ALBERT

Description: BLyS and its major receptor BAFF-R have been shown to be critical for development and homeostasis of normal B lymphocytes, and for cell growth and survival of neoplastic B lymphocytes, but the biologic mechanisms of this ligand/receptor-derived intracellular signaling pathway(s) have not been completely defined. We have discovered that the BAFF-R protein was present in the cell nucleus, in addition to its integral presence in the plasma membrane and cytoplasm, in both normal and neoplastic B cells. BAFF-R interacted with histone H3 and IKKβ in the cell nucleus, enhancing histone H3 phosphorylation through IKKβ. Nuclear BAFF-R was also associated with NF-κB/c-Rel and bound to NF-κB targeted promoters including BLyS, CD154, Bcl-xL, IL-8, and Bfl-1/A1, promoting the transcription of these genes. These observations suggested that in addition to activating NF-κB pathways in the plasma membrane, BAFF-R also promotes normal B-cell and B-cell non-Hodgkin lymphoma (NHL-B) survival and proliferation by functioning as a transcriptional regulator through a chromatin remodeling mechanism(s) and NF-κB association. Our studies provide an expanded conceptual view of the BAFF-R signaling, which should contribute a better understanding of the physiologic mechanisms involved in normal B-cell survival and growth, as well as in the pathophysiology of aggressive B-cell malignancies and autoimmune diseases.

Description: Aggressive non-Hodgkin lymphomas (NHL), such as Diffuse Large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), are one of a small number of important human cancers increasing in incidence in the US over the last four decades. Although these lymphomas are now potentially curable, almost half the treated patients still develop relapsed/refractory disease with poor survival outcomes, indicating an urgent need for better therapeutic approaches with improved efficacy. Double–hit lymphomas (DHL) constitute a particularly aggressive subtype of DLBCL and are characterized by a chromosomal breakpoint affecting the MYC/8q24 locus in combination with another recurrent breakpoint, usually a t(14;18)(q32;q21) involving BCL2. Prognosis for DHL is extremely poor, indicating a need for new treatment options as well. Multi-drug combination therapies are expected to potentiate therapeutic responses and delay disease recurrence by blocking adaptive resistance responses. Carfilzomib (CFZ), a novel second-generation proteasome inhibitor, and the novel Selective Inhibitor of Nuclear Exports (SINE) KPT-330 are effective treatments for refractory lymphomas. We hypothesized that combining the two would be even more successful because the drugs target different cellular pathways and processes in malignant B-cells. CFZ inhibits chymotrypsin-like, protein-degrading activity in the ubiquitin-proteasome pathway while KPT-330 irreversibly inhibits the major nuclear export receptor, chromosome region maintenance 1, CRM1, also termed XPO1. This study investigates whether the effects of CFZ and KPT-330 in combination are synergistic and thus more effective, reducing side effects and chemoresistance. Two DLBCL cell lines, DOHH-2 and OCI-Ly10, two DH-DLBCL cell lines, CJ and U-2973, and two MCL cell lines, Mino and Jeko-1, were used. Working with these cell lines, we identified and analyzed the increase in the efficacy of the combination over single agents alone through MTS proliferation and Annexin-V binding apoptosis assays. Growth inhibitory combination indexes for each cell line were calculated, and the Chou-Talalay Method was used to quantify additive effects (CI=1), synergistic effects (CI1). Results indicated strong synergistic behavior of the combinatorial therapy, with many of the dose combinations indexes falling below 1. Apoptosis assays also showed a high degree of synergy when combining CFZ with KPT-330. The efficacy of CFZ and KPT-330 in combination signifies the combination’s potential as a foundation for future studies, treatments, and clinical trials. Disclosures: Kirk: Onyx Pharmaceuticals: Employment, Equity Ownership. Kauffman:Karyopharm Therapeutics Inc.: Employment. Shacham:Karyopharm : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Description: The incidence of diffuse large B-cell lymphoma (DLBCL) is rising, a situation that highlights the need for improved therapy with greater efficacy and fewer side effects. The fundamental clinical problem is that standard frontline combination chemotherapy for DLBCL (R-CHOP) achieves lasting remissions but is not curative. Development of a new, effective, low-toxicity frontline therapy against DLBCL that incorporates targeted agents is much needed. Constitutive activation of the NF-kB, NFAT, and STAT3 (NNS) pathways is a hallmark of DLBCL, particularly the activated B-cell–like (ABC) subtype. Although these pathways have been studied intensively recently, the precise mechanisms controlling these pathways in DLBCL is still uncertain. Elucidating the key mechanism(s) controlling these pathways should improve our understanding of the pathophysiology of DLBCL as well as the therapeutic strategy against this deadly disease. MicroRNAs (miRNA) are short 18-23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Aberrant expression of miRNAs has been implicated in numerous diseases, including cancers. MiRNA-155 (miR- 155) has shown to be overexpressed in aggressive hematologic malignancies, including DLBCL. The oncogenic nature of this miRNA was confirmed in Em-miR-155 transgenic mice, where the mice develop aggressive pre B cell neoplasms within 3-4 weeks. However, the role miR-155 in the pathophysiology of DLBCL is still unclear. We performed gene expression profiling (GEP) in 24 representative DLBCL cell lines (17 GCB- and 7 ABC-subtype). Our data show that miR-155 expression is significantly higher in ABC vs. GCB DLBCL cell lines (p-value

Description: Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin’s lymphoma B cells (NHL-B), a lymphoma with increasing incidence over the last few decades. MCLs are classified into at least two subgroups, “classic” type of MCL and “blastoid-variant”- an even more aggressive type of MCL. Both subgroups are incurable, unusually refractory to standard chemotherapy combinations and associated with poor prognosis. New therapeutic agents with greater efficacy and less toxicity are necessary in MCL. We have shown previously that in MCL cells, the key transcription factor NF-kB, is constitutively active and maintains lymphoma cells survival. We also demonstrated that treating the MCL cells with the proteasome inhibitor bortezomib (Velcade) inhibits constitutive NF-kB activation, leading to G1 cell cycle arrest and apoptosis. Recently, a novel small molecular weight compound called WP-1066, a derivative of AG490, was synthesized by screening a synthetic library for agents that block stat3 activation. WP-1066 has shown to have anti-tumor activity in MCL cells through the inhibition of IL-6 mediated stat3 activation and NF-kB inhibition. A more effective compound called WP-1130, a derivative of WP-1066, was synthesized and is shown to be more potent than WP-1066 in MCL cell lines. Single agents are rarely effective in treating a disease like MCL; therefore, we hypothesized that the combination of bortezomib and WP-1130 would likely be more effective in MCL. We showed that MCL cells, both “classic” and “blastoid-variant”, treated in-vitro with bortezomib in conjunction with WP-1130 resulted in a synergistic growth inhibition and apoptosis induction. The drug concentrations use for bortezomib and WP-1130 that produce the synergistic effects were in the low nanomolar (nM) and micromolar (uM) ranges, respectively. Bortezomib at a concentration of 10 nM induces approximately 15% MCL apoptosis after 48 hr treatment when compare to untreated controls, while WP-1130 at a concentration of 1 uM induces 5% apoptosis. The combination of bortezomib and WP-1130 at the same concentrations induces 60% of MCL cell apoptosis. Bortezomib and WP-1130 showed efficacy in both classic and blastoid-variant MCL cell lines. The apoptotic effects in these cells were correlated with the down-regulation of bcl-2 and the up-regulation of bax proteins. The status of constitutive NF-kB was also examined after drug treatments. While a single agent treatment with low drug concentrations had only minimal effect on NF-kB inhibition, the combination of the two drugs dramatically inhibits NF-kB activation. These two drugs also synergize to inhibit cyclin D1 (a molecular signature of MCL), and c-myc (an oncogene commonly over-expressed in lymphoma B cells). Agents such as bortezomib and WP-1130, that can pharmacologically modulate key intracellular targets such as constitutively expressed NF-kB and cyclin D1 in MCL cells, may be more effective therapeutic agents for the treatment of MCL.

Description: The tumor necrosis factor (TNF) family (TNF-R; CD40; BAFF-R) plays a key role in neoplastic as well as normal B cell growth and survival mechanisms. TNF receptor-associated factor-6 (TRAF-6) is an adapter molecule that regulates several important signaling pathways critical for cell growth and cell survival. It is a member of seven closely related TRAF proteins that serve as signaling molecules, coupling to TNF-receptor superfamily to intracellular signaling, particularly in the CD40 Signalosome. TRAF6 has shown to be over-expressed and play an important role in cell growth and cell survival through the activation of the key transcription factor NF-kB in aggressive non-Hodgkin’s lymphoma B cells (NHL-B), common B cell neoplasm that have been increasing in recent years. Although much of TRAF-6 functions have focused primarily as an adaptor molecule in signaling pathways in the cytoplasm, the role of TRAF-6 in other cellular compartments has not been investigated. Here, we demonstrate, by confocal microscopy as well as cellular fractionation studies that TRAF-6 resides not only in the cytoplasm but also in the nucleus of lymphoma B cells. Immunoprecipitation studies show that TRAF6 is auto-ubiquitinated in the cytoplasm but not in the nucleus, suggesting that nuclear TRAF6 functions differently than cytoplasmic TRAF6. Chromatin immunoprecipitation (ChiP) cloning assays using anti-TRAF6 polyclonal antibody reveal over 200 clones, one of which contains a 130 bp fragment belonging to the proximal 5′ end of the c-myb oncogene promoter. Further experiments demonstrate that nuclear TRAF6 co-localized with SUMO1 and c-myb, suggesting that TRAF-6 may enter the nucleus through SUMO1 interaction and serve as an E3 sumo ligase, in addition to its known adapter role in cytoplasmic signaling. Over-expression studies show that TRAF6 enhances c-myb sumoylation in lymphoma B cells, where this oncogene is over-expressed. C-myb correlates with TRAF6 protein and mRNA expressions in NHL-B cells, suggesting that TRAF6 may be involved in the modulation of c-myb expression through sumoylation, regulating key genes that are regulated by c-myb. Small interfering RNA (siRNA) targeting c-myb results in inhibition of lymphoma cell survival, suggesting that SUMO1/TRAF6/c-myb interactions are important in cell survival pathways in aggressive NHL-B. Such pathways could represent novel targets for the development of therapeutic agents for aggressive B cell lymphomas.

Description: CD40 is a member of TNF receptor family, which plays important roles in the proliferation, survival and differentiation of lymphocytes. Constitutively active CD40 signaling is one of the hallmarks of aggressive B cell lymphoma development and mediates pivotal functions through its downstream pathways, involving NF-kBs. Recently, our group has reported the nuclear localization of CD40, opening a new paradigm in the functional role of CD40 in non-Hodgkin lymphomas of B cell origin (NHL-B). In this study, we show that CD40 co-localizes and immuno-precipitates with nuclear pore complex (NPC) proteins, which suggests NPC proteins are involved in the classic karyopherin pathway that facilitates the nuclear translocation of CD40 protein. Importantly, our co-immunoprecipitation assays and confocal microscopy have demonstrated that, in NHL-B cells, nuclear localized CD40 interacts with the NF-kB protein-c-Rel, but not p65. Chromotin-IP assay further indicates that CD40 forms complexes with c-Rel on the promoters of several proliferation and survival genes-CD154, Blys/BAFF and Bfl-1/A1 in various NHL-B cell lines. Co-transfection of CD40 and c-Rel in NHL-B cells synergistically enhance Blys promoter-driven luciferase activity while CD40 and p65 do not show such synergy. In normal B cells, transfection of c-Rel has synergistically enhanced the proliferation of B lymphocyte after CD40 ligand stimulation, which results in accumulation of CD40 in the nucleus. Furthermore, co-transfection of c-Rel and wild type CD40 also synergizes the proliferation of NHL-B cells, while co-transfection of c-Rel and nuclear localization signal (NLS) mutated CD40 does not. This study suggests that the nuclear CD40 plays an important role in lymphoma growth and survival mechanisms through its interaction with c-Rel, an oncogene that is amplified in many cases of NHL-B that may contribute to the pathophysiology of the disease process. Modulating the nuclear function of CD40 could provide a new targeted therapeutic approach for lymphoma therapy.

Description: Mantle Cell Lymphoma (MCL) is a relatively uncommon, aggressive form of B-cell non-Hodgkin’s lymphoma (NHL), defining approximately 5-8% of adult NHL in the United States. Although individual survival times have increased in recent years, prognosis remains among the poorest of NHL, with a median survival of 5 to 7 years. Despite an initial response to chemotherapy, MCL cells remain residually in the marrow and digestive tract, making MCL prone to recur leading to short survival prognosis. The characteristic re-emergence of MCL is likely related to drug resistance generated from the tumor microenvironment (TME). In particular, stromal cells found in bone marrow and secondary lymphoid sites promote the movement and proliferation and possibly drug resistance from cross-talk between stromal and lymphoma cells. While studies show a correlation between stromal cells and tumor survival in various forms of NHL, the mechanisms are poorly understood and its role in MCL has not extensively explored. Based on our experimental studies on an extensive spectrum of representative patient samples, we have shown that in vitro culturing of primary leukemic phase MCL cells (15 cases) usually leads to the spontaneous formation of activated CD68+ monocytic macrophages and stromal cells. The MCL tumor cells tended to form rosettes, clustering around and adhering to these cultured lymphoma-associated macrophages (LAM). The LAM formed in culture coexisted with rosetted MCL tumor cells for up to 4 months. During this period, a small subset of MCL cells showed limited mitotic figures, suggesting that these cells were proliferating and possibly becoming incipient immortalized MCL cells. When these cells were weaned from the adherent LAM, they began to expand autonomously and proliferate as an autonomous cell line. Using this methodology, we have developed 3 MCL cell lines (PF-1, PF-2, and PF-3) from 15 leukemic phase MCL cases. PF-1 MCL cell line represents classic typical MCL cells, while PF-2 and PF-3 MCL cell lines represent the in situ indolent MCL cells that are CD200 positive. When these cells were xeno-transplanted into SCID mice, lymphoma cell uptakes were disseminated throughout the lymphatic system, including the spleen, lymph nodes, and GI tract, representing excellent in vivo models for MCL. Next, we delineate the biologic functions of LAM in the MCL microenvironment milieu. Our data showed that when we initially removed the accessory cells (monocytes, etc.) from the MCL primary culture, no LAM were formed and the purified MCL cells died out in

Description: Abstract 1305 Cancer cells exist in a stressed environment, mainly due to lack of nutrients and oxygen, particularly during chemotherapy, and rely on metabolic homeostatic regulatory mechanisms for protection against these lethal challenges. Increasing glucose metabolism and continuous reactive oxygen species (ROS) production is one strategy of metabolic adaptation utilized by tumor cells to relieve this stress. Thioredoxin interacting protein (TXNIP) is a negative regulator for both redox thioredoxin (ROS production) and cellular glucose uptake, not well understood but found to be repressed in various cancers, including diffuse large B-cell lymphomas (DLBCL), the most common form of non-Hodgkin lymphoma that continues increasing in incidence and remains incurable in many cases, primarily due to development of chemo-resistance. The molecular mechanisms by which TXNIP expression is down-regulated during cancer progression and chemo-resistance development have not been completely elucidated. Since key gene silencing events have now been identified in the pathogenesis of DLBCL, recent therapeutic interest has focused on dysregulated histone modifications as potentially important therapeutic targets, for developing strategies that can reactivate silenced tumor suppressor genes. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), is a highly conserved histone methyltransferase that targets lysine-27 of histone H3 (H3K27). Studies in human tumors show that EZH2 is frequently over-expressed in a wide variety of tumors, including lymphomas. More importantly, recent studies using whole-genome sequencing in primary DLBCL, identified frequent mutations in the EZH2 gene that leads to critical gene silencing in DLBCL pathophysiology. Our study showed that EZH2 is either over-expressed or mutated in representative DLBCL cell lines and primary DLBCL cells, and that down-regulation of EZH2 with siRNA leads to the reactivation of TXNIP, with subsequent inhibition of tumor cell growth and survival mediated through both thioredoxin and glucose metabolism in DLBCL. We also found that histone deacetylation (HDAC) is also involved in EZH2-mediated silencing of TXNIP in DLBCL. Pharmacologic agents aimed at reactivating TXNIP genes include histone methylation inhibitor 3-Deazaneplanocin A (DZNep) that targets EZH2, as well as HDAC inhibitor Vorinostat. DZNep is currently the only histone methylation inhibitor that is commercially available. Our data indicated that DZNep is highly effective in inhibiting cell growth in various DLBCL cell lines, particularly in chemo-resistant DLBCL cell lines. Vorinostat, on the other hand, has been a good drug and is currently in clinical trial for relapsed DLBCL and has been FDA approved for treating cutaneous T-cell lymphoma patients. Our data showed synergistic activity of DZNep and Vorinostat in reactivating TXNIP gene expression and inhibiting DLBCL cell growth and survival. We also discovered that EZH2 controls constitutive NF-κB activity through both, the canonical and alternative NF-κB pathways in DLBCL. This function of EZH2 is independent of its histone methyltransferase activity. These findings reveal that EZH2 and NF-κB, the two oncogenic factors display functional crosstalk in DLBCL cells. Our findings have indicated that deregulated EZH2 leads to constitutive NF-kB activation and to epigenetic silencing of TXNIP, resulting in uncontrolled tumor cell growth and survival mediated through both thioredoxin and glucose metabolism in DLBCL, and that targeting this pathway represents a novel, rational, and effective therapeutic approach to selectively reverse chemoresistance in DLBCL patients, particularly relapsed/refractory patients. Disclosures: No relevant conflicts of interest to declare.

Description: Aberrant nuclear factor κB (NF-κB) signaling has been found to be of particular importance in diffuse, large B-cell lymphoma (DLBCL) cell survival and proliferation. Although the canonical NF-κB signaling pathway has been studied in some detail, activation of the alternative NF-κB pathway in DLBCL is not well characterized. Important insights into the regulation of the alternative NF-κB pathway in B lymphocytes has recently revealed the regulatory importance of the survival kinase NIK (NF-κB–inducing kinase) in genetically engineered murine models. Our studies demonstrate that both the canonical and alternative NF-κB pathways are constitutively activated in DLBCL. We also demonstrate that NIK kinase aberrantly accumulates in DLBCL cells due to constitutive activation of B-cell activation factor (BAFF)–R (BR3) through interaction with autochthonous B-lymphocyte stimulator (BLyS) ligand in DLBCL cells. Activation of BR3 in DLBCL induces recruitment and degradation of tumor necrosis factor receptor-associated factor 3, which results in NIK kinase accumulation, IκBα phosphorylation, and NF-κB p100 processing, thereby resulting in continuous activation of both NF-κB pathways in DLBCL cells, leading to autonomous lymphoma cell growth and survival. These results further elucidate mechanisms involved in abnormal NF-κB activation in DLBCL, and should contribute to better future therapeutic approaches for patients with DLBCL.

Description: The nuclear factor of activated T cells (NFAT) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for cell growth and survival in hematopoietic cells. Recent experimental evidence has implicated the calcineurin/NFAT signaling pathway in the pathogenesis of various malignancies, including diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanism(s) underlying NFATc1 regulation of genes controlling lymphoma cell growth and survival is still unclear. In this study, we demonstrate that the transcription factor NFATc1 regulates gene expression in DLBCL cells through a chromatin remodeling mechanism that involves recruitment of the SWItch/Sucrose NonFermentable chromatin remodeling complex ATPase enzyme SMARCA4 (also known as Brahma-related gene 1) to NFATc1 targeted gene promoters. The NFATc1/Brahma-related gene 1 complex induces promoter DNase I hypersensitive sites and recruits other transcription factors to the active chromatin site to regulate gene transcription. Targeting NFATc1 with specific small hairpin RNA inhibits DNase I hypersensitive site formation and down-regulates target gene expression. Our data support a novel epigenetic control mechanism for the transcriptional regulation of growth and survival genes by NFATc1 in the pathophysiology of DLBCL and suggests that targeting NFATc1 could potentially have therapeutic value.