Chemo-Resistance in Diffuse Large Cell Lymphoma: Novel Drug Combinations Targeting NFAT/NF-Kb Growth/Survival/Chemo-Resistance Signaling Pathways in Validated Novel Experimental Systems

Abstract Abstract 1428 Acquired chemo-resistance (ACR) is currently the most important cause of treatment failure and early mortality in DLBCL, arguably the most important unmet need in lymphoma therapy today. Diffuse Large B cell Lymphoma (DLBCL), the most common human lymphoma, comprises a genetically and clinically diverse group of aggressive B cell non-Hodgkin lymphomas (NHL-B), among a small group of important human cancers increasing in incidence in the US over the last four decades. NHL-B are the fifth most common cancers in the USA (>62,000 new cases/20,000 deaths) expected in 2011. The molecular biologic and genetic basis of the patho-physiology of these important lymphoid tumors is still mostly unresolved. This is due primarily to the lack of valid patho-biologic experimental models allowing for identification of the key patho-physiologic molecular/genetic mechanisms involved in chemo-resistance, resulting in mostly unsuccessful empiric new drug salvage trials, rather than efficient drug-targeting key growth/survival/chemo-resistance (GSC) pathways essential for effective salvage therapies. We have been developing such novel translational experimental DLBCL systems (>25 DLBCL cell lines derived from relapsed DLBCL patients) and novel agents as the conceptual basis of this model. We have distinguished a set of cell lines that are more resistant to chemo-therapy and identified that the transcription factor p52 component of the alternative NF-kB pathway is highly expressed in DLBCL cell lines that show the highest chemo-resistance characteristics. Down-regulation of p52 sensitizes resistant cells to chemotherapy. This is of particular interest since previous studies have not as yet established definitive role(s) for the alternative NF-kB pathway, particularly p52, in chemo-resistance development. We have discovered that the second generation proteasome inhibitor, Carfilzomib can target the alternative NF-kB-p52 pathway by down-regulating the TNF-receptor family BAFF-R, resulting in lymphoma cell growth inhibition and apoptosis induction. NFATc1, another important multifunctional regulatory molecule (transcription factor (TF), chromatin remodeler, etc), that we have shown to be intrinsically involved with NF-kBs in most DLBCL, and whose involvement in DLBCL is becoming increasingly important on multiple levels, that was recently confirmed genetically, identifying NFATc1 expression as a candidate oncogene in ABC DLBCLs. We have also discovered that GSK3b, a key upstream natural inhibitor of NFATc1, is constitutively phosphorylated in DLBCL cells and can negatively regulate NFATc1 activation. The PKC beta II inhibitor Enzastaurin, affectively inhibits pGSK3b, leading to NFATc1 inactivation and inhibiting cell growth/survival in a broad range of DLBCL cell lines, both GCB and ABC subtypes, with IC: 50 values in the low uM ranges. Enzastaurin strongly synergizes with Carfilzomib to inhibit DLBCL cell growth and induce apoptosis, particularly in chemo-resistant DLBCL cells. Carfilzomib alone enhances pGSK3b and NFATc1 activation, while Enzastaurin abolishes CFZ-induced pGSK3b and NFATc1, suggesting a mechanism for the synergistic activity of the drugs. Novel drug combinations with agents that target multiple growth, survival, and chemo-resistance pathways, such as Carfilzomib and Enzastaurin, represent promising, emerging therapeutic options for reversing chemo-resistance in relapsed/refractory DLBCL patients. Disclosures: No relevant conflicts of interest to declare.

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NIK Is Involved In the Activation of the Classical and Alternative NF-κB Pathways In Diffuse Large B Cell Lymphoma

Blood ◽

10.1182/blood.v116.21.3099.3099 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3099-3099

Author(s):

Lina Odqvist ◽

Margarita Sánchez-Beato ◽

Santiago Montes-Moreno ◽

Ken H Young ◽

Francesco Acquadro ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Alternative Pathway ◽

B Cell Lymphoma ◽

Classical Pathway ◽

Strong Positive Correlation ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Abstract 3099 Deregulated NF-κB activity plays a role in the lymphoma pathogenesis, and has been proposed to constitute a cardinal feature of some subtypes of diffuse large B cell lymphoma (DLBCL). The NF-κB-Inducing Kinase (NIK) is essential for the activation of the alternative NF-κB pathway by inducing the phosphorylation of the NF-κB member p100, which leads to its processing to p52 and its subsequent nuclear translocation. A role for NIK in the classical NF-κB pathway as well has been shown, suggesting NIK as an attractive therapeutic target in lymphomas. Here, we study the frequency and extent of alternative and classical NF-κB activation in diffuse large B cell lymphoma, and the implication of NIK in both pathways. The activation of the classical and alternative NF-κB pathways was present in 28 and 34% of DLBCL cases respectively, as assessed by nuclear expression of p50 (classical pathway) and p52 (alternative pathway) by immunohistochemistry in a series of 301 samples. Activation of both NF-κB pathways was observed in germinal centre B-cell like (GC) and activated B-cell like (ABC) subtypes, with a slight predominance, although not significant, in ABC subtype. In contrast, the levels of p52 and p50 were significantly higher in ABC-DLBCL cell lines than those of GC subtype. The activation of both pathways was mostly overlapped and there was a strong positive correlation between nuclear p52 and p50 (p<0.001). Eighteen % of the cases expressed both p50 and p52 while only 8 and 16% expressed exclusively p50 or p52, respectively. Activation of the alternative NF-κB pathway was strongly associated with Epstein-Barr virus (EBV), since 93% of EBV+ cases expressed nuclear p52 (p<0.001). In our study, no TRAF3 deletions were detected in a panel of 25 DLBCL samples, although absence of TRAF3 was observed in one DLBCL cell line. Since NIK acts as a bottleneck in the activation of the alternative pathway but has also been described to play a role in the classical pathway, we wanted to analyze the effect of the knockdown of NIK on both pathways. Using small interference RNA in two lymphoma cell lines, we observed that the silencing of NIK had an effect on both pathways, decreasing the processing of p100 as well as p105. Taken together, our results show that the activation of NF-κB distinguishes a subset of DLBCL cases, comprising both ABC and GC subtypes, suggest a frequent overlap between the classical and alternative NF-κB pathway in DLBCL, and identify a possible role for NIK in the activation of both pathways. Disclosures: No relevant conflicts of interest to declare.

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Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽

10.1182/blood.v120.21.2417.2417 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2417-2417

Author(s):

Olga Ritz ◽

Jochen K Lennerz ◽

Karolin Rommel ◽

Karola Dorsch ◽

Elena Kelsch ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Ectopic Expression ◽

B Cell Lymphoma ◽

Proximal Promoter ◽

Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

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5-Aza-2-Deoxycytidine Regulates Wnt Beta-Catenin Pathway in B Cell Lymphoma

Blood ◽

10.1182/blood.v126.23.4810.4810 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4810-4810

Author(s):

Xinyu Li ◽

Lingyu Geng ◽

Xiangxiang Zhou ◽

Kang Lu ◽

Peipei Li ◽

...

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Cell Lines ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Beta Catenin ◽

Cell Functions ◽

Protein Expressions

Abstract Introduction: The Wnt/beta-catenin pathway is aberrantly activated in B cell lymphomas, unphosphorylated beta-catenin accumulates and translocates into the nucleus, regulates the expression of c-myc, cyclinD1 and many other target genes which govern fundamental cell functions, such as proliferation, cell cycle regulation and apoptosis. Methylation is a highlight of epigenetic regulation research which also occurred in lymphoma, but the concrete mechanism of how the demethylation drug 5-aza-2-deoxycytidine affect Wnt/beta-catenin pathway is still unknown. This study was designed to illuminate the implications on Wnt/beta-catenin pathway via demethylation 5-aza in B cell lymphoma. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from samples of 30 primary CLL patients. The PBMCs contained more than 90% of CD19+ B lymphocytes, which were detected by flow cytometry and were referred to as primary CLL cells. The activation of Wnt/beta-catenin pathway and DNMT-1 of B cell lymphoma cells lines (MEC-1, LY8, Jeko-1, Grant519, mino and sp53) and the 30 patients were detected by qPCR and western blot. The expressions of beta-catenin in 20 cases of B cell lymphoma tissues were measured by IHC. The B cell lines and PBMCs from 10 primary CLL patients were given 5-aza-2-deoxycytidine in different concentrations, the effects in the pathway and apoptosis were observed by WB and flow cytometry. Results: The expressions of beta-catenin, c-myc, cyclinD1 and DNMT-1 were aberrantly higher in all cell lines we used ( MEC-1,LY8, Jeko-1, Grant519, mino and sp53 Fig.1-A,B), most primary CLL patients (Fig.1-C), and B cell lymphoma tissues (Fig.1-D). The protein expressions of beta-catenin in MEC-1 were higer than primary CLL patients. 0, 0.5, 1.0, 2.0¦ÌM 5-aza-2-deoxycytidine were given to the B cells lines and PBMCs from primary CLL patients for 48h, beta-catenin were found accumulated, but c-myc and cyclinD1 in the downstream were reduced (Fig.2-A,B,C,D). For further understanding of aberrant accumulation ofbeta-catenin, we extracted the nuclear protein of MEC-1, nuclear beta-catenin protein expressions were found decreased and cytoplasmic were increased (Fig.2-E). After 5-aza treatment, the apoptosis rate increased and caspase pathway were activated (Fig.2-A,F). Conclusions: The enhanced expressions of beta-catenin, c-myc, cyclinD1 in the B cell lines and the B cell lymphoma samples indicated the Wnt/beta-catenin was aberrantly activated. After 5-aza treatment with the cell lines (MEC-1, Jeko-1, LY8) and primary CLL cells, the abnormal accumulation of beta-catenin protein was observed which was discrepancy with previous reports, but the decrease of c-myc and cyclinD1 suggested the pathway was inhibited, apoptosis also occurred. The increase of totalbeta-catenin protein was supposed to be an stress reaction of the 5-aza treatment, however, the redundant beta-catenin protein in B cell lymphoma was speculated to be combined with demethylated genes and resulted in dormancy of this pathway. Our results indicated that 5-aza played a demethylation role through Wnt/beta-catenin pathway in B cell lymphoma. The data are of interest in the context of epigenetic-based therapy in B cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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The Combination Of 2-DG and Metformin Inhibits The mTORC1 Pathway and Suppresses Aggressive B Cell Lymphoma Growth and Survival

Blood ◽

10.1182/blood.v122.21.1665.1665 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1665-1665 ◽

Cited By ~ 2

Author(s):

Chengfeng Bi ◽

Kai Fu ◽

Chunsun Jiang ◽

Xin Huang ◽

Wing Chung Chan ◽

...

Keyword(s):

Cell Growth ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Combined Treatment ◽

Control Cell ◽

B Cell Lymphoma ◽

Protein Translation ◽

Growth And Survival ◽

Aggressive B Cell Lymphoma

Abstract mTORC1 (mammalian target of rapamycin complex 1) plays a central role in integrating nutrient and growth factor inputs to control cell growth in all eukaryotes and is commonly deregulated in human cancers. Inhibition of mTORC1 is a promising strategy in lymphoma therapy. However, only a few drugs, such as rapamycin and its analogs (rapalogs), have been approved for treatment in a limited number of cancer types, due to their incomplete and nonspecific inhibition of mTORC1 as well as their limited effects toward the 4EBP1 pathway. 4EBP1 pathway regulates protein translation which is considered to be crucial in cancer cell survival and proliferation. In this study, we used the glycolysis inhibitor 2-deoxyglucose (2-DG) together with the mitochondrial respiratory inhibitor metformin to treat aggressive B cell lymphoma cells in vitro and in vivo. We found that the combined treatment inhibited mTORC1 and its major downstream targets, including 4EBP1. As a result, combined treatment significantly inhibited tumor cell growth and survival by the inhibition of 5’ cap-dependent translation involving lymphoma associated oncogenes such as MCL-1, BCL-XL and Cyclin D1. Moreover, the combination of 2-DG and metformin suppressed tumor growth in B cell lymphoma xenograft mouse models. Although the combined treatment dramatically decreased cellular ATP levels, mTORC1 inhibition was independent of AMPK activity but instead resulted from inhibitory effects on Rag-GTPases, which are upstream activators of mTORC1. Given that both 2-DG and metformin have been used in clinical diagnosis or treatment for decades, the combination of the two drugs hold promise as a new strategy to treat aggressive B cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

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Pre-Clinical Evaluation of a Novel DNA Crosslinking Agent, Bo-1055 in B-Cell Lymphoma

Blood ◽

10.1182/blood.v124.21.5483.5483 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5483-5483 ◽

Cited By ~ 2

Author(s):

Eloisi Caldas Lopes ◽

Fabian Correa ◽

Elizabeth Peguero ◽

Srikanth R. Ambati ◽

Jae Hung Shieh ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Crosslinking Agent ◽

B Cell Lymphoma ◽

Water Soluble ◽

Therapeutic Window ◽

Hematopoietic Stem ◽

Normal Human

Abstract Some B-cell lymphoma including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) remain incurable using conventional chemotherapeutic approaches. Therefore it is important that new treatment strategies be developed. We have evaluated the efficacy of a number of novel, third generation, DNA-directed alkylating agents that have DNA specific binding domain by linking DNA-affinic molecules to N-mustard pharmacophore via a urea, carbamate or hydrazinecarboxamide linker (Kapuriya et al. Bioorganic & Medicinal Chem. 19:471–485; 2011). One of these, the water-soluble ureidomustine BO-1055, was screened for toxicity against a panel of human lymphoma cell lines including MCL (JEKO-1, Z-138, HBL-2), DLBCL (OCILy10, OCILy19) and a spontaneous murine B-cell lymphoma. We also screened BO1055 against a panel of normal human cells including mesenchymal stromal cells (IC50 >10uM), bone marrow-derived endothelium (IC50 >10uM) lung basal epithelium, bronchial epithelium and myofibroblasts (IC50s >10uM) and purified cord blood CD34+ cells in suspension culture or colony-forming assay (for hematopoietic progenitor cells) (IC50 >10uM) and in cobblestone area-forming assay (for hematopoietic stem cells) (IC50 9.1uM). The mean IC50±SD (uM) in MCL cell lines was JEKO-1 (0.266±0.27), Z-138 (0.182±0.15), HBL2 (0.161±0.34), In DLBCL lines OCILy10 (0.117±0.21), OCILy19 (0.287±0.17) and murine B-cell lymphoma (0.463±0.39). Our results indicated that BO-1055 has a significant therapeutic window (50-100-fold) between its toxicity against human B-cell lymphomas compared to various normal human cell types. We evaluated BO-1055 cardiotoxicity in the HL-1 cardiomyocyte line and observed a 227-fold less cytotoxicity compared to Doxorubicin. Treatment with BO-1055 resulted in accumulation of cells in S-phase and up-regulation of proteins involved in DNA repair [MRE11, p-P95/NBS1 (ser343), RAD50, p-ATR (ser428)] while Bcl-6, an important B-cell lymphoma biomarker, was down-regulated. Xenograft experiments in NSG mice bearing JEKO-1 GFP/luciferase+ tumors treated with BO-1055 (30mg/kg) 3x/week showed complete tumor remission after 2 weeks of treatment as monitored by luciferase image. Our results suggest that BO-1055 has potent activity against B-cell lymphoma and present a strong rationale for its further therapeutic development as an alkylating agent due to his low toxicity against normal tissue and high toxicity against hematopoietic tumors. Disclosures No relevant conflicts of interest to declare.

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Imbalance in Alternative Splicing of MCL-1 Inhibits Apoptosis in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v124.21.5424.5424 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5424-5424

Author(s):

Nicolle H Rekers ◽

Laura M Moesbergen ◽

Nathalie J Hijmering ◽

Wim Vos ◽

Joost Oudejans ◽

...

Keyword(s):

Alternative Splicing ◽

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Apoptosis Resistance ◽

Expression Levels ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL) remains eventually fatal in 30-40% of the patients, despite intensive chemotherapy (CHOP) in combination with rituximab. A subgroup of chemotherapy-refractory DLBCL is characterized by high expression levels of both pro- and anti-apoptotic genes, including MCL-1. Alternative splicing of the MCL-1 gene results in a Bcl-2-like anti-apoptotic MCL-1L protein and a BH3-only pro-apoptotic MCL-1S protein. In the present study, we investigated if a switch in alternative splicing of MCL-1 is involved in apoptosis-resistance in primary lymphoma cells of 20 DLBCL patients and 5 DLBCL cell lines. RT-MLPA analysis revealed that MCL-1L and MCL-1S are both expressed in all tested DLBCL samples and DLBCL cell lines, however expression levels varied strongly. An imbalance between the expression levels of MCL-1L and MCL-1S to an anti-apoptotic status was observed in DLBCL patient cells and DLBCL cell lines, especially in activated B-cell like (ABC)-DLBCL, compared to tonsillar germinal center B-cells. MCL-1 mRNA expression was confirmed at protein level using immunohistochemistry and western blot analysis. Co-immunoprecipitation demonstrated that MCL-1L inhibited apoptosis by binding of Bak in MCL-1L positive DLBCL cell lines. Knockdown of MCL-1L with siRNA analysis resulted in induction of apoptosis in both GCB- and ABC-DLBCL cell lines and also in increased sensitivity to the conventional chemotherapeutical drugs etoposide. Downregulation of MCL-1L using flavopiridol induced apoptotic cell death of MCL-1L-positive DLBCL cells with low Bcl-2 expression. In summary, a switch in alternative splicing of MCL-1 occurs in a subgroup of DLBCL leading to an increase in the level of anti-apoptotic MCL-1L that contributes to therapy-resistance. These preclinical data suggest that targeting of MCL-1L might be a therapeutic option for MCL-1L positive DLBCL. Disclosures No relevant conflicts of interest to declare.

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Inhibition of Fatty Acid Synthase Suppresses c-Met Receptor Kinase and Induces Apoptosis in Diffuse Large B Cell Lymphoma.

Blood ◽

10.1182/blood.v114.22.4787.4787 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4787-4787

Author(s):

Shahab Uddin ◽

Azhar Hussain ◽

Prashant Bavi ◽

Khawla Al-Kuraya

Keyword(s):

Fatty Acid ◽

B Cell ◽

Cell Lines ◽

Fatty Acid Synthase ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Specific Chemical ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Abstract 4787 Targeted approaches are expected to revolutionize cancer treatment in near future. Fatty acid synthase (FASN), the enzyme responsible for de novo synthesis of fatty acids has emerged as a potential therapeutic target for several cancers however its role in diffuse large B-cell lymphoma (DLBCL) has not been fully elucidated.. Therefore, we investigated the expression of FASN in tissue micro array cohort of 301 DLBCL patients. FASN was found to be expressed in 62.6% (162/259) DLBCL samples and was seen in highly proliferative tumors manifested by high Ki67 (p<0.0001). Significant association was found between tumors expressing high FASN and c-Met tyrosine kinase (p<0.0002) as well as p-AKT (p=0.0309). In vitro, pharmacological FASN inhibition and SiRNA targeted against FASN triggered caspase dependent apoptosis and suppressed expression of c-Met kinase in DLBCL cell lines which further highlighted the molecular link between FASN and c-Met kinase. Finally, simultaneous targeting of FASN and c-Met with specific chemical inhibitors induced a synergistically stimulated apoptotic response in DLBCL cell lines. These findings provide evidence of an active role of FASN in DLBCL evolution by specifically regulating tyrosine kinases related to malignant transformation strongly suggest that targeting FASN may have therapeutic value in treatment of DLBCL. Disclosures: No relevant conflicts of interest to declare.

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Targeting XIAP Via Degradation of MDM-2 By MX69 in Aggressive Lymphomas

Blood ◽

10.1182/blood-2018-99-118480 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5379-5379

Author(s):

Sumera Khan ◽

Kyle Runckel ◽

Cory Mavis ◽

Matthew J. Barth ◽

Francisco J. Hernandez-Ilizaliturri

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Single Agent ◽

B Cell Lymphoma ◽

Parp Cleavage ◽

Synergistic Activity ◽

In Vitro Exposure ◽

Induction Of Apoptosis

Abstract Background: The addition of Rituximab to front-line therapy has improved clinical outcomes in diffuse large B-cell lymphoma (DLBCL), but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for Rituximab associated chemotherapy cross-resistance our group developed and characterized several Rituximab resistance cell lines (RRCL). We previously demonstrated using SiRNA interference, that X-linked inhibitor of apoptosis (XIAP) is critical for chemotherapy sensitivity and survival in RRCL. MX69, a dual inhibitor of Mdm2 and XIAP that indirectly downregulates XIAP, is undergoing pre-clinical testing. MX69 affects XIAP levels by its effects on the ubiquitination and degradation of endogenous MDM-2, resulting in decrease XIAP translation and activation of caspase 3, 7 and 9 as well as PARP cleavage leading to apoptosis of cancer cells. In our current work, we pharmacologically inhibited XIAP in lymphoma pre-clinical models using MX69. Materials and Methods: A panel of Burkitt's Lymphoma (BL, including RRCL), germinal center B-cell (GCB)-DLBCL (including RRCL), activated B-cell (ABC)-DLBCL, Mantle cell Lymphoma (MCL) and Pre-B cell Leukemia cell lines were exposed to MX69 as a single agent (0-80uM) over 24, 48, 72 hrs and IC50 concentrations were calculated for each cell line. Changes in Mdm2, p53, XIAP and PARP expressions were determined following MX69 exposure (at IC50 doses) for 24 hrs. Induction of apoptosis was evaluated by Annexin V/propidium iodine staining. Subsequently, cell lines were exposed to MX69 (0-80 uM), in combination with Doxorubicin (0-1uM), Cytarabine(0-50uM), Vincristine (0-10nM), Etoposide(0-50uM), Carboplatin (0-20uM), Ixazomib (0-1.5uM), Ibrutinib (0-20uM) and Venetoclax (0-10uM) for 48 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. Results: In vitro, MX69 single agent exposure induced cell death in a dose and time-dependent manner in all cell lines tested. Western blotting studies confirmed downregulation of Mdm2, XIAP and changes in P53 and PARP, following in vitro exposure to MX69. Induction of apoptosis was observed by flow cytometry in all cell lines tested. The combination of MX69 with Doxorubicin, Cytarabine, Vincristine, Ixazomib, Carboplatin, Etoposide, Ibrutinib, and Venetoclax resulted in significant synergistic activity. The strongest CI of synergy was observed when cell lines were exposed to MX69 and Venetoclax, Ixazomib, Etoposide or Ibrutinib. Conclusion: Our data suggests that in vitro exposure of a wide variety of B-cell lymphoma cell lines (including BL, DLBCL, MCL or RRCL) to MX69 resulted in anti-tumor activity. Perhaps related to its anti-tumor effects, MX69 inhibited XIAP levels. These findings are similar to prior SiRNA XIAP knockdown experiments. Strong synergistic activity was observed when XIAP was combined with various chemotherapy agents and small molecules inhibitors (such as Venetoclax, ixazomib or ibrutinib). Ex vivo experiments using primary tumor cells isolated from lymphoma patients and lymphoma mouse models are been planned. Targeting Mdm2 and XIAP can be an attractive therapeutic strategy in patients with Rituximab-sensitive or -resistant B-cell lymphoma. Disclosures No relevant conflicts of interest to declare.

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Entinostat (SNDX-275), a Novel DAC Inhibitor, Is Highly Effective in Rituximab- [Chemotherapy]-Sensitive or Rituximab-[Chemotherapy]-Resistant Lymphomas and Has Synergistic Anti-Tumor Activity When Combined with Bortezomib.

Blood ◽

10.1182/blood.v114.22.3727.3727 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3727-3727 ◽

Cited By ~ 1

Author(s):

Umeer Ashraf ◽

Myron S. Czuczman ◽

Cory Marvis ◽

John Gibbs ◽

Francisco J Hernandez-Ilizaliturri

Keyword(s):

B Cell ◽

Tumor Cells ◽

Cell Lines ◽

Primary Tumor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Resistant Cell ◽

Synergistic Activity ◽

Kinetic Assay ◽

Anti Tumor Activity

Abstract Abstract 3727 Poster Board III-663 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins, leading to regulation of gene transcription and other cellular processes. Entinostat (SNDX-275) is a novel and potent DAC inhibitor that is selective for class I DACs and is currently undergoing pre-clinical and clinical testing in Hodgkin lymphoma (HL). Potent synergistic anti-tumor activity has been observed by combining less potent DAC inhibitors with bortezomib in pre-clinical models. In our efforts to develop more therapeutic options for refractory/resistant B-cell lymphoma, we evaluated the effects of Eentinostat as a single agent and in combination with bortezomib against B-cell non-Hodgkin's lymphoma (NHL) cell lines and primary NHL cells. Studies were conducted in a panel of 12 NHL cell lines representing various subtypes of B-cell lymphoma (i.e. DLBCL/ABC, DLBCL/GCB, Burkitt's, transformed and MCL), which included: rituximab-[chemotherapy]-sensitive cell lines (RSCL, Raji, RL and DHL-4), rituximab-[chemotherapy]-resistant cell lines (RRCL, Raji-4RH, Raji-2R, RL-4RH, and DHL-4 4RH), and primary lymphoma cells isolated from patients with various subtypes of NHL and HL. Patient-derived tumor cells were isolated from fresh specimens by negative selection using magnetic beads. NHL cells and patient-derived primary cells were exposed to entinostat at different doses (0.01 to 100uM) either alone or in combination with CDDP (1 to 100μM), doxorubicin (4 to 16μM), vincristine (1 to 5μM), or bortezomib (1 to 10nM). Anti-tumor activity was measured after a 24 or 48 hr incubation. In cell lines, changes in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay measuring activity at 4 hr intervals for 24 and 48 hrs. For patient-derived primary NHL cells, changes in ATP content (apoptosis) was determined using the cell titer glow assay. Entinostat was highly active in all the cell lines tested including rituximab-[chemotherapy]-resistant cell lines. The IC50 of Entinostat in the majority of the cells tested was 0.5 to 5uM at 48 hrs. Similar findings were observed in primary tumor cells derived from lymphoma patients. In addition, synergistic activity was observed by combining entinostat and bortezomib in both NHL cell lines, as well as in primary NHL/HL tumor specimens. A lesser degree of augmented anti-tumor activity was also observed when entinostat was combined with cisplatin or doxorubicin (but not vincristine). In summary, our data suggests that entinostat is a novel and potent DAC inhibitor with a wide therapeutic spectrum. Entinostat is capable of inducing cell death against various subtypes of B-cell lymphoma cell lines including RSCL, RRCL, as well as patient-derived primary tumor cells and augments the anti-tumor effects of bortezomib and other chemotherapeutic agents. Given the isoform selectivity of entinostat, the results indicate that HDAC1 and 2 may be the key targets of DAC inhibitors in HL and NHL cells. Ongoing studies are evaluating the mechanisms responsible for the synergistic effects of entinostat plus chemotherapy and will be updated at the annual meeting. Current findings strongly suggest that entinostat added to bortezomib and/or other chemo agents may become a novel and potent strategy in the treatment of aggressive and indolent NHL and HL in the future. Disclosures: No relevant conflicts of interest to declare.

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