression of PTGDS in DLBCL. Upregulation of PTGDS mRNA in DLBCL cells was identified based on Oncomine database (Fig.1A). Then, the expression level of PTGDS protein in tumor tissue (n=53) was validated to be higher in comparison with control group (Fig.1B). Furthermore, survival analysis revealed that PTGDS high expression was associated with reduced overall survival of DLBCL patients (Fig.1C). Moreover, high level of soluble PTGDS protein was detected in the serum of DLBCL patients (n=53, Fig.1D). High expression of PTGDS was also confirmed in DLBCL cells by western blotting (Fig.2A). The biological function of PTGDS in progression of DLBCL was further verified. Gene ontology and KEGG analysis revealed that PTGDS was enriched in cellular process and biological regulation (Fig.2B). After the treatment with rhPTGDS, increased proliferation of DLBCL cell was observed in a dose-dependent manner (Fig.2D), and overexpression of PTGDS also promoted cell growth (Fig.2E). Furthermore, knockdown of PTGDS (shPTGDS) significantly restrained cell proliferation (Fig.2F), promoted cell cycle arrest (Fig.2G) and cell apoptosis (Fig.2H). AT56 suppressed the proliferation of DLBCL cells in a dose- and time-dependent manner (Fig. 3A). Additionally, with the treatment of AT56, DLBCL cells exhibited induced G0/G1 phase arrest (Fig. 3B) and increased cell apoptosis (Fig. 3C). As Bendamustine and Adriamycin were found to decrease the mRNA level of PTGDS (Fig. 3D), we further observed that AT56 enhanced sensitivity to them in cell proliferation (Fig. 3E) and cell apoptosis (Fig. 3F). Next, we explored the underlying mechanism of PTGDS in DLBCL progression. The expression of Wnt pathway molecules, such as p-LRP6, β-catenin, p-GSK3-β, Lef-1, p-STAT3, were decreased with treatment of AT56 in dose-dependent manner (Fig. 4A). Besides, STAT3 inhibitor WP1066 was found to restore the proliferation promotion (Fig. 4B) caused by PTGDS overexpression. Moreover, we validated that Wnt pathway activator Wnt3a could restore the phenotype changes caused by AT56, including cell proliferation (Fig. 4C), cell apoptosis (Fig. 4D) and cell cycle (Fig. 4E). Glycosylation, a kind of post-translational modification, could significantly alter protein function and then cellular characteristics. Protein Blast analysis indicated the potential glycosylation sites (Fig. 5A) on PTGDS protein (Ser29, Asn51 and Asn78). Furthermore, Tunicamycin was used to inhibit the N-glycosylation of protein and molecular weight of PTGDS changed from 37kD to 21kD (Fig.5B). Besides, the deglycosylation could promote the translocation of PTGDS into the nucleus (Fig.5C-D), indicating the potential role of glycosylated PTGDS in DLBCL. Conclusions : Our investigations identified for the first time the aberrant overexpression of PTGDS in DLBCL, which was associated with poor prognosis. AT56 exerted anti-tumor effect via inhibiting Wnt pathway. Besides, PTGDS protein in DLBCL cells were highly glycosylated and deglycosylation promoted its translocation into nucleus, indicating the mechanism of PTGDS in DLBCL. Further investigation is warranted to substantiate PTGDS as a promising therapeutic target in DLBCL. Disclosures No relevant conflicts of interest to declare.

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P–543 Inhibition of cell proliferation and extracellular matrix formation in human uterine leiomyomas by 5-aza–2’-deoxycitidine via Wnt/ β-catenin pathway

Human Reproduction ◽

10.1093/humrep/deab130.542 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

M C Carbajo-García ◽

A Corachán ◽

M Segura ◽

J Monleón ◽

J Escrig ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Extracellular Matrix ◽

Cell Proliferation ◽

Protein Expression ◽

Hormonal Treatment ◽

Dependent Manner ◽

Dnmt Inhibitors ◽

Qrt Pcr ◽

Dose Dependent

Abstract Study question Is DNA methylation reversion through DNA methyltransferases (DNMT) inhibitors, such as 5-aza–2’-deoxycitidine, a potential therapeutic option for treatment of patients with uterine leiomyomas (UL)? Summary answer 5-aza–2’-deoxycitidine reduces proliferation and extracellular matrix (ECM) formation by inhibition of Wnt/ β-catenin pathway on UL cells, suggesting DNMT inhibitors as an option to treat UL. What is known already: UL is a multifactorial disease with an unclear pathogenesis and inaccurate treatment. Aberrant DNA methylation have been found in UL compared to myometrium (MM) tissue, showing hypermethylation of tumor suppressor genes, which contributes to the development of this tumor. The use of DNMT inhibitors, such as 5-aza–2’-deoxycytidine (5-aza-CdR), has been suggested to treat tumors in which altered methylation pattern is related to tumor progression, as occurs in UL. Based on this, we aimed to evaluate whether DNA methylation reversion through 5-aza-CdR reduces cell proliferation and ECM formation in UL cells, being a potential option for UL medical treatment. Study design, size, duration Prospective study comparing UL versus MM tissue and human uterine leiomyoma primary (HULP) cells treated with/without 5-aza-CdR at 0 µM (control), 2 µM, 5 µM and 10 µM for 72 hours. UL and MM tissue were collected from women without any hormonal treatment for the last 3 months (n = 16) undergoing myomectomy or hysterectomy due to symptomatic leiomyoma pathology. Participants were recruited between January 2019 and February 2020 at Hospital Universitario y Politecnico La Fe (Spain). Participants/materials, setting, methods Samples were collected from Caucasian premenopausal women aged 31–48 years, with a body mass index of < 30 and without hormonal treatment. DNMT1 gene expression was analysed in UL vs MM tissue by qRT-PCR and activity of DNMT was measured in UL and MM tissue and cells by ELISA. 5-aza-CdR effect on proliferation was assessed by CellTiter test and Western blot (WB), apoptosis and ECM analyzed by WB and Wnt/ β-catenin pathway by qRT-PCR and WB. Main results and the role of chance: DNMT1 gene expression was increased in UL compared to MM tissue (fold change [FC]=2.49, p-value [p]=0.0295). Similarly, DNMT activity was increased in both UL compared to MM tissue and HULP cells versus MM cells (6.50 vs 3.76 OD/h/mg, p = 0.026; 211.30 vs 63.67 OD/h/mg, p = 0.284, respectively). After 5-aza-CdR treatment, cell viability of HULP cells was reduced in a dose dependent manner, being statistically significant at 10 µM (85.25%, p = 0.0001). Accordantly, PCNA protein expression was significantly decreased at 10 µM in HULP cells (FC = 0.695, p = 0.034), demonstrating cell proliferation inhibition. Additionally, 5-aza-CdR inhibited ECM protein expression in HULP cells in a dose-dependent manner being statistically significant at 10 µM for COLLAGEN I (FC = 0.654, p = 0.023) and PAI–1 (FC = 0.654, p = 0.023), and at 2 µM and 10 µM for FIBRONECTIN (FC = 0.812, p = 0.020; FC = 0.733, p = 0.035; respectively). Final targets of Wnt/ β-catenin pathway were decreased after 5-aza-CdR treatment, protein expression of WISP1 was significantly inhibited at 10 µM (FC = 0.699, p = 0.026), while expression levels of Wnt/ β-catenin target genes C-MYC (FC = 0.745, p = 0.028 at 2 µM; FC = 0.728, p = 0.019 at 10 µM) and MMP7 (FC = 0.520, p = 0.003 at 5 µM, FC = 0.577, p = 0.007 at 10 µM) were also significantly downregulated in HULP-treated cells vs untreated cells. Limitations, reasons for caution: This study has strict inclusion criteria to diminish epigenetic variability, thereby we should be cautious extrapolating our results to general population. Besides, this is a proof of concept with the inherent cell culture limitations. Further studies are necessary to determine 5-aza-CdR dose and adverse effects on UL in vivo. Wider implications of the findings: 5-aza-CdR treatment reduces cell proliferation and ECM formation through Wnt/ β-catenin pathway inhibition, suggesting that inhibition of DNA methylation could be a promising new therapeutic approach to treat UL. Trial registration number Not applicable

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Hif-1α Inhibitors Could Successfully Inhibit the Progression of Differentiated Thyroid Cancer in Vitro

Pharmaceuticals ◽

10.3390/ph13090208 ◽

2020 ◽

Vol 13 (9) ◽

pp. 208

Author(s):

Min-Hee Kim ◽

Tae Hyeong Lee ◽

Jin Soo Lee ◽

Dong-Jun Lim ◽

Peter Chang-Whan Lee

Keyword(s):

Cell Proliferation ◽

Thyroid Cancer ◽

Cell Lines ◽

Cancer Progression ◽

Hypoxia Inducible Factor ◽

Soft Agar ◽

Dependent Manner ◽

Thyroid Cancer Cell Lines ◽

Dose Dependent

Hypoxia-inducible factor (HIF)-1α plays an important role in cancer progression. In various cancers, including thyroid cancer, overexpression of HIF-1α is related to poor prognosis or treatment response. However, few studies have investigated the role of HIF-1α inhibition in thyroid cancer progression. We evaluated the utility of the HIF-1α inhibitor IDF-11774 in vitro utilizing two thyroid cancer cell lines, K1 and BCPAP. Both cell lines were tested to elucidate the effects of IDF-11774 on cell proliferation and migration using soft agar and invasion assays. Here, we found that a reduction of HIF-1α expression in BCPAP cells was observed after treatment with IDF-11774 in a dose-dependent manner. Moreover, cell proliferation, migration, and anchorage-independent growth were effectively inhibited by IDF-11774 in BCPAP cells but not in K1 cells. Additionally, invasion of BCPAP but not K1 cells was controlled with IDF-11774 in a dose-dependent manner. Our findings suggest that promoting the degradation of HIF-1α could be a strategy to manage progression and that HIF-1α inhibitors are potent drugs for thyroid cancer treatment.

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PIM as a Rational Target for B-Cell Lymphomas.

Blood ◽

10.1182/blood.v114.22.3946.3946 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3946-3946

Author(s):

Cristina Gomez-Abad ◽

Helena Pisonero ◽

Juan F Leal ◽

Giovanna Roncador ◽

Jose A. Martinez-Climent ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Specific Inhibitor ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Micromolar Range ◽

Large B Cell ◽

Stat Pathway

Abstract Abstract 3946 Poster Board III-882 INTRODUCTION The Pim kinases are a family of serine/threonine kinases composed by three members: Pim1, Pim2 and Pim3, involved in the phosphorylation and regulation of several proteins that are essential for cell cycle progression, metabolism or apoptosis (BAD, p21, p27KIP, AKT, Mdm2 and cMyc, among them). Overexpression, translocation or amplification of Pim family have been described in many human cancers, including B-cell Non Hodgkin's Lymphoma, Multiple Myeloma, Prostate cancer and Pancreatic cancer. In addition, 50% of patients diagnosed with diffuse large B-cell Lymphoma (DLBCL) present somatic mutations in Pim1. Despite of its important role in cancer progression, very few chemical inhibitors have been described in the literature, being effective all of them in the high micromolar range. PURPOSE Validating PIM as a rational therapeutic target in B-cell lymphoma, developing tools for patient stratification and pharmacodynamic studies on PIM inhibition. MATERIAL AND METHODS Gene expression profiling and Copy Number data were obtained from a series of 94 B-cell Non-Hodgkin Lymphoma patients (DLBCL, FL, MALT, MCL and NMZL). The effect of Pim inhibition was checked on cell lines by using a novel specific inhibitor for the Pim family (ETP-39010). Newly produced antibodies and RT-PCR primers and protocols were standarized. RESULTS Gene expression data revealed high Pim isoforms expression in a subset of patients with Mantle cell lymphoma (MCL), and Diffuse Large B-cell lymphoma (DLBLC)-ABC type. CGH analysis focused on chromosomal regions containing Pim family and its main regulatory upstream pathway (JAK/STAT) was performed. Heterozygous gains of Pim1 (6p21.2) and Pim3 (22q13.33) were identified in 13.6% of DLBCL patients and in 4.2% of MCL. Alterations in JAK/STAT pathway were also detected in 59.1% of DLBCL patients, and 37.5% of MCL patients presented any alteration in JAK/STAT pathway, being frequent losses of JAK2 chromosomal region. Analysis of additional pathways involved in the up-stream regulation of Pim family disclosed heterozygous gains of PIK3C3 in 40.9% of DLBCL patients, and gains of PIK3CA in 45.9% of MCL patients. Lymphoma cell lines (15) derived from both MCL (9) and ABC-DLBLC (6) subtype, have been analyzed by qRT-PCR and Western-blot, showing variable expression levels of Pim1, Pim2 and Pim3. IC50 obtained for the ETP-39010 compound is in the low micromolar range for the MCL (0.7-8.7 micromolar) and DLBCL-ABC (0.8-10.3 micromolar) cell lines. Since Pim kinase family phosphorilate multiple sites of Bad and AKT, we have checked the inhibition of its phosphorilation as molecular biomarkers for the ETP-39010 effect. Our data show an inhibition of at least 20% of pBad (S112) and almost a complete inhibition of pAKT (S473) 4h after treatment. In addition, cell cycle arrest at G1 and induction of apoptosis were observed 24h after the treatment. CONCLUSION Pim family genes are a rational therapeutic target in MCL and DLBCL-ABC lymphoma subtypes. Stratification and pharmacodynamic markers have been developed for PIM inhibition using a novel specific inhibitor compound -ETP-39010-. Disclosures: No relevant conflicts of interest to declare.

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Inhibition Of PI3K Classia Kinases Using GDC0941 Overcomes Protection Of Multiple Myeloma Cells In The Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v122.21.3169.3169 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3169-3169

Author(s):

Hugh Kikuchi ◽

Amofa Eunice ◽

Maeve McEnery ◽

Farzin Farzaneh ◽

Stephen A Schey ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Proliferation ◽

Bone Resorption ◽

Cell Lines ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Pi3k Pathway ◽

Dependent Manner ◽

Dose Dependent

Abstract Despite of newly developed and more efficacious therapies, multiple myeloma (MM) remains incurable as most patient will eventually relapse and become refractory. The bone marrow (BM) microenvironment provides niches that are advantageous for drug resistance. Effective therapies against MM should ideally target the various protective BM niches that promote MM cell survival and relapse. In addition to stromal mesenchymal/myofibroblastic cells, osteoclasts play a key supportive role in MM cell viability. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity. Increased osteoclast activity is characteristic in these patients and targeting osteoclast function is desirable to improve therapies against MM. Osteoclasts need to form an F-actin containing ring along the cell margin that defines a resorbing compartment where protons and degradative enzymes are secreted for dissolution of bone mineral. Remodelling of F-actin and vesicle secretion are regulated by the class IA PI3K pathway during osteoclastic bone resorption. Additionally, it has recently been shown that inhibition of the class IA PI3K pathway in MM cells with GDC0941 induces apoptosis-mediated killing. We hypothesised that GDC0941 could be used as a therapeutic agent to overcome MM-induced osteoclast activation. GDC0941 inhibited maturation of osteoclasts derived from BM aspirates from MM patients in a dose dependent manner. This correlated with decreased bone resorption of osteoclasts cultured on dentine discs. Exposure of mature osteoclasts to GC0941 resulted in abnormal organisation of larger F-actin rings, suggesting a negative effect on the dynamics of the actin cytoskeleton required for bone resorption. We also found that GDC-0941 can prevent protection of the MM cell lines MM1.S and MM1.R by osteoclasts against killing. GDC-0941 alone blocked MM cell proliferation independently of the presence of BM stromal cells and synergised with other therapeutic agents including Lenalidomide, Pomalidomide, Bortezomid and Dexamethasone. We also found that in the presence of MM cells, Dexamethasone (a drug commonly used alone or in combination with new drugs against MM) induced the proliferation of BM stromal cells and adhesion of MM cells on this protective stroma in a dose dependent manner. Dexamethasone is highly effective at MM cell killing when cells are cultured alone. However, we found that at low doses (below 1 uM) and in the presence of BM stromal cells, Dexamethasone could induce MM cell proliferation. GDC0941 enhanced Dexamethasone killing even in the presence of BM stromal cells by blocking Dexamethasone-induced stromal cell proliferation and adhesion of MM cells on the stroma. Targeting individual the PI3K Class IA isoforms alpha, beta, delta or gamma proved to be a less efficient strategy to enhance Dexamethasone killing. Previous work has shown that efficacy of targeting individual PI3K Class I A isoforms would be low for activation of caspases in MM cells as it would be dependent on relative amounts of isoforms expressed by the MM patient. GDC-0941 also inhibited the proliferation of MM1.R and RPMI8266 MM cell lines, which are less sensitive to treatment to Dexamethasone. Co-culture of MM cells with BM stromal cells induced the secretion of IL-10, IL-6, IL-8, MCP-1 and MIP1-alpha. The dose-dependant increased proliferation of Dexamethasone-treated MM cells in the presence of the BM stroma correlated with the pattern of secretion of IL-10 (a cytokine that can induce B-cell proliferation) and this was blocked by the combination of Dexamethasone with GDC0941. GDC-0941 alone or in combination with Dexamethasone was more efficacious at inducing MM cell apoptosis in the presence of the BM stroma cells vs treatment of MM cells alone. These are very encouraging results as they suggest that GDC-0941 in combination with Dexamethasone would be potentially highly efficacious for targeting MM cells in the BM microenvironment. We are currently performing in vivo data using C57BL/KaLwRij mice injected with 5T33-eGFP MM cells that will be discussed at the meeting. We propose that MM patients with active bony disease may benefit from treatment with GDC0941 alone or in combination with currently used therapeutic drugs against MM. Disclosures: No relevant conflicts of interest to declare.

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MicroRNA Expression Profiling of Diffuse Large B-Cell Lymphoma Reveals Differences between Germinal Center-Like and Activated B Cell-Like Subtypes.

Blood ◽

10.1182/blood.v106.11.3002.3002 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3002-3002

Author(s):

Charles H. Lawrie ◽

Shamit Soneji ◽

Christopher D. Cooper ◽

Chris Hatton

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Expression Profile ◽

Germinal Center ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Large B Cell ◽

Clinical Cases

Abstract MicroRNAs (miRNA) are a recently discovered class of short non-coding RNA molecules that negatively regulate gene expression. They have been shown to play a critical role in many biological functions. In humans about 320 miRNAs have been identified, some of which are expressed in a cell-specific and developmental stage-specific manner. Recently it has been shown that the expression profile of miRNAs can be used to subtype clinical cases (and cell lines) according to diagnosis with a greater degree of accuracy than traditional gene expression analysis. The identity of miRNAs associated with different lymphoma types however remains poorly defined. Previous expression studies have revealed the presence of at least two subtypes of diffuse large B-cell lymphoma (DLBCL) representing the postulated cell of origin; those that are germinal center B cell derived (GCB-type) and those that are activated B-cell derived (ABC-type). The latter subtype has been linked with poor prognostic outcome. It is not known whether these subtypes are also defined at the miRNA level. Therefore we examined the miRNA expression profile of DLBCL cell lines of defined subtypes as well as sub-populations of B-lymphocytes by microarray analysis. Consistent with recent publications, we found that mir-19a, 19b and 17-5p (part of mir-17-92 cluster) were up-regulated in cell lines but not in normal lymphocyte populations. Furthermore, cluster analysis showed that GCB-type cell lines (SUD-HL4, SUD-HL6 & SUD-HL10) have a distinct miRNA profile from ABC-type cell lines (OCI-Ly3 & OCI-Ly10). Most notably, high levels of expression of mir-155, mir-181b and mir-325 were found in ABC-type cell lines whilst high levels of mir-181a were found in GCB-type cell lines. We looked at expression of mir-155, 181a, 143, 145, 378 and 16 in these cell lines as well as clinical cases of DLBCL by RNase-protection assay. Consistent with the microarray data, we found that mir-155 was expressed in ABC-type cell lines but not GCB-type cell lines whilst the converse was true for mir-181a. Clinical cases showed similar patterns of expression but have still to be sub-typed according to immunohistochemical markers. Although still preliminary, our data suggests that miRNA profiling may be a useful tool in predicting the subtype of DLBCL cases and hence clinical outcome.

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Inhibition of Human Plasmacytoma Cell Growth by a Novel JAK Kinase Inhibitor.

Blood ◽

10.1182/blood.v104.11.644.644 ◽

2004 ◽

Vol 104 (11) ◽

pp. 644-644

Author(s):

Renate Burger ◽

Steven Legouill ◽

Yu-Tzu Tai ◽

Reshma Shringarpure ◽

Klaus Podar ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Growth ◽

Cell Lines ◽

Hormone Receptors ◽

Critical Role ◽

Myeloma Cell ◽

Thymidine Uptake ◽

Dependent Manner ◽

Jak Kinase ◽

Dose Dependent

Abstract Novel strategies in cancer therapy aim at inhibiting distinct signal transduction pathways that are aberrantly activated in malignant cells. Protein tyrosine kinases of the JAK family are associated with a number of cytokine and cytokine-like hormone receptors and regulate important cellular functions such as proliferation, survival, and differentiation. Constitutive or enhanced JAK activation has been implicated in neoplastic transformation and abnormal cell proliferation in various hematological malignancies. In multiple myeloma (MM), JAK kinases play a critical role because of their association with cytokine receptors of the IL-6/gp130 family. A novel small-molecule inhibitor was developed that shows a 100 to 1,000-fold selectivity for JAK1, JAK2, JAK3, and TYK2 relative to other kinases including Abl, Aurora, c-Raf, FGFR3, GSK3b, IGF-1R, Lck, PDGFRa, PKBb, and Zap-70. Growth of MM cell lines and primary patient cells was inhibited by this compound in a dose-dependent manner. The IL-6 dependent cell line INA-6 and derived sublines were sensitive to the drug, with IC50’s of less than 1 mM, in [3H]-thymidine uptake and a colorimetric, tetrazolium compound (MTS) based assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI). Importantly, INA-6 and patient tumor cell growth was also inhibited in the presence of bone marrow stromal cells, which by themselves remained largely unaffected. Growth suppression of INA-6 correlated with a significant and dose-dependent increase in the percentage of apoptotic cells, as evaluated by Apo2.7 staining after 48 hours of drug treatment. In addition, the compound blocked IL-6 induced phosphorylation of STAT3, a direct downstream target of JAK kinases and important transcription factor triggering anti-apoptotic pathways. In other myeloma cell lines, the drug overcame the protective effect of gp130 cytokines on dexamethasone induced apoptosis. In MM1.S cells, it completely blocked IL-6 induced phosphorylation of SHP-2 and AKT, both known to mediate the protective effects of IL-6. In contrast, AKT phosphorylation induced by IGF-1 remained unchanged, demonstrating selectivity of the compound. These studies show that disruption of JAK kinase activity and downstream signaling pathways inhibits myeloma cell growth and survival as well as circumvents drug resistance, thereby providing the conceptual basis for the use of JAK kinase inhibitors as a novel therapeutic approach in MM.

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

Blood ◽

10.1182/blood.v108.11.829.829 ◽

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 < .0001) whereas the constitutively expressed β isoform was increased in BCR DLBCLs (p < .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.

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