cHL Cell Research Articles

Macrophages and dendritic cells as actors in the immune reaction of classical Hodgkin lymphoma.

BackgroundThe inflammatory infiltrate plays a pivotal role in classical Hodgkin lymphoma (cHL). Here, we focussed on the role of macrophages (MΦ) and dendritic cells (DC).MethodsMΦ and DC infiltration was investigated in 106 cHL specimens using immunohistochemistry and cytokine expression was analyzed in a subset by real-time PCR. Human peripheral blood-derived monocytes, DC, MΦ stimulated with GM-CSF (MΦGM-CSF, pro-inflammatory MΦ-1-model) or M-CSF (MΦM-CSF, immunomodulatory MΦ-2-model) were incubated with cHL cell line (L1236, HDLM2) supernatants (SN). DC maturation or MΦ polarization were investigated by flow cytometry. Furthermore, the impact of DC or MΦ on cHL cell proliferation was analyzed by BrdU/CFSE assay.ResultsIn cHL tissues mature myeloid (m)DC and MΦ predominated. High numbers of CD83+ mDC and low numbers of CD163+ MΦ were associated with improved disease specific survival. In numerous cHL specimens increased levels of both pro- and anti-inflammatory cytokines and of IL13 and GM-CSF were observed compared to reactive lymphadenopathies. Maturation of DC and induction and maintenance of an immunomodulatory MΦ phenotype were promoted by SN derived from cHL cell lines. TNFα neutralization in SN resulted in a significant inhibition of mDC maturation. DC and pro-inflammatory MΦ inhibited the proliferation of cHL cells.ConclusionAdopting an immunomodulatory phenotype is a potential mechanism for how MΦ promote immune evasion in cHL. Mature DC, in contrast, might participate in antitumoral immunity.

FOXO1 repression contributes to block of plasma cell differentiation in classical Hodgkin lymphoma

AbstractThe survival of classical Hodgkin lymphoma (cHL) cells depends on activation of NF-κB, JAK/STAT, and IRF4. Whereas these factors typically induce the master regulator of plasma cell (PC) differentiation PRDM1/BLIMP-1, levels of PRDM1 remain low in cHL. FOXO1, playing a critical role in normal B-cell development, acts as a tumor suppressor in cHL, but has never been associated with induction of PC differentiation. Here we show that FOXO1 directly upregulates the full-length isoform PRDM1α in cHL cell lines. We also observed a positive correlation between FOXO1 and PRDM1 expression levels in primary Hodgkin-Reed-Sternberg cells. Further, we show that PRDM1α acts as a tumor suppressor in cHL at least partially by blocking MYC. Here we provide a link between FOXO1 repression and PRDM1α downregulation in cHL and identify PRDM1α as a tumor suppressor in cHL. The data support a potential role for FOXO transcription factors in normal PC differentiation.

Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

RNA interference (RNAi) is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4), which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA) to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3) were designed to target the coding sequence (CDS) of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55%) in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.

Selective JAK2 Inhibition Specifically Decreases Hodgkin Lymphoma and Mediastinal Large B-cell Lymphoma Growth In Vitro and In Vivo

Classical Hodgkin lymphoma (cHL) and primary mediastinal large B-cell lymphoma (MLBCL) share similar histologic, clinical, and genetic features. In recent studies, we found that disease-specific chromosome 9p24.1/JAK2 amplification increased JAK2 expression and activity in both cHL and MLBCL. This prompted us to assess the activity of a clinical grade JAK2 selective inhibitor, fedratinib (SAR302503/TG101348), in in vitro and in vivo model systems of cHL and MLBCL with defined JAK2 copy numbers. We used functional and immunohistochemical analyses to investigate the preclinical activity of fedratinib and associated biomarkers in cell lines and murine xenograft models of cHL and MLBCL with known 9p24.1/JAK2 copy number. Chemical JAK2 inhibition decreased the cellular proliferation of cHL and MLBCL cell lines and induced their apoptosis. There was an inverse correlation between 9p24.1/JAK2 copy number and the EC50 of fedratinib. Chemical JAK2 inhibition decreased phosphorylation of JAK2, STAT1, STAT3, and STAT6 and reduced the expression of additional downstream targets, including PD-L1, in a copy number-dependent manner. In murine xenograft models of cHL and MLBCL with 9p24.1/JAK2 amplification, chemical JAK2 inhibition significantly decreased JAK2/STAT signaling and tumor growth and prolonged survival. In in vitro and in vivo studies, pSTAT3 was an excellent biomarker of baseline JAK2 activity and the efficacy of chemical JAK2 inhibition. In in vitro and in vivo analyses, cHL and MLBCL with 9p24.1/JAK2 copy gain are sensitive to chemical JAK2 inhibition suggesting that clinical evaluation of JAK2 blockade is warranted.

Insulin-Like Growth Factor 1 Receptor Is a Prognostic Factor in Classical Hodgkin Lymphoma

The interaction between the tumor cells in classical Hodgkin lymphoma (cHL) and the microenvironment includes aberrant activity of receptor tyrosine kinases. In this study we evaluated the expression, functionality and prognostic significance of Insulin-like growth factor-1 receptor (IGF-1R) in cHL. IGF-1R was overexpressed in 55% (44/80) of cHL patients. Phosphorylated IGF-1R was detectable in a minority of the IGF-1R positive tumor cells. The overall survival (OS, 98%) and 5-year progression-free survival (PFS, 93%) was significantly higher in IGF-1R positive cHL patients compared to IGF-1R negative patients (OS 83%, p = .029 and PFS 77%, p = .047, respectively). Three cHL cell lines showed expression of IGF-1R, with strong staining especially in the mitotic cells and expression of IGF-1. IGF-1 treatment had a prominent effect on the cell growth of L428 and L1236 cells and resulted in an increased phosphorylation of IGF1R, Akt and ERK. Inhibition of IGF-1R with cyclolignan picropodophyllin (PPP) decreased cell growth and induced a G2/M cell cycle arrest in all three cell lines. Moreover, a decrease in pCcd2 and an increase in CyclinB1 levels were observed which is consistent with the G2/M cell cycle arrest. In conclusion, IGF-1R expression in HRS cells predicts a favorable outcome, despite the oncogenic effect of IGF-1R in cHL cell lines.

Genotoxicity Assessment of Erythritol by Using Short-term Assay

Erythritol is a sugar alcohol that is widely used as a natural sugar substitute. Thus, the safety of its usage is very important. In the present study, short-term genotoxicity assays were conducted to evaluate the potential genotoxic effects of erythritol. According to the OECD test guidelines, the maximum test dose was 5,000 μg/plate in bacterial reverse mutation tests, 5,000 μg/ml in cell-based assays, and 5,000 mg/kg for in vivo testing. An Ames test did not reveal any positive results. No clastogenicity was observed in a chromosomal aberration test with CHL cells or an in vitro micronucleus test with L5178Y tk+/− cells. Erythritol induced a marginal increase of DNA damage at two high doses by 24 hr of exposure in a comet assay using L5178Y tk+/− cells. Additionally, in vivo micronucleus tests clearly demonstrated that oral administration of erythritol did not induce micronuclei formation of the bone marrow cells of male ICR mice. Taken together, our results indicate that erythritol is not mutagenic to bacterial cells and does not cause chromosomal damage in mammalian cells either in vitro or in vivo.

Hodgkin-Reed-Sternberg Cells in Classical Hodgkin Lymphoma Show Alterations of Genes Encoding the NADPH Oxidase Complex and Impaired Reactive Oxygen Species Synthesis Capacity

The membrane bound NADPH oxidase involved in the synthesis of reactive oxygen species (ROS) is a multi-protein enzyme encoded by CYBA, CYBB, NCF1, NCF2 and NCF4 genes. Growing evidence suggests a role of ROS in the modulation of signaling pathways of non-phagocytic cells, including differentiation and proliferation of B-cell progenitors. Transcriptional downregulation of the CYBB gene has been previously reported in cell lines of the B-cell derived classical Hodgkin lymphoma (cHL). Thus, we explored functional consequences of CYBB downregulation on the NADPH complex. Using flow cytometry to detect and quantify superoxide anion synthesis in cHL cell lines we identified recurrent loss of superoxide anion production in all stimulated cHL cell lines in contrast to stimulated non-Hodgkin lymphoma cell lines. As CYBB loss proved to exert a deleterious effect on the NADPH oxidase complex in cHL cell lines, we analyzed the CYBB locus in Hodgkin and Reed-Sternberg (HRS) cells of primary cHL biopsies by in situ hybridisation and identified recurrent deletions of the gene in 8/18 cases. Immunohistochemical analysis to 14 of these cases revealed a complete lack of detectable CYBB protein expression in all HRS cells in all cases studied. Moreover, by microarray profiling of cHL cell lines we identified additional alterations of NADPH oxidase genes including CYBA copy number loss in 3/7 cell lines and a significant downregulation of the NCF1 transcription (p=0.006) compared to normal B-cell subsets. Besides, NCF1 protein was significantly downregulated (p<0.005) in cHL compared to other lymphoma cell lines. Together this findings show recurrent alterations of the NADPH oxidase encoding genes that result in functional inactivation of the enzyme and reduced production of superoxide anion in cHL.

Preclinical Analyses Of The Chemical JAK2 Inhibitor, SAR302503, In Classical Hodgkin Lymphoma and Primary Mediastinal Large B-Cell Lymphoma

Classical Hodgkin lymphoma (cHL) and primary mediastinal large B-cell lymphoma (MLBCL) are diseases which share certain clinical, pathologic and genetic features. We previously characterized chromosome 9p24 amplification as a disease-specific structural alteration in cHL and MLBCL and identified the immunoregulatory genes, PD-L1 and PD-L2, and JAK2 as key targets of the 9p24 amplification. In a panel of informative cHL and MLBCL cell lines with defined 9p24 copy number, JAK2 amplification increased JAK2 protein expression and activity and enhanced sensitivity to chemical JAK2 inhibition with commercially available tool compounds. Given the importance of JAK/STAT signaling as a survival pathway in primary cHL and MLBCL and the pathway's additional role in augmenting PD-1 ligand transcription, we postulated that JAK2 was a promising rational therapeutic target in cHLs and MLBCLs with 9p24 amplification.SAR302503 (fedratinib), formerly TG101348, is a selective, potent and oral JAK2 inhibitor that has demonstrated clinical activity in myeloproliferative disorders with activating JAK2 mutations. For these reasons, we have evaluated the preclinical activity of SAR302503 in in vitro and in vivo model systems of cHL and MLBCL with known 9p24/JAK2 copy number. In in vitro analyses, SAR302503 inhibited the cellular proliferation of cHL and MLBCL cell lines and induced their apoptosis. Of note, there was an inverse correlation between 9p24/JAK2 copy number and the EC50 of SAR302503, indicating that cHLs and MLBCLs with JAK2 amplification exhibited increased sensitivity to chemical JAK2 inhibition. In this series of cHLs and MLBCLs of defined JAK2 copy number, JAK2 copy gain was associated with higher baseline phosphorylated JAK2 (pJAK2) and increased abundance of phosphorylated STAT family members including pSTAT1, pSTAT3 and pSTAT6. Consistent with the copy number-dependent anti-proliferative effects of SAR302503, the compound decreased pJAK2, pSTAT1, 3 and 6 in a copy-number dependent manner in cHLs and MLBCLs. These effects were apparent within 2 hours of SAR302503 treatment.We next developed a comprehensive phosphoJAK/STAT immunohistochemical signature to assess baseline pathway activity and sensitivity to targeted chemical JAK2 inhibition. In cHL cell lines with 9p24 amplification, SAR302503 treatment abrogated pJAK2, pSTAT1 and pSTAT3 immunohistochemical staining at early and late timepoints. Furthermore, chemical JAK2 inhibition decreased PD-L1 transcript abundance in 9p24-amplified cHL and MLBCL cell lines and reduced the abundance of downstream JAK2 targets including MYC and PIM1 in cHLs with high JAK2 copy number.After demonstrating the activity and specificity of SAR302503 in in vitro assays, we evaluated the JAK2 inhibitor in murine xenograft models of cHL and MLBCL with 9p24/JAK2 amplification (Karpas 1106 [MLBCL] and HDLM2 [cHL]). In systemic MLBCL (Karpas 1106) and subcutaneous cHL (HDLM2) xenograft models, tumor growth was monitored via bioluminescent imaging (Karpas 1106) and mass measurements (HDLM2) and SAR302503 treatment was begun following the establishment of ≅ 100 mm3 tumors. In both models, pSTAT3 was analyzed as a pharmacodynamic marker following 5 days of treatment and SAR302503-treated animals had markedly decreased tumor cell pSTAT3 expression. In the Karpas 1106 MLBCL murine model, SAR302503 treatment significantly prolonged overall survival (p = .0002). In the HDLM2 cHL xenograft model, the JAK2 inhibitor significantly decreased subcutaneous tumor growth (p< .0001) and pSTAT3 expression in tumor cells (p = .0004). Transcriptional profiling confirmed that the HDLM2 tumors from SAR302503-treated animals exhibited coordinately decreased expression of STAT3 targets and downregulation of a functionally validated JAK2 gene set. Taken together, these data indicate that SAR302503 specifically decreases cHL and MLBCL growth in a 9p24/JAK2 copy number-dependent manner in vitro and in vivo and highlight the utility of pSTAT3 immunohistochemistry as a biomarker of pathway responsiveness. Clinical evaluation of JAK2 pathway inhibition in patients with cHL and MLBCL with known 9p24/JAK2 copy number status is warranted. Disclosures:Hao:Sanofi: Research Funding. Off Label Use: Drug is not yet approved but is being evaluated in myelofibrosis. Shipp:Sanofi: Research Funding.

Downregulation Of FOXO1 Prevents Induction Of The Tumor Suppressor BLIMP-1 In Classical Hodgkin Lymphoma

In contrast to other B cell lymphomas, Hodgkin and Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), have mainly lost their B cell identity. Recently, we reported that the transcription factor FOXO1, indispensable for B cell development and differentiation, is downregulated in HRS cells and expression of FOXO1 results in growth arrest and apoptosis in cHL cell lines.To find molecular targets of FOXO1 we performed gene expression profiling of 5 cHL cell lines expressing constitutively active FOXO1 fused to ligand binding domain of estrogen receptor (FOXO1(A3)ER).We found that FOXO1 activation led to downregulation of genes, which are normally upregulated in cHL, such as CD30/TNFRSF8 and the proto-oncogene MYC. On the other hand, FOXO1 activated expression of germinal center-specific genes including BCL6, AICDA, BACH2, and GCET2. The most surprising finding was induction of BLIMP-1/PRDM1, the master regulator of plasma cell differentiation and tumor suppressor in activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL). A positive correlation of FOXO1 and BLIMP-1 expression in microdissected HRS cells further indicated a role for FOXO1 in BLIMP-1 regulation.Of note, HRS cells do not undergo plasma cell differentiation despite constitutive expression of NF-κB, IRF4, and STAT3, known inducers of BLIMP-1. However, BLIMP-1 levels remain low in HRS cells thereby preventing terminal differentiation. The mechanisms leading to BLIMP-1 repression in cHL are poorly understood. We found that in most cHL cell lines, FOXO1 specifically upregulated BLIMP-1α representing the full-length isoform of BLIMP-1. In addition, luciferase reporter assay showed that FOXO1 activates the BLIMP-1α promoter in the cHL cell line L428. Overexpression of BLIMP-1α using a lentiviral vector strongly inhibited growth of cHL cell lines. In line with the fact that BLIMP-1 and MYC form a negative feedback loop, we found that ectopic BLIMP-1α expression resulted in downregulation of MYC protein levels, which most likely contributes to the anti-tumor effect of BLIMP-1α. On the other hand, MYC inhibition in cHL cell lines led to BLIMP-1 upregulation. Thus, our data indicate that FOXO1, BLIMP-1 and MYC constitute a negative feed-forward regulatory loop, which is controlled by FOXO1.Searching for additional mechanisms of BLIMP-1α downregulation in cHL we found that BLIMP-1α promoter was hypermethylated in two cHL cell lines but not in microdissected HRS cells. This indicates that hypermethylation is not critical for BLIMP-1α repression.Interestingly, the functionally impaired BLIMP-1 variant BLIMP-1β, which is normally not detected in normal B cells, was upregulated in cHL cell lines. BLIMP-1β is also expressed in ABC-DLBCL and has been suggested to block BLIMP-1α in a dominant-negative manner. We speculate that BLIMP-1β attenuates function of residual BLIMP-1α in cHL. However, further investigation is required.Taken together, we show for the first time that FOXO1 induces expression of BLIMP-1. We identified BLIMP-1α as a tumor suppressor which downregulates expression of the proto-oncogene MYC in cHL. Therefore, FOXO1 might exert its tumor suppressor function at least in part by upregulation of BLIMP-1α.Further work on BLIMP-1α regulation by FOXO1 and investigation of a potential role of FOXO1 in plasma cell differentiation is warranted. Disclosures:No relevant conflicts of interest to declare.

Specific PML Isoforms and SATB1 Affect HLA Expression In Classical Hodgkin Lymphoma

IntroductionHodgkin Reed-Sternberg (HRS) cells are characterized by a general loss of B cell phenotype, whereas antigen presenting properties are commonly retained. HLA class I and class II are expressed in most EBV+ cHL cases, with an even enhanced expression in some cases. The mechanisms underlying enhanced or lost HLA expression are unknown. Promyelocytic leukemia protein (PML) is the main component of nuclear bodies (NBs) and it organizes the chromatin structure into loops by anchoring matrix attachment regions to the nuclear matrix. The PML transcript can be processed into seven different isoforms. Downregulation of PML isoform III or V results in a reduced HLA-A and HLA-G expression in Jurkat T cells, whereas PML enhances HLA class II expression by upregulating the class II transactivator (CIITA). Special AT-rich region binding protein 1 (SATB1) has been shown to be associated with PML-NBs in the HLA region. Downregulation of SATB1 in Jurkat cells results in an enhanced expression of HLA-A, HLA-G and HLA-H. AimTo determine if specific PML isoforms and SATB1 regulate HLA expression in cHL cell lines. MethodsWe stained HLA class I, HLA class II, PML-NBs and SATB1 in 54 EBV+ cHL cases and in 27 EBV- cHL cases. We analyzed HLA class I, HLA class II, PML isoforms I to V and SATB1 mRNA expression levels by qRT-PCR in cHL cell lines (n=6). Next, we inhibited SATB1 and PML expression in the cHL cell line L1236 using shRNA constructs. The effects of the shRNA constructs on HLA protein expression levels were assessed by Western blot and flow cytometry. ResultsWe observed a normal HLA class I membrane expression in 40% of EBV+ and 20% of EBV- cases. There was a stronger than normal HLA class I protein expression in approximately 40% of EBV+ cHL and not in EBV- cHL patients. The number of PML-NBs was positively correlated to the level of HLA class I expression (p<0.01), whereas the percentage of SATB1 positive cells was inversely correlated with the level of HLA class I expression in EBV+ cHL cases (p<0.05). We observed normal HLA class II expression in 50% of EBV+ and 50% of EBV- cases. A stronger than normal HLA class II expression was observed in 4 EBV+ and 1 EBV- cHL patients. The number of PML-NBs and the percentage of SATB1 positive cells were not correlated with classical HLA class II expression in EBV+, EBV- or the total cHL patient group.We further investigate the association of PML, SATB1 and HLA class I and II in six cell lines. At the mRNA level, significant positive correlations were found between PML-III and HLA-C and between PML-III and β2M. Borderline significant positive correlations were found between PML-V and CIITA and between PML-V and HLA-DPB1. Suggestive, though not significant, correlations were observed of various PML isoforms and SATB1 for several HLA genes. The limited number of cell lines, precluded more definitive conclusions for the other comparisons.To further study the role of specific PML isoforms and SATB1 in regulation of HLA gene expression, we inhibited PML-III, PML-V or SATB1 in L1236 by shRNA-based knockdown. The PML-III shRNA induced a ∼80% reduction at the PML-III mRNA level, but did not affect membranous HLA class I expression and possibly affected HLA class II expression levels in L1236. The shRNA for PML-V was not effective and needs to be optimized and experiments with a pan-PML shRNA are ongoing. The SATB1 shRNA1 induced a ∼75% at the SATB1 mRNA level and a ∼80% reduction at the protein level, while SATB1 shRNA2 showed a ∼75% reduction and a ∼25% reduction, respectively. Inhibition of SATB1 by shRNA1 significantly upregulated HLA class II expression (P=0.02) and no significant effect on HLA class I expression. Inhibition of SATB1 by shRNA2 showed no significant effect on HLA class I and II expression. ConclusionPML and SATB1 protein levels are associated with HLA class I in HRS cells. Various negative associations of SATB1 with HLA genes at the mRNA level support a possible regulatory effect. This was indeed observed upon inhibition of SATB1 in L1236 that induced enhanced HLA-DR/DP/DQ expression. The PML regulation appears to be complex with different PML isoforms showing different associations with specific HLA genes. Disclosures:No relevant conflicts of interest to declare.

Prognostic Significance Of a 4-Microrna Signature Targeting JAK2 In Classical Hodgkin Lymphoma

IntroductionThe behavior of classical Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the Hodgkin/Reed Sternberg (HRS) cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. One of the genetic lesions most frequently found in HRS cells involves members of the JAK/STAT signaling pathway. However, few studies have identified microRNAs (miRNAs) that target JAK2 in cHL. In a previous study, our group identified miR-135a as a JAK2 regulator and showed the prognostic impact of this miRNA in a small set (n=89) of cHL patients (Navarro A. et al. Blood 2009). In the present study, we have examined novel miRNAs targeting JAK2 and evaluated the prognostic impact of their expression levels in 170 lymph nodes of cHL patients. MethodsTargetScan and miRbase were used to identify JAK2-related miRNAs. The conserved miRNAs with higher scores were selected and further validated by Renilla-Luciferase assay and Western Blot. We performed a Renilla-Luciferase assay with a modified expression vector psiChecK-2 containing the complete 3'UTR region of JAK2 cloned in the 3'UTR region of Renilla luciferase gene. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in the HDMYZ cHL cell line together with 1μM of modified psicheck2 vector using Lipofectamine 2000, and luminescence was read at 24 hours. Further validation of the miRNA target was performed by Western Blot in three cHL cell lines (L428, L-1236 and HDMYZ). The expression levels of the miRNAs identified as targeting JAK2 were then assessed in lymph nodes from 170 cHL patients (median age, 33 years; male, 51%; EBV, 43.8%; HIV, 12%) diagnosed and treated in a single institution. Moreover, 15 reactive lymph nodes (RLN) were used as normal controls. RNA was purified from formalin-fixed paraffin-embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation kit. The miRNA expression was analyzed using TaqMan microRNA assays. Statistical analyses were performed using R v2.13 and PASW Statistics 18. ResultsThe bioinformatic analysis identified seven microRNAs as possible regulators of JAK2 (miR-34a, miR-101, miR-135a, miR-204, miR-216a, miR-216b and miR-375). The Renilla-Luciferase assay confirmed that in addition to miR-135a, miR-101 (61%; p=0.01), miR-204 (46%; p=0.003) and miR-216a (64.8%; p=0.02) also targeted JAK2. These findings were confirmed by Western Blot analysis of JAK2 levels after transfection with pre-miRNAs (median % of protein reduction in the 3 cell lines of 21.1%, 46.7% and 24%, respectively). The analysis of these miRNAs in RLN showed that miR-101, miR-135a and miR-204 were significantly downregulated in lymph nodes of cHL patients (p<0.001, p<0.001 and p=0.02 respectively) in comparison with RLN used as control. In contrast, miR-216 did not show significant differences in comparison with RLN. Interestingly, the analysis of the expression levels of these 4 miRNA in the patient lymph nodes had an impact on prognosis. First, low expression levels of miR-135a was associated with lower rate of complete response to first line treatment (76% vs 89%, p=0.012) and a trend was observed for miR-216(p=0.077). Secondly, patients with low expression levels of miR-101 or miR-204 had shorter disease-free survival (DFS) (151 vs 109 months, p=0.020; and 149.2 vs 105.5 months, p=0.055). Finally, patients with low levels of miR-135a, miR-204 or miR-216 had shorter overall survival (OS) (158 vs 136 months; p=0.039; and 157.5 vs 110 months, p=0.025; and 165 vs 140 moths, p=0.011 respectively). Since all four miRNAs showed prognostic impact (DFS, OS or treatment response), we decide to analyze the prognostic impact of the combination of the 4 miRNAs. Patients were then classified in two subgroups according to the expression levels of all four miRNAs. Patients with low expression of more than 2 miRNAs had shorter OS (163 vs 132 months; p=0.012). The multivariate analysis identified the combination of the four miRNAs as an independent prognostic marker of OS (OR, 7.6; 95% CI, 1.1-57.2; p=0.048) together with Age≥45years (OR, 5.9; 95% CI, 2.4-14.4; p<0.001) and B symptoms (OR, 2.6; 95% CI, 1.09-6.3; p=0.03). ConclusionsMiR-101, miR-135a, miR-204 and miR-216 regulate JAK2 and are independent prognostic factors in cHL. AcknowledgmentsSDCSD of School of Medicine of UB. AC is an APIF-UB fellow. Disclosures:No relevant conflicts of interest to declare.

C-JUN N-terminal kinase (JNK) is activated and contributes to tumor cell proliferation in classical Hodgkin lymphoma.

c-JUN N-terminal Kinase (JNK) is activated/phosphorylated by upstream MAPK kinases (MKK), and, in turn, phosphorylates and activates its major substrate c-JUN, a member of the activator protein-1 (AP-1) transcription factors. c-JUN is overexpressed and activated in Hodgkin and Reed Sternberg cells (HRS) of classical Hodgkin lymphoma (cHL), however, the mechanism of its activation remains unknown. JNK activation was immunohistochemically assessed in 60 cases of HL and in a control group of 151 B-cell non-Hodgkin lymphomas. The biologic effects of JNK activation in cultured HRS cells were investigated using colony formation, cell growth and viability assays and cell cycle analysis by flow cytometry. Western blotting was used to assess protein levels. p-JNK was expressed in 90% of HL, 83% of Burkitt lymphomas, 28% of mantle cell lymphomas, 23% of diffuse large B-cell lymphomas, 19% of follicular lymphomas, and 18% of extranodal marginal zone lymphomas of MALT type. None of the 48 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma and 18 cases of plasma cell myeloma showed JNK phosphorylation (P < 001, Kruskall-Wallis test). Pharmacological inhibition of JNK activity in cultured HRS cells resulted in a significant decrease of cell growth, which was associated with cell cycle arrest at the G2/M phase. The cell cycle effects were linked to deactivation of c-JUN and upregulation of its known target, the cyclin-dependent kinase inhibitor p21. JNK is highly activated in HRS cells, and may contribute to uncontrolled cell cycle progression and proliferation of tumor cells in cHL.

Matrix Metalloproteinase-9 Expression by Hodgkin-Reed-Sternberg Cells Is Associated with Reduced Overall Survival in Young Adult Patients with Classical Hodgkin Lymphoma

Previous studies have investigated the prognostic relevance of MMP9 in classical Hodgkin lymphoma (cHL), with negative results. However, we have found that MMP9 immunoistochemical expression by Hodgkin-Reed-Sternberg cells is associated with reduced overall survival in a subset of young adult Brazilian patients diagnosed with cHL. Additionally, we have observed that MMP9 expression by neoplastic cells in cHL is associated with EBV positivity. These results may support a rational basis for additional studies on the role of this metalloproteinase as a target for therapy in classical Hodgkin lymphoma.

Bortezomib down-modulates the survival factor interferon regulatory factor 4 in Hodgkin lymphoma cell lines and decreases the protective activity of Hodgkin lymphoma-associated fibroblasts

Bortezomib is a proteasome inhibitor active in classical Hodgkin lymphoma (cHL) cell lines, but poorly active in the clinic when used as a single agent, suggesting that the microenvironment could protect from drug efficacy. Therefore, we investigated the effects of bortezomib activity in the presence of HL-associated fibroblasts (HL-AFs) and sCD40L. We found that co-cultivation with human HL-AFs or the addition of sCD40L during bortezomib treatment protected cHL cells from apoptosis and cytotoxicity and rescued the down-regulation of the survival factor interferon regulatory factor 4 (IRF4). In contrast, bortezomib treatment before co-cultivation with HL-AFs inhibited in a dose-dependent manner cHL cell adhesion to HL-AFs and completely overcame HL-AF protection against drug activity. Consistently, we found that bortezomib treatment down-regulated the surface expression of CD49d and CD44, which mediate the adhesion of cHL cells to HL-AFs, and of CD54 and CD40, which mediate the adhesion to CD40L+ rosetting T-cells. These preclinical findings suggest that the low in vivo activity of bortezomib as a single agent may be due to a protective influence of the microenvironment. However, inclusion of bortezomib in the cHL drug regimen, by reducing IRF4 expression and interactions with the microenvironment, could increase the efficacy of current chemotherapeutic treatment of relapsed/refractory cHL.

Efficacy of an adapted granzyme B-based anti-CD30 cytolytic fusion protein against PI-9-positive classical Hodgkin lymphoma cells in a murine model

Tumors develop when infiltrating immune cells contribute growth stimuli, and cancer cells are selected to survive within such a cytotoxic microenvironment. One possible immune-escape mechanism is the upregulation of PI-9 (Serpin B9) within cancer cells. This serine proteinase inhibitor selectively inactivates apoptosis-inducing granzyme B (GrB) from cytotoxic granules of innate immune cells. We demonstrate that most classical Hodgkin lymphoma (cHL)-derived cell lines express PI-9, which protects them against the GrB attack and thereby renders them resistant against GrB-based immunotherapeutics. To circumvent this disadvantage, we developed PI-9-insensitive human GrB mutants as fusion proteins to target the Hodgkin-selective receptor CD30. In contrast to the wild-type GrB, a R201K point-mutated GrB construct most efficiently killed PI-9-positive and -negative cHL cells. This was tested in vitro and also in vivo whereby a novel optical imaging-based tumor model with HL cell line L428 was applied. Therefore, this variant, as part of the next generation immunotherapeutics, also named cytolytic fusion proteins showing reduced immunogenicity, is a promising molecule for (targeted) therapy of patients with relapsing malignancies, such as cHL, and possibly other PI-9-positive malignancies, such as breast or lung carcinoma.

PU.1 is a potent tumor suppressor in classical Hodgkin lymphoma cells

AbstractPU.1 has previously been shown to be down-regulated in classical Hodgkin lymphoma (cHL) cells via promoter methylation. We performed bisulfite sequencing and proved that the promoter region and the −17 kb upstream regulatory element of the PU.1 gene were highly methylated. To evaluate whether down-regulation of PU.1 is essential for the growth of cHL cells, we conditionally expressed PU.1 in 2 cHL cell lines, L428 and KM-H2. Overexpression of PU.1 induced complete growth arrest and apoptosis in both cell lines. Furthermore, in a Hodgkin lymphoma tumor xenograft model using L428 and KM-H2 cell lines, overexpression of PU.1 led to tumor regression or stable disease. Lentiviral transduction of PU.1 into primary cHL cells also induced apoptosis. DNA microarray analysis revealed that among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428 cells after PU.1 induction. Stable knockdown of p21 rescued PU.1-induced growth arrest in L428 cells, suggesting that the growth arrest and apoptosis observed are at least partially dependent on p21 up-regulation. These data strongly suggest that PU.1 is a potent tumor suppressor in cHL and that induction of PU.1 with demethylation agents and/or histone deacetylase inhibitors is worth exploring as a possible therapeutic option for patients with cHL.

The histone Deacetylase Inhibitor Givinostat in Combination with Sorafenib Induces Reactive Oxygen Species (ROS) Generation and Exerts Potent Antitumor Effects in NOD/SCID Mice with Hodgkin Lymphoma Cell Line Xenografts

AbstractAbstract 3711 INTRODUCTION:Patients with refractory or relapsed classical Hodgkin Lymphoma (cHL) represent an unmet medical need and would benefit from the development of new therapies. Histone deacetylases (HDACs) and the RAF/MEK/ERK pathway are aberrantly controlled in cHL and influence a broad repertoire of tumor processes, suggesting a rationale for therapeutically targeting these pathways. We targeted these pathways using the HDAC inhibitor Givinostat (Italfarmaco S.p.A., Milan, Italy), and the RAF/MEK/ERK inhibitor Sorafenib (Nexavar, Bayer, Germany, EU) in order to investigate in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS:Three cHL cell lines, including HDLM-2, L-540 and HD-MyZ, were used to investigate the effects of Givinostat and Sorafenib, used alone or in combination, by means of in vitro assays evaluating cell growth and cell survival. Additionally, live cell imaging was used to asses the production of reactive oxygen species (ROS), and Western blotting (WB) to assess modulating effects of the two-drug combination on MAPK, PI3K/AKT, HDACs as well as the apoptotic pathways. The efficacy of Givinostat/Sorafenib combination was finally confirmed in NOD/SCID mice with cHL cell line xenografts. RESULTS:While Givinostat and Sorafenib as single agents exerted a limited activity against cHL cells, the combined Givinostat/Sorafenib treatment was associated with potent dephosphorylation of MAPK and PI3K/Akt pathways and significantly increased H3 and H4 acetylation due to a nearly complete inhibition of class I and II HDACs. Furthermore, these events were associated with a time-dependent synergistic cell growth inhibition (70% to 90%) in all Givinostat/Sorafenib-treated cHL cells. Upon Givinostat/Sorafenib exposure, HDLM-2 and L-540 cell lines showed significantly (P ≤.0001) increased levels of apoptosis (90 ± 2% and 96 ± 1%, respectively) and mitochondrial dysfunction (up to 70%, P≤.0001), as compared with single agents. Apoptosis induced by Givinostat/Sorafenib combination failed to induce processing of caspase-8, −9, −3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, suggesting the occurrence of caspase-independent apoptosis. Besides downregulating the expression of the anti-apoptotic protein Mcl-1 and ERK1/2 phosphorylation, Givinostat/Sorafenib strongly increased expression of the BH-3 only protein Bim, compared to single treatments. These findings were dependent on a potent, early and time-dependent ROS generation (up to 60%, P≤.0001) that was synergistically induced by Givinostat/Sorafenib treatment. Additionally, pretreatment of cHL cells with the ROS inhibitor YCG063 prevented the generation of ROS as well as mitochondrial membrane depolarization along with cell death induced by the two-drug combination, suggesting that ROS generation is the triggering event in Givinostat/Sorafenib induced-cell death. In vivo Givinostat/Sorafenib treatment significantly reduced the growth of L-540 and HD-MyZ nodules, resulting in an average 35% to 65% tumor growth inhibition (P ≤.0001) compared to single treatments, in the absence of any toxicity. Interestingly, as compared to controls or treatment with single agents, the combined Givinostat/Sorafenib treatment significantly increased in vivo Bim expression (7- to 21-fold increase, P ≤.0001), resulting in a marked tumor necrosis (3- to 5-fold increase, P ≤.0001). CONCLUSIONS:The combined Givinostat/Sorafenib treatment demonstrates a potent preclinical in vitro and in vivo activity against cHL cell lines by targeting aberrant expression of HDACs and MAPK. Antitumor activity of this combination involves ROS generation and Bim upregulation and provides a rationale for clinical studies using this combination in refractory/relapsed cHL patients. Disclosures:No relevant conflicts of interest to declare.

Expression, Prognostic Value and Function of IGF1R in Hodgkin Lymphoma.

AbstractAbstract 2640 Background:Classical Hodgkin's lymphoma (cHL) is a B cell lymphoma characterized by a minority of malignant Hodgkin Reed-Sternberg (HRS) cells that are surrounded by a vast majority of reactive cells. These reactive cells provide a microenvironment that supports survival and growth of the HRS cells. The interaction between cancer cells and microenvironment includes aberrant activity of multiple receptor tyrosine kinases (RTK). The insulin-like growth factor-1 receptor (IGF1R) is a RTK that is involved in many malignancies and plays a crucial role in proliferation, transformation, apoptosis protection and chemotherapy-resistant. In this study we evaluated the expression, prognostic significance and function of IGF1R in cHL. Design and Methods:Immunohistochemistry was applied on 80 cHL cases. IGF1R expression was correlated with clinical data in a cHL patient cohort using Cox-regression, Chi-square and one-way ANOVA statistics. Western blot was used to determine IGF1R expression in four cHL cell lines (L428, L1236, KMH2 and UHO-1) and the IGF1 production was measured by ELISA. The Alamar blue assay was performed to determine the effect of IGF1 and cyclolignan picropodophyllin (PPP, a highly selective IGF1R inhibitor) treatment on cell growth. Flowcytometry was performed to analyze cell cycle distribution after PPP treatment. Results:Expression of IGF1R was observed in all or the vast majority of HRS cells in positive cases, whereas in negative cases almost all HRS cells were negative. Expression of IGF1R was detected in 55% (44/80) of the cHL patients. There was a significant difference in IGF1R expression with subtype (p=0.0157), with a high percentage of positive cases in the nodular-sclerosis cases (38/61) and a low percentage of positive cases in mixed-cellularity cases (1/9). There was no association between IGF1R expression and age, EBV status or Ann Arbor Stage. The 5-year progression-free survival (PFS) was 95% in IGF1R positive patients, whereas in IGF1R negative patients the 5-years PFS was 77% (p=0.018).All four cHL cell lines showed expression of IGFR1, with the highest levels observed for L428 cells. The IGF1 production level was similar for all four cell lines. IGF1 treatment had the most prominent effect (31% increase) on the proliferation of L428 cells, whereas only a small effect was seen in the other three cell lines. Thus L428 cells were most sensitive to IGF1 treatment, consistent with its high IGF1R expression level. After 72 hours of PPP treatment (2μM) both the viability and proliferation decreased in all 4 cHL cell lines. The strongest effects were observed for L428 cells and UHO-1 cells with a reduction in cell growth of 48% and 43% respectively. The reduction in cell growth was 30% in KM-H2 cells and 26% in L1236. Treatment with PPP induced a G2/M cell cycle arrest in all 4 cell lines. After 24 hours incubation with PPP (2μM), the G2/M cell population increased from 8% to 45% in L428, from 10% to 64% in UHO-1, from 4% to 48% in KMH2 and from 3% to 38% in L1236. Conclusions:Expression of IGF1R in the HRS cells was observed at a high frequency in the nodular sclerosis subtype and correlates with a favorable 5-years PFS. Functional studies in cHL showed a marked decrease in cell growth and an increase in the percentage of cells in the G2/M phase in all four cHL cell lines upon inhibition of IGF1R. Disclosures:No relevant conflicts of interest to declare.

Role of early B-cell factor 1 (EBF1) in Hodgkin lymphoma

A hallmark of classical Hodgkin lymphoma (cHL) is that the B-cell-derived Hodgkin and Reed-Sternberg (HRS) tumor cells have largely lost the B-cell-typical gene expression program. The factors causing this 'reprogramming' of HRS cells are only partly understood. As early B-cell factor 1 (EBF1), a major B-cell transcription factor, is downregulated in HRS cells, we analyzed whether this downregulation contributes to the lost B-cell phenotype and tested the consequences of EBF1 re-expression in cHL cell lines. EBF1 re-expression caused an upregulation of B-cell genes, such as CD19, CD79A and CD79B, although the B-cell genes FOXO1 and PAX5 remained lowly expressed. The re-expression of CD19, CD79A and CD79B occurred largely without demethylation of promoter CpG motifs of these genes. In the cHL cell line L-1236 fitness decreased after EBF1 re-expression. These data show that EBF1 has the ability to reintroduce part of the B-cell signature in cHL cell lines. Loss of EBF1 expression in HRS cells therefore contributes to their lost B-cell phenotype. Notably, in the cHL cell line KM-H2 destructive mutations were found in one allele of EBF1, indicating that genetic lesions may sometimes have a role in impairing EBF1 expression.

Analyzing primary Hodgkin and Reed-Sternberg cells to capture the molecular and cellular pathogenesis of classical Hodgkin lymphoma

The pathogenesis of classical Hodgkin lymphoma (cHL), the most common lymphoma in the young, is still enigmatic, largely because its Hodgkin and Reed-Sternberg (HRS) tumor cells are rare in the involved lymph node and therefore difficult to analyze. Here, by overcoming this technical challenge and performing, for the first time, a genome-wide transcriptional analysis of microdissected HRS cells compared with other B-cell lymphomas, cHL lines, and normal B-cell subsets, we show that they differ extensively from the usually studied cHL cell lines, that the lost B-cell identity of cHLs is not linked to the acquisition of a plasma cell-like gene expression program, and that Epstein-Barr virus infection of HRS cells has a minor transcriptional influence on the established cHL clone. Moreover, although cHL appears a distinct lymphoma entity overall, HRS cells of its histologic subtypes diverged in their similarity to other related lymphomas. Unexpectedly, we identified 2 molecular subgroups of cHL associated with differential strengths of the transcription factor activity of the NOTCH1, MYC, and IRF4 proto-oncogenes. Finally, HRS cells display deregulated expression of several genes potentially highly relevant to lymphoma pathogenesis, including silencing of the apoptosis-inducer BIK and of INPP5D, an inhibitor of the PI3K-driven oncogenic pathway.