Primary Mediastinal B-cell Lymphoma Cell Research Articles

Exportin 1-mediated nuclear/cytoplasmic trafficking controls drug sensitivity of classical Hodgkin's lymphoma.

Exportin 1 (XPO1) is the main nuclear export receptor that controls the subcellular trafficking and the functions of major regulatory proteins. XPO1 is overexpressed in various cancers and small inhibitors of nuclear export (SINEs) have been developed to inhibit XPO1. In primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin's lymphoma (cHL), the XPO1 gene may be mutated on one nucleotide and encodes the mutant XPO1E571K . To understand the impact of mutation on protein function, we studied the response of PMBL and cHL cells to selinexor, a SINE, and ibrutinib, an inhibitor of Bruton tyrosine kinase. XPO1 mutation renders lymphoma cells more sensitive to selinexor due to a faster degradation of mutant XPO1 compared to the wild-type. We further showed that a mistrafficking of p65 (RELA) and p52 (NFκB2) transcription factors between the nuclear and cytoplasmic compartments accounts for the response toward ibrutinib. XPO1 mutation may be envisaged as a biomarker of the response of PMBL and cHL cells and other B-cell hemopathies to SINEs and drugs that target even indirectly the NFκB signaling pathway.

Obinutuzumab (GA101) vs. rituximab significantly enhances cell death, antibody-dependent cytotoxicity and improves overall survival against CD20+ primary mediastinal B-cell lymphoma (PMBL) in a xenograft NOD-scid IL2Rgnull (NSG) mouse model: a potential targeted agent in the treatment of PMBL.

Primary mediastinal large B-cell lymphoma (PMBL), a distinct mature B-cell lymphoma, expresses CD20 and has recently been successfully treated with the combination of a type I anti-CD20 monoclonal antibody, rituximab, with multiple combination chemotherapy regimens. Obinutuzumab is a glycoengineered type II anti-CD20 monoclonal antibody (mAb), recognizing a unique CD20 extracellular membrane epitope with enhanced antibody dependent cellular cytotoxicity (ADCC) vs rituximab. We hypothesize that obinutuzumab vs rituximab will significantly enhance in-vitro and in-vivo cytotoxicity against PMBL. PMBL cells were treated with equal dose of obinutuzumab and rituximab for 24 hours (1–100 μg/ml). ADCC were performed with ex-vivo expanded natural killer cells at 10:1 E: T ratio. Mice were xenografted with intravenous injections of luciferase expressing Karpas1106P cells and treated every 7 days for 8 weeks. Tumor burden was monitored by IVIS spectrum system. Compared with rituximab, obinutuzumab significantly inhibited PMBL cell proliferation (p = 0.01), promoted apoptosis (p = 0.05) and enhanced ADCC (p = 0.0002) against PMBL. Similarly, in PMBL xenografted NOD scid gamma mice, obinutuzumab significantly enhanced survival than rituximab when treated with equal doses (p = 0.05). Taken together our results suggest that obinutuzumab significantly enhanced natural killer cytotoxicity, reduced PMBL proliferation and prolonged the overall survival in humanized PMBL xenografted NOD scid gamma mice.

Polatuzumab Vedotin, an Antibody-Drug Conjugate Targeting CD79b, Is a Highly Active Agent Against Burkitt Lymphoma and Primary Mediastinal B-Cell Lymphoma

Introduction Mature B-cell lymphoma (MBL) accounts for approximately 60% of all non-Hodgkin lymphoma (NHL) in children and adolescents (Cairo et al, Blood, 2007). MBL in children and adolescents is comprised of approximately 75-80% Burkitt lymphoma (BL), 15-20% diffuse large B cell lymphoma (DLBCL) and 1-2% primary mediastinal B-cell lymphoma (PMBL). However, despite the differences in histology they are treated uniformly in children and adolescents (Cairo, BJH, 2016). The use of short and intense multiagent chemotherapy alone and in combination with rituximab (R) results in ≥90% event-free survival (EFS) in children and adolescents with BL and DLBCL (Cairo et al, JCO, 2012; Goldman/Cairo Leukemia, 2013; BJH, 2014). However, similar chemotherapy approaches in adolescents and young adults with PMBL have only resulted in a 60-70% EFS (Gerrard/Cairo et al, Blood, 2013). Furthermore, patients with induction failure, recurrent or refractory MBL have a dismal prognosis with ≤30% overall survival (OS) with current re-induction platforms and autologous stem cell transplantation (Cairo et al, BJH, 2018). Newer therapeutic strategies are urgently needed for recurrent/refractory patients with MBL, especially those with the BL subtype and newly diagnosed and refractory patients with PMBL (Cairo, BJH, 2019). We previously demonstrated that Obinutuzumab (O), a new type II anti-CD20 monoclonal antibody, is an active agent against both BL and PMBL (Awasthi/Cairo et al, BJH, 2015). Additionally, we have previously demonstrated high expression of CD79b in children and adolescents with BL and PMBL (Miles/Cairo et al, BJH 2007). Polatuzumab vedotin (PV), an anti-CD79b antibody-drug conjugate, recently approved by the FDA in adults with recurrent/refractory DLBCL has been demonstrated to be an active agent alone and in combination with chemotherapy in all molecular forms of adult DLBCL (Pfeifer et al, Leukemia, 2015; Corrinna et al, Lancet Oncology, 2015; Morschauser et al, Lancet Onc, 2019; Tilly et al, Lancet Onc, 2019). However, the efficacy of PV alone or in combination with R or O against other subtypes of B-NHL, including BL and PMBL, is relatively unknown. Objective To determine the in-vitro cytotoxicity and changes in in-vivo survival in a BL NSG xenograft model of PV alone or in combination with O vs R against R-sensitive and R-resistant BL and PMBL. Methods We compared the cytotoxicity of PV±R (10 ug/ml) vs PV±O (10 ug/ml) against R-sensitive BL (Raji) and R-resistant BL (Raji 4RH) and PBML (Karpas-1106p) alone or with ex-vivo expanded NK cells (Chu/Cairo et al, Can Imm Res, 2015) (10:1 E:T) by the DELFIA cytotoxicity assay. PV and O were generously supplied by Genentech and Roche, respectively. Furthermore, NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) were xenografted with luciferase positive BL (Raji, Raji 4RH) and PMBL cells (Karpas-1106p) and treated with PV (5 mg/kg) + O (20 mg/kg) vs PV + R (20 mg/kg) vs PV vs IgG isotype control vs PV + O + NK vs PV + R + NK vs PV + NK vs O + NK vs R + NK vs PBS (without tumor) and tumor burden was compared between treatment groups using the IVIS bioluminescence system and probability of OS by KM method as we have previously described (Chu/Cairo et al, Can Imm Res, 2015). Results PV + O + NK induced significantly more in-vitro cytotoxicity against BL Raji compared to PV + R + NK (p<0.007) vs PV, R or O + NK (p<0.0001), BL (Raji 4RH [R-resistant BL]) PV + O vs PV + R (p<0.004) and O, R or PV (p<0.002) and against PMBL (Karpas-1106p) PV + O + NK vs PV + R + NK (p<0.006) vs O, R, or PV + NK (p<0.001) (Fig. 1A, 1B, 1C, respectively). Most importantly, PV + O + NK vs PV + R + NK vs PV + NK + IgG isotype control significantly enhanced probability of survival in BL (Raji) and BL (Raji 4RH) NSG xenografts (p<0.03 and (p<0.05), (95.5 vs 50.4 days) (185 vs 47 days), respectively (Fig. 2A and 2B, respectively). Conclusions PV alone and in combination with O significantly induces cytotoxicity against BL, R-resistant BL and PMBL. Most importantly, PV + O + NK cells result in a significantly improved OS in BL and R-resistant BL NSG xenografts. These preclinical results suggest that PV should be considered for investigation in patients with both recurrent/refractory BL and PMBL and in patients with newly diagnosed disease with either high risk features or those with poor responses to induction therapy. Authors AA, MB & AA are all considered co-primary first authors Disclosures Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Cairo:Jazz Pharmaceuticals: Other: Advisory Board, Research Funding, Speakers Bureau; Osuka: Research Funding; Miltenyi: Other: MTA.

Abstract 1537: Polatuzumab Vedotin alone or in-combination with anti-CD20 antibody significantly enhanced overall survival in xenografted NSG mice against rituximab sensitive and resistant Burkitt Lymphoma (BL) and Primary Mediastinal B-cell Lymphoma (PMBL)

Abstract Background: Patients with relapsed or refractory B-cell Non-Hodgkin Lymphoma (B-NHL) have an unfavorable prognosis with few treatment options (Goldman/Cairo et al. Leukemia, 2013). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. Polatuzumab Vedotin (PV) has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al. Can. Res. 2009). We previously observed that obinutuzumab vs. rituximab (anti-CD20 mAb) significantly increased overall survival against xenografted NSG mice in BL (Awasthi/Cairo et al., BJH 2015). However, synergistic effects of PV with obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV alone or in-combination with rituximab/obinutuzumab against rituximab-sensitive/resistant BL (Raji & Raji4RH) and PMBL (Karpas1106P) cell lines. Methods: Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were xenografted with intravenous injections of Luc+ BL and PMBL cells. Mice were divided into following groups: PBS, isotype control, PV, anti-CD79B and MMAE (5mg/kg) and or/with obinutuzumab/RTX (20mg/kg) for 8 weeks with 106 ex.PBNK cells. Animals were monitored for tumor burden progression/regression and survival for up to 12-30 weeks via BLI using the IVIS spectrum system as previously described (Awasthi/Cairo et al BJH, 2015). Results: The probability of OS in B-NHL xenografts NSG mice receiving PV alone was significantly increased compared to anti-CD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Furthermore, The probability of OS of B-NHL xenogrfats NSG mice receiving PV+obinutuzumab+NK significantly increased compared to PV+rituximab+NK in Raji (95.5 vs. 50.4 days, p=0.03 ) and Raji4RH (185 vs.47 days, p=0.05). Moreover, obinutuzumab+NK vs. rituximab+NK also significantly increased over all survival both in Raji (92.5 vs. 52 days, p=0.05) and Raji4RH (80.5 vs. 40.0 days, p=0.04) respectively. Conclusion: Our preliminary data indicates that PV significantly increased overall survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with obinutuzumab+NK-cells significantly enhance improved OS in BL and PMBL NSG xenografts compared to obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Janet Ayello, Christian Klein, Mitchell S. Cairo. Polatuzumab Vedotin alone or in-combination with anti-CD20 antibody significantly enhanced overall survival in xenografted NSG mice against rituximab sensitive and resistant Burkitt Lymphoma (BL) and Primary Mediastinal B-cell Lymphoma (PMBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1537.

Abstract 2381: Blinatumomab enhanced anti-tumor activity against rituximab sensitive and resistant Burkitt Lymphoma (BL) and Primary Mediastinal B-cell Lymphoma (PMBL)

Abstract Background: Childhood, Adolescent and Young Adult (CAYA) B-NHL represents the third most common malignancies in children under the age of 15yrs (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012).The prognosis of mature-B-NHL (which include BL/PMBL) has significantly improved over the last 40 years through the use of short and intense multi-agent chemo-immunotherapy, however; a subset of patients with relapsed/refractory disease has chemoimmunotherapy resistant disease and a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al.Blood. 2007; Cairo et al. JCO.2012, Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). It is therefore critical to investigate and develop targeted translational strategies in BL/PMBL to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. Blinatumomab, targeting CD19, has demonstrated encouraging clinical activity against pre-B-ALL (Topp et al, Leukemia, 2018) and highly expressed in BL/PMBL. Objectives: To determine in-vitro activity of blinatumomab against rituximab sensitive/resistant BL and PMBL cell lines. Methods: BL; Raji, Raji-4RH, and PMBL: Karpas1106p/MedB-1 were cultured in RPMI with 10 or 20% FBS. Tumor cells were incubated with/without blinatumomab (generously supplied by Amgen) for 4 hr. with T-cells. CD3+ isolated and expanded T-cells were used for cytotoxicity assay. Cytotoxicity was determined by DELFIA®cytotoxicity assay at 10:1 E: T ratio and cytokines secretion was measured by multiples ELISA kit. CD3+/CD107a+, granzyme b and perforin T-cell expression level were measured by flow-cytometry. Results: Blinatumomab+T-cells compared to T-cells only elicited a significant increased cytotoxicity against, Raji 58.18±7.6% vs 27.3.81±11.2% (p=0.007), Raji-4RH 67.4±8.0% vs 27.6±2.5%, (p=0.004), Karpas1106p, 75.7±3.06 % vs. 17.86± 4.82% (p=0.003) and MedB1, 65.17±13.3% vs18.1±2.03%, respectively (p=0.05). Blinatumomab treated T-cells also increased IFN-γ secretion compared to IL2-T cells, Raji 1 (p=0.002) and Raji-4RH (p=0.02) respectively. Furthermore, CD107a, granzyme b and perforin expression were significantly enhanced with blinatumomab treated/activated T-cells against; Raji (p=0.03, p=0.003 & p=0.009), Raji-4RH (p=0.03, p=0.02 & p=0.03) and Karpas1106p (p=0.006, p=0.03 & p=0.02) respectively. Conclusion: Blinatumomab significantly enhances T-mediated in-vitro cytotoxicity and cytokine secretion against BL/PMBL. Further, blinatumomab treated and activated T-cells significantly enhanced CD107a, granzyme b and perforin expression. These preliminary studies demonstrate that BiTE (CD3/CD19) would be a novel agent to investigate as immunotherapy therapy in patients with relapse refectory BL/PMBL. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Janet Ayello, Mitchell S. Cairo. Blinatumomab enhanced anti-tumor activity against rituximab sensitive and resistant Burkitt Lymphoma (BL) and Primary Mediastinal B-cell Lymphoma (PMBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2381.

STAT6 is a cargo of exportin 1: Biological relevance in primary mediastinal B-cell lymphoma

Primary mediastinal B-cell lymphoma (PMBL) is a distinct B-cell lymphoma subtype with unique clinicopathological and molecular features. PMBL cells are characterised by several genetic abnormalities that conduct to the constitutive activation of the Janus kinase 2/signal transducer and activator of transcription 6 (JAK2/STAT6) signalling pathway. Among recurrent genetic changes in PMBL, we previously reported that the XPO1 gene encoding exportin 1 that controls the nuclear export of cargo proteins and RNAs, is mutated (p.E571K) in about 25% of PMBL cases. We therefore hypothesized that STAT6 could be a cargo of XPO1 and that STAT6 cytoplasm/nucleus shuttle could be altered in a subset of PMBL cells. Using immunocytochemistry techniques as well as the proximity ligation assay, we showed that STAT6 bound XPO1 in PBML cell lines and in HEK-293 cells genetically engineered to produce STAT6. Moreover, XPO1-mediated export of STAT6 occurs in cells expressing either a wild-type or the E571K mutated XPO1 protein.

Ruxolitinib significantly enhances in vitro apoptosis in Hodgkin lymphoma and primary mediastinal B-cell lymphoma and survival in a lymphoma xenograft murine model.

Hodgkin lymphoma (HL) and primary mediastinal B-cell lymphoma (PMBL) share similar molecular features by gene expression profiling. Frequent gains of chromosome 9p exhibit higher Janus Kinase 2 (JAK2) transcript levels with increased JAK2 activity, suggesting aberrant activity of JAK2 and STAT pathways. This signaling pathway alteration may in part play an important role in the pathogenesis and/or chemoradiotherapy resistance in HL and PMBL. Ruxolitinib is a potent and selective JAK1/JAK2 inhibitor, with activity against myeloproliferative neoplasms (MPNs) including those harboring the JAK2V617F mutation. We investigated the in vitro and in vivo efficacy of ruxolitinib and changes in downstream signaling pathways in HL and PMBL. We demonstrated that ruxolitinib significantly inhibited STAT signaling in both HL and PMBL with constitutively active JAK2 signaling. We also observed that ruxolitinib significantly induced in vitro anti-proliferative effects (p < 0.05) and increased programmed cell death (p < 0.05) against both HL and PMBL cells. Importantly, ruxolitinib significantly inhibited tumor progression by bioluminescence (p < 0.05) and significantly improved survival in HL (p = 0.0001) and PMBL (p < 0.0001) xenograft NSG mice. Taken altogether, these studies suggest that ruxolitinib may be a potential adjuvant targeted agent in the therapeutic approach in patients with high risk HL and PMBL.

Abstract 2657: PD-L1 blockade enhances T cell cytotoxicity against primary mediastinal B cell lymphoma

Abstract Background: Primary Mediastinal B cell Lymphoma (PMBL) represents 2-4% of Non-Hodgkin Lymphomas (NHL) in adolescents and young adults (AYA) (Gerrard/Cairo et al., Blood, 2013). Disease progression, relapse and long-term toxicity remain a concern for patients treated on current chemo-immunotherapy and mediastinal radiotherapy. Alternative therapeutic regimens are urgently needed, especially in patients without an early response to therapy. Programmed Death 1 (PD-1) is a negative co-stimulatory receptor critical for suppression of T-cell activation, with binding of PD-1 and Programmed Death Ligand 1 (PD-L1) resulting in T cell exhaustion (Postow/Wolchok et al., J Clin Oncol, 2015). Gain in 9p associated with amplification of PD-L1 has been reported in up to 60% of PMBL specimens, leading to overexpression of PD-L1 and potential immune cell evasion of PMBL (Rosenwald/Staudt et al., J Exp Med, 2003; Twa/Steidl et al., Blood, 2014). Blockade of PD-1/PD-L1 interaction, therefore, constitute a potentially promising alternative for treatment of resistant PMBL. Objective: In the current study, we sought to investigate whether PD-L1 blockade enhances T cell responses in PD-L1 expressing PMBL. Methods: PD-L1 expression on Karpas 1106p PMBL treated with or without IFN γ was investigated by western blotting and flow cytometry analyses. T cells were isolated from human PBMC followed by activation and expansion using anti-CD2, anti-CD3 and anti-CD28 Biotinylated MACSiBead particles. T cell activation was confirmed by CD25 and CD69 expression using flow cytometry. Activated T cells were incubated with or without anti-PD-L1 (Clone 6E11, Genentech) at a dose of 10 ug/ml together with Karpas 1106p cells treated with or without IFN γ. Cell proliferation was assessed with MTS assays after incubation for 24 hours. Results: We demonstrated that Karpas 1106p cells express a low level of PD-L1. However, following IFN γ treatment (48 hours) there was a significant increase in PD-L1 expression. Anti-PD-L1 had no significant effect on T cell mediated inhibition of cell proliferation in Karpas 1106p cells with low PD-L1 expression. However, anti-PD-L1 and T cell treatment significantly inhibited cell proliferation in IFN γ treated Karpas 1106p cells when compared to T cell treatment alone (32±18% vs. 3±3% at E:T=5:1, p=0.048). Conclusion: PD-L1 blockade enhances T cell cytotoxicity against PMBL, which is dependent on PD-L1 expression level on PMBL cells. Future in-vivo NSG xenograft studies are ongoing. Citation Format: Tishi Shah, Wen Luo, Aradhana Awasthi, Janet Ayello, Jessica Hochberg, Mitchell Cairo. PD-L1 blockade enhances T cell cytotoxicity against primary mediastinal B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2657. doi:10.1158/1538-7445.AM2017-2657

Recurrent mutations of the exportin 1 gene (XPO1) and their impact on selective inhibitor of nuclear export compounds sensitivity in primary mediastinal B-cell lymphoma.

Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We investigated the prevalence, specificity, and clinical relevance of mutations of XPO1, which encodes a member of the karyopherin-β nuclear transporters, in a large cohort of PMBL. PMBL cases defined histologically or by gene expression profiling (GEP) were sequenced and the XPO1 mutational status was correlated to genetic and clinical characteristics. The XPO1 mutational status was also assessed in DLBCL, Hodgkin lymphoma (HL) and mediastinal gray-zone lymphoma (MGZL).The biological impact of the mutation on Selective Inhibitor of Nuclear Export (SINE) compounds (KPT-185/330) sensitivity was investigated in vitro. XPO1 mutations were present in 28/117 (24%) PMBL cases and in 5/19 (26%) HL cases but absent/rare in MGZL (0/20) or DLBCL (3/197). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in GEP-defined PMBL and was associated with shorter PFS. Age, International Prognostic Index and bulky mass were similar in XPO1 mutant and wild-type cases. KPT-185 induced a dose-dependent decrease in cell proliferation and increased cell-death in PMBL cell lines harboring wild type or XPO1 E571K mutant alleles. Experiments in transfected U2OS cells further confirmed that the XPO1 E571K mutation does not have a drastic impact on KPT-330 binding. To conclude the XPO1 E571K mutation represents a genetic hallmark of the PMBL subtype and serves as a new relevant PMBL biomarker. SINE compounds appear active for both mutated and wild-type protein. Am. J. Hematol. 91:923-930, 2016. © 2016 Wiley Periodicals, Inc.

Ruxolitinib Significantly Prolongs Survival in Both a Primary Mediastinal B-Cell Lymphoma (PMBL) and Hodgkin Lymphoma (HL) Xenograft NSG Mouse Model: Ruxolitinib May be a Potential Adjuvant Agent in the Treatment of PMBL and of HL

BACKGROUND: Primary Mediastinal large B-cell lymphoma (PMBL) and Hodgkin lymphoma (HL) are two of the most common malignancies among adolescents and young adults (AYA). PMBL and HL share similar molecular features by gene expression profiling and pathogenesis (Rosenwald et al., J Exp Med., 2003). HL represents only approximately 4-5% of all cancers in children younger than 15 years of age, but is the most common cancer in the 15-35 yrs AYA group. While the prognosis is excellent with AYA HL, there are significant late effects secondary to chemoradiotherapy and therefore new targeted agents are needed to avoid these morbid late effects in HL (Hochberg/Cairo et al., Br. J. Haem., 2009). Frequent gains of chromosome 9p exhibit higher Janus Kinase 2 (JAK2) transcript levels with increased JAK2 activity (Bentz et al., Genes Chromosomes Cancer, 2001), suggesting aberrant activity of JAK2 and Signal Transducer and Activator of Transcription (STAT) pathways, which may in part play an important role in the pathogenesis of HL and PMBL. Ruxolitinib is a potent and selective JAK1/JAK2 inhibitor against myeloproliferative neoplasms (MPNs) that consistently exhibits dysregulation of the JAK1/JAK2 pathway, including those MPNs with a JAK2V617F mutation. Ruxolitinb also inhibits JAK2/STAT5 signaling in vitro and in a murine model of MPN (Quintas-Cardama et al., Blood, 2010).OBJECTIVES: We hypothesize that ruxolitinib may potentially function as targeted therapeutic agent for both PMBL and HL and therefore, investigated the efficacy of ruxolitinib in PMBL and HL cells xenografted into NSG mice.METHODS: Cell proliferation and apoptosis analysis were assessed using MTS and Caspase-3/7 assay (Promega), respectively. The expression of protein was examined by immunoblotting and statistical significance was determined by Student t-test. Karpas-1106P PMBL cell line and L-428 HL cell line were stably transfected with a firefly luciferase expression plasmid (ffluc-zeo), kindly provided by Laurence Cooper MD, PhD. The six to eight weeks old Female NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were from Jackson laboratory. The ffluc-zeo NSG mice were irradiated (2.5 Gy) and then were subcutaneously injected with 1x106 ffluc-zeo Karpas-1106P or L-428 tumor cells. Tumor burden and tumor progression were monitored by bioluminescence imaging (BLI) using the Xenogen IVIS-200 (Caliper Life Sciences) for up to 60 days. Mice were orally gavaged with either vehicle or ruxolitinib (45.0mg/kg or 90.0mg/kg) (generously provided by Incyte Corporation, Wilmington, DE, USA) for 21 days. Survival rates were analyzed by the Kaplan-Meier method and differences evaluated by log-rank test using the Prism Version 6.0 software.RESULTS: We observed that ruxolitinib significantly decreased the phosphorylation of STAT3, -5 and -6 in Karpas-1106P PMBL and L-428 HL cells. The reduction in cell proliferation by 10uM ruxolitinib are 41% in Karpas-1106P (p<0.04) and 19% in L-428 (p<0.03), respectively. In addition, ruxolitinib significantly induced Caspase-3/7 activity (p<0.05) and increased cleavage of PARP and caspase-3 fragments (p<0.001). In PMBL and HL xenografted mice models, we observed a significant decrease in tumor luminescence intensity following ruxolitinib treated PMBL (45.0mg/kg and 90.0mg/kg, p<0.05) and HL xenografted NSG mice at day 21 and 28 (45.0mg/kg, p<0.05) compared to control mice (Figure 1A). Ruxolitinib (45.0mg/kg) treated xenograft NSG mice had a significantly prolonged survival in PMBL (p<0.0001) and HL (p=0.0001) compared to control mice (Figure 1B).CONCLUSIONS: Ruxolitinib significantly inhibited JAK2 enzymatic activity by the abrogation of the phosphorylation of STAT3, STAT5 and STAT6, and ruxolitinib showed significant anti-proliferative effects. Ruxolitinib (45mg/kg) significantly inhibited tumor progression and significantly prolonged survival in PMBL and HL xenografted NSG mice compared to control mice. Ruxolitinib may be a potential adjuvant agent in the treatment of PMBL and HL. Future studies will focus on whether the addition of ruxolitinib to chemoimmunotherapy improves the survival in a NSG PMBL and HL xenograft mouse models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ibrutinib Alone and in Combination with Dexamethasone and Carfilzomib Significantly Inhibits Cell Proliferation in Primary Mediastinal B-Cell Lymphoma (PMBL): Ibrutinib May be a Future Targeted Agent in Combination Therapy in Patients with PMBL

BACKGROUND: Primary Mediastinal B-Cell Lymphoma (PMBL) is a rare form of B-cell non-Hodgkin lymphoma (Lones/Cairo et al, JCO 2000). We have previously reported a significant decrease in EFS among pediatric and adolescent patients with PMBL compared with other stage III non-PMBL diffuse large B-cell lymphoma (DLBCL) patients following FAB/LMB 96 therapy, suggesting that new therapeutic targeted agents are needed (Gerrard/Cairo et al, Blood 2013). Activation of the B-cell receptor (BCR) signaling pathway has now emerged as a central oncogenic pathway that promotes growth and survival in both normal and malignant B-cells. The cascade of signaling pathways downstream of the BCR includes BTK, MAPK, NF-κB and AKT. PMBL has a constitutively activated NF-kB pathway (Rosenwald et al, JEM 2003). Ibrutinib is a selective and covalent BTK inhibitor that inhibits chronic active BCR signaling and downstream signaling including the NF-kB pathway (Herman et al, Blood 2014) and is an active agent in ABC-DLBCL, which also has an activated NF-kB pathway (Griner et al, PNAS 2014). Preclinical studies of ibrutinib in CLL and MCL suggested that the inhibitory effects on cell proliferation were seen in the range of 1.0uM to 25.0uM (Herman et al, Blood 2011; Cinar et al, Leuk. Res 2013). Despite these relatively high IC50 values in vitro, ibrutinib has been highly effective in the treatment of patients with refractory CLL and MCL (Byrd et al, NEJM 2013 and Wang et al, NEJM 2013). Ibrutinib was approved by the FDA (IMBRUVICA, USPI) for patients with MCL in November 2013 or CLL in February 2014, who have received at least one prior therapy. However, the antitumor activity against PMBL of ibrutinib alone and in combination with other agents is currently unknown.OBJECTIVES: We hypothesize that ibrutinib in combination with other agents may be a future targeted agent in the treatment of PMBL. Therefore, we investigated the effect of ibrutinib alone and in combination with dexamethasone, rituximab and carfilzomib on cell proliferation and apoptosis in a PMBL cell line.METHODS: The Karpas-1106P PMBL cell line (DSMZ, Germany) was treated with ibrutinib alone (0-10uM, generously provided by Janssen R&D LLC) and in combination with dexamethasone (1uM), rituximab (20ug/ml) and carfilzomib (5nM) for 5 days treatment. The MTS cell proliferation assay (Promega) and western blot analysis were performed. Significant differences were determined by Student's t -test.The IC50 values were determined with CompuSyn software (Chou and Martin, ComboSyn, 2005).RESULTS: We observed a significant down-regulation of phosphorylated BTK after 5 days with ibrutinib treatment in Karpas-1106P cell line with 1uM ibrutinib (0.178 ± 1.63, p<0.0001) and the expression of total BTK protein was also significantly decreased with 1uM ibrutinib following 5 days of treatment (0.803±0.190, p<0.005) compared to control (1.000±0.00). We also observed a significant dose-dependent decrease in cell proliferation (0-10uM, p<0.0001) in ibrutinib-treated Karpas-1106P PMBL cell line. In combination with dexamethasone, the ibrutinib IC50 values for inhibition of cell proliferation were significantly decreased from 0.71uM ± 0.172 (ibrutinib alone) to 0.04uM ± 0.007 (p<0.005) with 1.0uM dexamethasone combination following 5 days of treatment. Second, in combination with rituximab experiment, the ibrutinib IC50 values was significantly decreased from 0.845uM ± 0.289 (ibrutinib alone) to 0.37uM ± 0.056 (p<0.05) with 20ug/ml rituximab following 5 days of treatment. Lastly, the ibrutinib IC50 values were significantly decreased from 0.72uM ± 0.185 (ibrutinib alone) to 0.002uM ± 0.001 (p<0.005) with 5nM carfilzomib combination following 5 days of treatment.CONCLUSIONS: Ibrutinib alone and in combination with dexamethasone, rituximab and carfilzomib significantly inhibited cell proliferation in the Karpas-1106P PMBL cell line. Ibrutinib's potential as an adjuvant agent in combination treatment in patients with PMBL is supported by these experiments. Future studies will focus on the in vivo effects of ibrutinib alone and in combination with dexamethasone and carfilzomib in a NOD/SCID PMBL xenograft mouse model. [Display omitted] DisclosuresGalardy:Mission Therapeutics: Research Funding.

Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines.

Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2–10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings highlight biallelic deletions as a major class of chromosomal lesion in PMBL cell lines, while endorsing the latter as preclinical models for hunting and testing new biomarkers and actionable targets.

FOXO1 downregulation contributes to the oncogenic program of primary mediastinal B-cell lymphoma.

Recently we have shown that the transcription factor FOXO1, highly expressed in B cells, is downregulated in classical Hodgkin lymphoma (cHL). As primary mediastinal B cell lymphoma (PMBL) has similarities with the cHL transcription program we investigated FOXO1 expression in this entity. By using immunohistochemistry we found that FOXO1 was absent or expressed at low levels in 19 of 20 primary PMBL cases. PMBL cell lines reproduce the low FOXO1 expression observed in primary cases. By analyzing gene expression profiling data we found that FOXO1 expression inversely correlated with JAK2 in PMBL cases. Targeting JAK2 activity by the small molecular weight inhibitor TG101348 resulted in upregulation of FOXO1 mRNA and protein expression in MedB-1 and U2940 cell lines, and the MYC inhibitor 10058-F4 increased FOXO1 mRNA in MedB-1 cells. Moreover, in MedB-1 cells FOXO1 expression was strongly upregulated by the inhibitor of DNA methylation 5-aza-2-deoxycytidine and by the histone deacetylase inhibitor trichostatin A. Since FOXO1 promoter was unmethylated, this effect is most likely indirect. FOXO1 activation in the FOXO1-negative Med-B1 cell line led to growth arrest and apoptosis, which was accompanied by repression of MYC and BCL2L1/BCLxL. Thus, FOXO1 repression might contribute to the oncogenic program and phenotype of PMBL.

Knock-down of BCL6 / STAT6 sensitizes primary B cell lymphoma cells for treatment with current therapeutic agents.

Primary mediastinal B cell lymphoma (PMBL) is characterized by specific molecular hallmarks including the expression of B Cell Lymphoma factor 6 (BCL6) and the presence of the activated Signal Transducers and Activators of Transcription factor 6 (STAT6). Recently we have shown that combined targeting of BCL6 and activated STAT6 by specific chemical inhibitors in PMBL resulted in additive efficacy regarding their negative effects on cell viability. Given that despite general efficient immuno-chemotherapy in PMBL the delayed treatment-related sequelae remains still a main challenge, we analyzed the role of BCL6 and activated STAT6 in the sensitivity of PMBL cells to the current treatment components. We found that the knock-down of BCL6 / STAT6 by siRNA sensitized the PMBL cells to the effects of R-CHOP components in two of three PMBL cell lines. In one cell line, MedB-1, which is marked by less expression of BCL6 and mutated STAT6, the knock-down of BCL6 / STAT6 did not enhance the efficiency of Doxorubicin, Rituximab, and Vincristin. Thus, the targeting of BCL6 and STAT6 in addition or prior to the treatment with components of the current immuno-chemotherapy may sensitize the PMBL tumor cells for drug effects, at least in parts of PMBL cases.

Obinutuzumab (GA101), a Novel Type II Glycoengineered CD20 Antibody, Significantly Induced Programmed Cell Death In Primary Mediastinal B-Cell Lymphoma (PMBL): Obinutuzumab May Be a Future Potential Targeted Agent For The Treatment Of PMBL

BackgroundPrimary Mediastinal large B-cell lymphoma (PMBL) is a rare form of Non Hodgkin Lymphoma (NHL) representing 2% of mature B-cell non-Hodgkin lymphoma in patients less than 18 years of age (Lones/Cairo et al., JCO, 2000; Burkhardt et al., BJH, 2005). PMBL has similar histo-pathological features along the biologic spectrum between Diffuse Large B-Cell Lymphoma (DLBCL) and classical HL (cHL) (Abramson et al., Blood, 2005). Gene expression studies suggested that the molecular signature of PMBL had a striking resemblance to the expression profile of cHL (Rosenwald et al., J Exp Med, 2003). We have recently reported that a significant decrease in EFS among children and adolescent PMBL patients compared with other stage III non-PMBL pediatric DLBCL patients following FAB/LMB 96 therapy, suggesting that children and adolescent PMBL may be an inherently different B-NHL (Gerrard/Cairo et al., Blood, 2013). Since over 95% of PMBL express CD20, targeting the CD20 receptor with a CD20 antibody is of high clinical interest. Obinutuzumab (GA101) is novel glycoengineered anti-CD20 targeted monoclonal antibody recognizing a unique CD20 type II epitope and it has been demonstrated to have greater efficacy in reducing tumor size, inducing remission and improving survival in other B-NHL xenograft models (Mössner et al., Blood, 2010). ObjectivesWe hypothesize that obinutuzumab may be a future potential targeted agent for the treatment of PMBL, and therefore, we investigated whether obinutuzumab significantly induces programmed cell death and reduces cell proliferation in PMBL. MethodsCD20+ PMBL, Karpas-1106P cells were obtained from the DSMZ and obinutuzumab was provided by C. Klein, Roche Glycart AG. Total RNA was prepared using Trizol reagent (Invitrogen) from Karpas-1106P cells and cDNA was synthesized using cDNA Synthesis Kit (Quantas). qRT-PCR was performed using CFX96 Real-time system with SsoFast Supermix (Bio-rad). Relative quantification (ddCt) of mRNA expression of CD20 genes was determined by normalizing to GAPDH. Antibodies for western blotting were obtained from Cell signaling Tech. and Santa Cruz Bio., respectively. MTS (Promega) and Caspase 3/7 assay (Promega) were employed for cell proliferation and apoptosis assay with treatment of obinutuzumab (1, 10, 20 and 100ug/ml) at every 24hours. Cell death was assayed by FACS (MoFlo XDP Sorter, Beckman Coulter) of FITC-Annexin V and propidium iodide (PI)–stained cells (BD Biosciences) according to the manufacturer's instruction. Statistical significance was determined by one-tailed Student t-test. ResultsWe observed a significantly higher expression of CD20 mRNA and protein in Karpas-1106P (>5.5-fold, p<0.01 and >2.5-fold, p<0.01) compared to the positive control, CD20+ Raji cells. There was a significant decrease of cell proliferation in obinutuzumab treated Karpas-1106P vs control cells at 24, 48 and 72 hours regardless of the dose of obinutuzumab with 23% reduction (p<0.05), 22% reduction (p<0.05), 24% reduction (p<0.05) and 24% reduction (p<0.05) with 1, 10, 20 and 100ug/ml of obinutuzumab treatment, respectively (Figure 1). A significant increase of Caspase 3/7 activity was observed at day 2 with >3.6-fold (p<0.03), >3.9-fold (p<0.03), >4.0-fold (p<0.02) and >4.2-fold increases (p<0.03) at escalating doses of obinutuzumab treatment, respectively compared to control (Figure 2). Treatment of PMBL cells with obinutuzumab (10 ug/ml) for 48 hours, CD20 expression were significantly decreased 26% (p<0.05) and 10% (p<0.05) in mRNA expression and protein levels, respectively. There was a significant increase of cell death in obinutuzumab treated Karpas-1106P compared to control (PBS) (37.8 vs 1.2%, p<0.02) using Annexin V-binding assay. Additionally, we assessed the expression of the pro-apoptotic gene, Bcl-xL and anti-apoptotic gene, Bax at both the mRNA and protein level. There were significant down-regulation of Bcl-xL (10% reduction, p<0.04) at both of the mRNA and protein levels, and a significant decrease in mRNA expression of Bax (30% reduction, p<0.02). [Display omitted] [Display omitted] ConclusionsObinutuzumab induced a significant increase in PMBL cell death and may be a future potential targeted agent for the treatment of PMBL lymphomas. Future studies will focus on the in vivo effects of obinutuzumab in a NOD/SCID PMBL xenograft mouse model. Disclosures:No relevant conflicts of interest to declare.

STAT6-mediated BCL6 repression in primary mediastinal B-cell lymphoma (PMBL)

Primary mediastinal B-cell lymphoma (PMBL) is characterized by aberrant activation of JAK/STAT-signaling resulting in constitutive presence of phosphorylated STAT6 (pSTAT6). In primary PMBL samples pSTAT6 is only expressed in a sub-population of lymphoma cells in a pattern that is reminiscent of that of the BCL6 oncogene. Double-fluorescence staining was carried out to determine the association between these two proteins in ten primary PMBL cases and three available PMBL cell line models. Surprisingly, only a minute fraction of double-positive nuclei was observed, while each sample contained considerable fractions of single-positive pSTAT6 and BCL6 nuclei. The intratumoral coexistence of BCL6+/pSTAT6- and BCL6-/pSTAT6+ subpopulations suggests a negative interaction between these factors. In silico screening of the STAT6 /BCL6 promoters for DNA consensus binding sites identified five STAT-binding-sites in the BCL6 promoter. We confirmed STAT6 binding to the BCL6 promoter in vitro and in vivo by band shift / super shift assays and chromatin immunoprecipitations. Using BCL6 luciferase reporter assays, depletion of STAT6 by siRNA, and ectopic overexpression of a constitutive active STAT6 mutant, we proved that pSTAT6 is sufficient to transcriptionally repress BCL6. Recently developed small molecule inhibitors 79-6 and TG101348 that increases BCL6 target gene expression and decreases pSTAT6 levels, respectively, demonstrate that a combined targeting results in additive efficacy regarding their negative effect on cell viability. The delineated pSTAT6-mediated molecular repression mechanism links JAK/STAT to BCL6-signaling in PMBL and may carry therapeutic potential.

JAK1/JAK2 Inhibitor, Ruxolitinib Inhibits Cell Proliferation and Induces Apoptosis in Hodgkin Lymphomas (HL) and Primary Mediastinal B-Cell Lymphomas (PMBL)

AbstractAbstract 4886 BACKGROUND:Hodgkin Lymphoma (HL) and Primary Mediastinal B-cell Lymphoma (PMBL) exhibit similar molecular features and pathogenesis. Both lymphoid malignancies shared similar cytogenetic abnormalities, namely 9p and 2p gains (Bentz et al., Genes Chromosomes Cancer, 2001; Joos et al., Int J Cancer 2003), and exhibit higher Janus Kinase 2 (JAK2) transcript levels with increased JAK2 activity (Green et al., Blood, 2010), suggesting aberrant activity of JAK2 and Signal Transducer and Activator of Transcription (STAT) pathways, which may play an important role in the pathogenesis of HL and PMBL. Ruxolitinib is a potent and selective JAK1/JAK2 inhibitor against myeloproliferative neoplasms (MPNs) that consistently exhibit dysregulation of the JAK1/JAK2 pathway, for reasons such as the presence of the JAK2 V617F mutation; this drug also inhibits JAK2/STAT5 signaling in vitro and in murine model of MPNs (Quintas-Cardama et al., Blood, 2010). Ruxolitinib is associated with marked and durable clinical benefits in patients with myelofibrosis (Verstovsek et al., NEJM, 2010). OBJECTIVE:Given the rationale for JAK2 inhibition in lymphoma, we designed the studies described here to evaluate the effects of Ruxolitinib on JAK2/STAT signaling pathways, cell proliferation and apoptosis in HDLM-2 (HL) and Karpas-1106P (PMBL) lymphomas. METHODS:Both HDLM-2 and Karpas-1106P cells were obtained from the DSMZ, Germany and maintained in RPMI with 20% FBS. Ruxolitinib was generously provided by Incyte Corporation, and for cytokine stimulation, Interlukin-4 (IL-4) was purchased from Invitrogen. Mono- or poly-clonal antibodies for western blotting were from Cell signaling Technology and Santa Cruz Biotech, respectively. For the effects on proliferative and apoptosis, Cell titer 96 Aqueous One solution cell proliferation assay (MTS) (Promega) and Caspase-Glo 3/7 assay (Promega) were used according to the manufacturer’s instruction. Briefly, HDLM-2 and Karpas-1106P cells (0.5×106/ml) were seeded into 24-well plated and treated with vehicle (DMSO) alone or Ruxolitinib at various concentrations for 48 and 72 hours and measured by Clarity Luminescence microplate reader (BioTek) and statistical significance on this study was determined by one-tailed Student t-test. RESULTS:Ruxolitinib treatment resulted in the dose dependent inhibition of JAK2-dependent pSTAT3 (IC50: 35nM for HDLM-2; IC50: 90nM for Karpas-1106P) and pSTAT5 (IC50: 28nM for HDLM-2; IC50: 98nM for Karpas-1106P) activation. In addition, the same inhibitor potency for pSTAT6 in both HDLM-2 and Karpas-1106P cells (IC50:20nM) was observed with Ruxolitinib. The level of STAT6 phosphorylation in Karpas-1106P cells was further enhanced significantly by 10ng/ml IL-4 treatment for 10 minutes and also increased by a dose-dependent reduction of Ruxolitinib (25–400nM).The effects of Ruxolitinib on cell proliferation by MTS assay demonstrated antiproliferative effects in a dose-dependent (1–100uM) manner (p<0.05) for up to 72 hours. Consistent with the anti-proliferative effect of Ruxolitinib, Ruxolitinib induced cell death was observed with increasing doses (1–100uM) (p<0.05) in both HL and PMBL cells. The cleavage of poly adenosine diphosphate ribose polymerase (PARP), another hallmark of apoptosis, was substantially increased by Ruxolitinib in the same dose-dependent manner. We examined the effects of Ruxolitinib on the expression of anti-apoptotic genes to enhance our understanding of the effect on apoptosis, expression of two anti-apoptotic genes, Bcl-xL and Mcl-1were inhibited in a dose-dependent manner 72hours after Ruxolitinib treatment of HDLM-2 cells. These results suggested that Ruxolitinib decreases cancer cell survival by inducing programmed cell death via down-regulating the expression of anti-apoptotic genes.Taken altogether, Ruxolitinib demonstrated efficay against HDLM-2 and Karpas-1106P cells with constitutively active JAK2 signaling and effectively blocked STAT signaling in both HL and PMBL. Ruxolitinib significantly induced antiproliferative effects as well as apoptosis in HL and PMBL. CONCLUSIONS:Ruxolitinib may be a future potential targeted agent for the treatment of HL and PMBL lymphomas, and in vivo efficacy of Ruxolitinib will be evaluated in NOD/SCID mouse models of HL and PMBL lymphomas. Disclosures:No relevant conflicts of interest to declare.

Abstract 5562: ML120B and Bortezomib (BTZ) inhibit both NF-κB and non NF-κB proteins in Primary Mediastinal B-cell Lymphoma (PMBL): Potential for future targeted therapy

Abstract Background: PMBL has a significantly poor prognosis compared to other DLBCLs (Lones/Cairo, JCO, 2000; Patte/Cairo, Blood 2007). New chemotherapeutic agents are urgently needed to improve the current treatment protocol and to improve overall survival rates. Several groups have demonstrated that the NF-κB signaling pathway levels are upregulated in PMBL (Savage, Blood, 2003; Rosenwald, J Exp Med, 2003; Feuerhake, Blood, 2005). We recently demonstrated that the NF-κB inhibitor bortezomib (BTZ) and/or ML120B (I B kinase, IKK, inhibitor) increase apoptosis in PMBL (Waxman/Cairo, et al., Ann Onc, 2008a). Objective: To elucidate the underlying molecular mechanism(s) by which NF-κB blockade contributes to apoptosis in PMBL, we performed proteomic analyses and western blotting of PMBL cells after NF-κB Inhibitor BTZ and ML120B exposure in-vitro. Methods: PMBL cell line Karpas-1106P cells were incubated with BTZ (5ng/ml), ML120B (10μg/ml, generously supplied by Millennium Pharmaceuticals), or both for 24 hours. NF-κB binding activity was measured by TransAm kit (Active Motif). CASPASE-3 was measured by Human Apoptosis 3-plex kit (Invitrogen). Proteomic analysis were performed with cell lysates utilizing iTRAQ labeling followed by LC-MS/MS. Ratios between untreated and treated were quantile normalized and log2 transformed. False-discovery rates (FDR) were used to estimate the error among the selected proteins. All proteins with an FDR &amp;lt;40% were considered differentially expressed. Several of these proteins were confirmed by western blot. Student t-test was used for statistical analysis. Results: The combination of BTZ and ML120B significantly increased CASPASE 3 formation (330±53 vs. 55±5 pg/mg protein) (p&amp;lt;0.006) and significantly reduced activation of NF-κB members, p50 (45% decrease (dec) vs. untreated control (unt); 0.051±0.0015; p&amp;lt; 0.001), p52 (50% dec vs. unt, 0.022±0.0002; p&amp;lt;0.001), c-rel (41% dec vs. unt, 0.020±0.0008; p&amp;lt;0.03), p65 (RelA) (52% dec vs. unt, 0.019±0.0008; p&amp;lt;0.001) and Rel B (43% dec vs. unt, 0.023±0.0002; p&amp;lt;0.001). There was a significant decrease in NF-κB proteins including HSP90 (−4.0F), PSAT1 (−3.3F) and SND1 (−3.9F) and c-myc downstream proteins SNRPB (−5.3F) and PFKP (−3.9F) by proteomic analysis and similar results confirmed by western blot. Conclusions: BTZ and ML120B inhibit both NF-κB and non NF-κB pathways which contribute to promotion of apoptosis in PMBL. These results could provide the basis for future translational studies in PMBL and other tumors with constitutive activation of the NF-κB pathway. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5562.

New Pathogenetic Insights Into Classical Hodgkin Lymphoma Revealed by Gene Expression Profiling of Microdissected Hodgkin/Reed-Sternberg Cells.

Abstract 266 Background.Previous gene expression profiling studies on cHL have been performed on whole tissue sections (mainly reflecting the prominent reactive background in which the few HRS cells are embedded), or on cHL cell lines. However, cultured HRS cells do not likely reflect primary HRS cells in all aspects, being derived from end-stage patients and from sites (e.g. pleural effusions or bone marrow) which are not typically involved by cHL and where HRS cells lost their dependence on the inflammatory microenvironment of the lymph node. Methods.∼1000–2000 neoplastic cells were laser-microdissected from hematoxylin/eosin-stained frozen sections of lymph nodes taken at disease onset from patients with cHL (n=16) or with various B-cell lymphomas (n=35), including primary mediastinal B-cell lymphoma (PMBL) and nodular lymphocyte-predominant Hodgkin lymphoma (nLPHL). After two rounds of in vitro linear amplification, mRNA was hybridized to Affymetrix HG-U133 Plus 2.0 chips. Expression profiles were likewise generated from sorted cHL cell lines and several normal mature B-cell populations. Results.Primary and cultured HRS cells, although sharing hallmark cHL signatures such as high NF-kB transcriptional activity and lost B-cell identity, showed considerable transcriptional divergence in chemokine/chemokine receptor activity, extracellular matrix remodeling and cell adhesion (all enriched in primary HRS cells), as well as in proliferation (enriched in cultured HRS cells). Unsupervised and supervised analyses indicated that microdissected HRS cells of cHL represent a transcriptionally unique lymphoma entity, overall closer to nLPHL than to PMBL but with differential behavior of the cHL histological subtypes, being HRS cells of the lymphocyte-rich and mixed-cellularity subtypes close to nLPHL cells while HRS cells of NS and LD exhibited greater similarity to PMBL cells. HRS cells downregulated a large number of genes involved in cell cycle checkpoints and in the maintenance of genomic integrity and chromosomal stability, while upregulating gene and gene signatures involved in various oncogenic signaling pathways and in cell phenotype reprogramming. Comparisons with normal B cells highlighted the lack of consistent transcriptional similarity of HRS cells to bulk germinal center (GC) B cells or plasma cells and, interestingly, a more pronounced resemblance to CD30+ GC B cells and CD30+ extrafollicular B cells, two previously uncharacterized subsets that are transcriptionally distinct from the other mature B-cell types. Conclusions.Gene expression profiling of primary HRS cells provided several new insights into the biology and pathogenesis of cHL, its relatedness to other lymphomas and normal B cells, and its enigmatic phenotype. Disclosures:No relevant conflicts of interest to declare.

Correlation Between Increases in Apoptosis in Primary Mediastinal B-Cell Lymphoma (PMBL) and NF-κB Pathway Blockade: Bortezomib (BTZ), Small Molecule IKK Inhibitor ML120B and Combination Therapy Significantly Decrease NF-κB Family Member Protein Activation

Primary mediastinal B-cell lymphoma (PMBL) is a rare subtype of diffuse large B-cell lymphoma (DLBCL) with a significantly lower event-free survival in children than other identically treated DLBCLs (Lones/Cairo, JCO, 2000). It has a unique gene expression profile with upregulation of a subset of NF-κB signal pathway genes (Rosenwald, J Exp Med, 2003). We previously reported significant increases in apoptosis in a PMBL cell line with NF-κB pathway blocking agents bortezomib (BTZ) and small molecule IKK inhibitor ML120B (supplied by Millennium Pharmaceuticals, MA) used alone (BTZ alone: 5.25%±1.43 increase in apoptosis vs. control; ML120B alone: 4.0%±1.13 increase in apoptosis vs. control, p<.0005) and in combination (ML120B+BTZ vs. ML120B single agent therapy; 17-fold increase in apoptosis, p<.01; ML120B+BTZ vs. BTZ single agent therapy; 4-fold increase in apoptosis, p<.01) (Waxman, Ann Onc, 2008a). We now report the effect of single and combination treatment in PMBL on expression of NF-κB family member protein expression and its correlation with apoptosis.PMBL line Karpas-1106P was incubated with BTZ (5 ng/ml), ML120B (10 μg/ml), and ML120B+BTZ for 24h. Activation of NF-κB transcription factors was measured by ELISA (TransAM Kit, Active Motif) and results were correlated with changes in apoptosis, determined by flow cytometry with Annexin V-FITC detection kit (BD Pharmingen) using identical drug doses and incubation times.In untreated PMBL, p50 had the highest activation (0.093±0.005OD/μg protein), followed in order of decreasing activation by P52 (0.045±0.0006), P65 (RelA) (0.040±0.0019), RelB (0.039±0.0016) and c-Rel (0.028±0.0036). ML120B monotherapy was associated with inhibition of the activation of P50 (32% decrease vs. untreated control, 0.064±0.0046; p<0.01), c-Rel (31% decrease vs. untreated control, 0.019±0.0004; p<0.05), P52 (16% decrease vs. untreated control, 0.038±0.0019; p<0.01) and P65 (RelA) (55% decrease vs. untreated control, 0.018±0.0006; p<0.001). BTZ monotherapy inhibited the activation of P52 (9% decrease vs. untreated control, 0.041±0.0006; p<0.01) and RelB (14% decrease vs. untreated control, 0.034±0.0009; p<0.05). ML120B+BTZ combination therapy strongly inhibited the activation of P50 (45% decrease vs. untreated control, 0.051±0.0015; p<0.001), P52 (50% decrease vs. untreated control, 0.022±0.0002; p<0.001) and RelB (43% decrease vs. untreated control, 0.023±0.0002; p<0.001).These results indicate that increases in apoptosis in PMBL previously reported for ML120B monotherapy, BTZ monotherapy, and ML120B+BTZ combination therapy are associated with inhibition of members of the NF-κB family of transcription factors. These results suggest that ML120B and BTZ increase apoptosis in PMBL by preventing activation of NF-κB transcription factors and subsequent transcription of anti-apoptotic genes that would otherwise promote cell survival. As ML120B+BTZ combination therapy leads to greater inhibition of P50 and P52 than single-agent therapy, there appears to be an additive effect when both agents are used together. Furthermore, as only ML120B inhibited c-Rel and RelA activation and only BTZ inhibited RelB activation, it is clear that each NF-κB blocker does not affect all 5 NF-κB transcription factor family members and gene expression profiling may therefore play a role in choosing optimal therapy. Finally, our findings suggest that ML120B and BTZ inhibit will also increase apoptosis in tumors other than PMBL with constitutive activation of the NF-κB pathway and these drugs should therefore be studied in other tumor types with upregulation of NF-κB genes.