Hodgkin Lymphoma Cell Line KM-H2 Research Articles

Establishment of the TBX-code reveals aberrantly activated T-box gene TBX3 in Hodgkin lymphoma.

T-box genes encode transcription factors which control basic processes in development of several tissues including cell differentiation in the hematopoietic system. Here, we analyzed the physiological activities of all 17 human T-box genes in early hematopoiesis and in lymphopoiesis including developing and mature B-cells, T-cells, natural killer (NK)-cells and innate lymphoid cells. The resultant expression pattern comprised six genes, namely EOMES, MGA, TBX1, TBX10, TBX19 and TBX21. We termed this gene signature TBX-code which enables discrimination of normal and aberrant activities of T-box genes in lymphoid malignancies. Accordingly, expression analysis of T-box genes in Hodgkin lymphoma (HL) patients using a public profiling dataset revealed overexpression of EOMES, TBX1, TBX2, TBX3, TBX10, TBX19, TBX21 and TBXT while MGA showed aberrant downregulation. Analysis of T-cell acute lymphoid leukemia patients indicated aberrant overexpression of six T-box genes while no deregulated T-box genes were detected in anaplastic large cell lymphoma patients. As a paradigm we focused on TBX3 which was ectopically activated in about 6% of HL patients analyzed. Normally, TBX3 is expressed in tissues like lung, adrenal gland and retina but not in hematopoiesis. HL cell line KM-H2 expressed enhanced TBX3 levels and was used as an in vitro model to identify upstream regulators and downstream targets in this malignancy. Genomic studies of this cell line showed focal amplification of the TBX3 locus at 12q24 which may underlie its aberrant expression. In addition, promoter analysis and comparative expression profiling of HL cell lines followed by knockdown experiments revealed overexpressed transcription factors E2F4 and FOXC1 and chromatin modulator KDM2B as functional activators. Furthermore, we identified repressed target genes of TBX3 in HL including CDKN2A, NFKBIB and CD19, indicating its respective oncogenic function in proliferation, NFkB-signaling and B-cell differentiation. Taken together, we have revealed a lymphoid TBX-code and used it to identify an aberrant network around deregulated T-box gene TBX3 in HL which promotes hallmark aberrations of this disease. These findings provide a framework for future studies to evaluate deregulated T-box genes in lymphoid malignancies.

Melittin Increases Cisplatin Sensitivity and Kills KM-H2 and L-428 Hodgkin Lymphoma Cells.

Hodgkin lymphoma (HL) is neoplasia with high cure rates. However, not all patients can be cured with the current treatment. Chemo-resistance of tumor cells is one factor involved in treatment failure. In addition to its pore-forming activity on lipid bilayer membranes, the toxin melittin from bee venom is an inhibitor of several cancer-related signaling pathways. Moreover, melittin analogs have been shown to inhibit the activity of ATP binding cassette (ABC) transporters which are known to play important roles in the chemo-resistance of tumor cells. Therefore, we tested the toxicity of melittin for HL cell lines KM-H2 and L-428 and whether melittin can increase the chemo-sensitivity of cisplatin-resistant HL cells. We found high toxicity of melittin for KM-H2 and L-428 cells. In co-cultures with normal blood cells, melittin preferentially killed KM-H2 and L-428 cells. In addition, we observed increased cisplatin sensitivity of chemo-resistant L-428 cells after treatment with melittin. ABC transporter activity was not reduced after treatment with melittin. Our data suggest that melittin or melittin analogs might be promising agents for the future development of treatment strategies for HL patients with resistant disease.

Recurrent somatic mutations of PTPN1 in primary mediastinal B cell lymphoma and Hodgkin lymphoma

Classical Hodgkin lymphoma and primary mediastinal B cell lymphoma (PMBCL) are related lymphomas sharing pathological, molecular and clinical characteristics. Here we discovered by whole-genome and whole-transcriptome sequencing recurrent somatic coding-sequence mutations in the PTPN1 gene. Mutations were found in 6 of 30 (20%) Hodgkin lymphoma cases, in 6 of 9 (67%) Hodgkin lymphoma-derived cell lines, in 17 of 77 (22%) PMBCL cases and in 1 of 3 (33%) PMBCL-derived cell lines, consisting of nonsense, missense and frameshift mutations. We demonstrate that PTPN1 mutations lead to reduced phosphatase activity and increased phosphorylation of JAK-STAT pathway members. Moreover, silencing of PTPN1 by RNA interference in Hodgkin lymphoma cell line KM-H2 resulted in hyperphosphorylation and overexpression of downstream oncogenic targets. Our data establish PTPN1 mutations as new drivers in lymphomagenesis.

Abstract 4694: The small molecule CHK1/CHK2 inhibitor PF-0477736 (Pfizer) demonstrates single agent activity in diffuse large B-cell lymphoma

Abstract Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the DNA damage response pathway. CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, including p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL with dysfunctional p53 axis (harboring p53 mutations and/or CDKN2A loss) have been recently shown to have a dismal outcome. In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a panel of B cell lymphoma cell lines and primary cells, and explore its mechanisms of action. Three Germinal center B cell (GCB) DLBCL derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2 were first screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but with an homozygous deletion of CDKN2A. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity (IC50 10 nM at 24 hrs). The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 25-500 nM induced cell death by apoptosis in a time and dose dependent manner. Of note PF-0477736 100-1000 nM demonstrated activity also in primary DLBCL cells. We found no correlations between baseline levels of CHK1/2 activation and outcome. In the sensitive cell lines inhibition of the downstream target CDC25c ser216 phosphorylation coupled with a marked increase in levels of the DNA damage marker γH2AX was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25 - 250 nM). PF-0477736 at the dose of 50-100 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) at 24 hrs. These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome, providing the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy. PF-0477736 was provided by Pfizer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4694. doi:1538-7445.AM2012-4694

The Small Molecule JAK1/JAK2 Inhibitor INCB16562 Shows Single Agent Activity and Strongly Synergizes with Bcl-2 Inhibitors in Lymphoma

Abstract 2731Constitutive activation of Janus Kinases has been shown to be an important mechanism supporting human lymphomagenesis. Aberrantly activated JAK/STAT signaling has been described in Hodgkin lymphoma (HL), Anaplastic Large T-cell lymphoma and Diffuse large B cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, making it an attractive target for therapy. In this study we report the activity profile of the JAK2 inhibitor INCB16562 in a panel of lymphoma cell lines (n=17) including Hodgkin (HL), Mantle Cell (MCL), Anaplastic Large T-cell (ALCL), and DLBCL. JAK-STAT pathway was found to be constitutively active (baseline p-STAT3 Tyr 705 detected by western immunoblotting) in the HL (HDLM-2, L.-428 and L-540), ALCL (SUP-M2, KARPAS-299, SUDHL-1) and the ABC derived DLBCL cell lines (HBL-1, U2932, TMD8, OCI-LY-3). In contrast, no activation of JAK-STAT was observed in the HL cell line KM-H2, in the MCL cell lines (Mino, Jeko-1, SP-53) and in the GCB-derived cell lines (SUDHL-4, SUDHL-6, BJAB). To assess the effect of INCB16562 on cell proliferation, cells were first incubated with increasing concentrations of INC16562 (from 0.1 to 10 μM) for 24, 48 and 72 hours (hrs) and cell viability was evaluated by MTS assay. The IC50 values at 48 hrs ranged from 1 to 9 μM. The ABC derived DLBCL cell line TMD8 showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with INCB16562 1μM for 24 hours. Of note, INCB16562 demostrated activity also in primary DLBCL cells. pSTAT3 negative cell lines were resistant to treatment (IC50 from 4.8 to 6.5 μM). However pretreatment level of pJAKs and pSTATs did not predict sensitivity to INCB16562. In fact inhibition of STAT3 phosphorylation alone was not sufficient to predict sensitivity to the drug in pSTAT3 expressing cells. Simultaneous inhibition of STAT3 and ERK phosphorylation was observed by western immunoblotting in the sensitive cell lines (TMD8, HBL-1, SUP-M2, KARPAS 299, SUDHL-1) (IC50 from 1 to 3 μM), whereas in the resistant cell lines (U2932, OCI-LY3, HD-LM2, L-540, L-428) a paradoxical hyperactivation of pERK was observed after incubation with INCB16562 (IC50 from 5–9 μM). Since it has been reported that sensitivity to JAK inhibition is associated to downregulation of bcl-2 family genes such as Bcl-xL and MCL-1, we investigated the baseline expression of these bcl-2 family members in our cell lines by western blot. Bcl-xL and MCL-1 were expressed at various levels, but there was no correlation between levels of baseline expression and outcome. Both Bcl-xL and MCL-1 were dowregulated in the sensitive cell lines following treatment with INCB16562. Furthermore The bcl-2 inhibitor ABT-737 synergistically enhanced the effect of INCB16562 particularly in HL cell lines (HDLM-2 and L-540) and DLBCL cells lines (HBL-1, TMD8, SUDHL-4). These data demonstrate that INCB16562 has a promising therapeutic value in lymphoma, and provide a rationale for combining INCB16562 with bcl-2 family inhibitors. Disclosures:No relevant conflicts of interest to declare.

Inactivating Gene Alterations of MHC Class II Transactivator CIITA Are Recurrent in Primary Mediastinal B Cell Lymphoma and Hodgkin Lymphoma

Abstract 437 INTRODUCTION:Escape from immune surveillance has been proposed to play an important role in cancer and in particular in the pathogenesis of a subgroup of lymphomas. Using massively parallel sequencing, we have recently identified recurrent rearrangements of the CIITA gene in classical Hodgkin lymphoma (HL) and primary mediastinal B cell lymphoma (PMBCL) (Steidl et al, Nature 2011). Specifically, a gene fusion in the HL cell line KM-H2, involving the genes CIITA and FLJ27352, suppressed expression of CIITA-regulated MHC class II genes in a dominant-negative manner. Therefore, we sought to further investigate if gene alterations targeting the CIITA locus are recurrent somatic events in HL and PMBCL that lead to loss of gene function and thereby reduce immunogenicity of the malignant cells. PATIENTS AND METHODS: To characterize CIITA gene alterations, we comprehensively studied the cell lines DEV (nodular lymphocyte predominance HL-derived) and KARPAS1106P (PMBCL-derived) by whole-transcriptome paired-end sequencing (RNA-seq) and single nucleotide polymorphism arrays (Affymetrix SNP 6.0). To identify single nucleotide mutations in the CIITA coding sequence we studied additional 7 mediastinal biopsy specimens of PMBCL by RNA-seq. Fluorescence in-situ hybridization (FISH) was used to determine and validate structural and copy number alterations of the CIITA locus. 23 samples of PMBCL, 3 PMBCL-derived cell lines and 9 HL cell lines were studied for mutations and small deletions in CIITA intron 1 by long-range PCR and direct sequencing. CIITA and HLA-DR expression levels were determined by quantitative reverse transcriptase PCR. RESULTS: We identified chromosomal rearrangements affecting both alleles of CIITA in DEV cells resulting in expression of CIITA-PDL2, CIITA-SOCS1 and SOCS1-CIITA fusion transcripts. In KARPAS1106P cells chromosome 16 deletions result in the loss of one entire CIITA allele and partial loss of the other allele. As a consequence, in both cell lines wildtype CIITA and HLA-DR expression was undetectable. Genomic breakpoints within the CIITA rearrangements, together with previously identified translocations, defined a 1.6Kb breakpoint cluster region in CIITA Intron 1. Further analysis of this breakpoint cluster region revealed small intronic deletions in 10 of 23 (43%) PMBCL cases while no such deletions were detected in 18 diffuse large B cell lymphoma and 15 reactive lymph node samples. Furthermore, sequencing revealed multiple deletions ranging from 1–1948 bp and a high incidence of single nucleotide mutations in the breakpoint cluster region of the deleted alleles. The base pair changes were enriched for C to T and G to A transitions over other transitions and transversions, indicative of a somatic hypermutation process. We found significantly lower CIITA expression in cases with CIITA rearrangement and/or small intronic deletions compared to cases without CIITA alterations (p=0.044). RNAseq revealed three cases with non-synonymous coding-sequence mutations including a truncating mutation in exon 4. These changes were somatic in 3 cases with available matching normal DNA. DISCUSSION:CIITA is the target of multiple genomic hits including chromosomal translocation, deletion and coding sequence mutations in HL and PMBCL. In two HL and PMBCL cell lines this leads to complete gene inactivation and loss of HLA class II expression. These data characterize CIITA alterations as a recurrent underlying genomic event of the previously described immunophenotype of reduced HLA class II expression in a subset of PMBCL cases. Our data show that an immune escape mechanism utilized by tumor cells can be directly linked to somatically acquired alterations in cancer genomes. Disclosures:Siebert:Abbott/Vysis: .

The Small Molecule CHK1/CHK2 Inhibitor PF-0477736 (Pfizer) Demonstrates Single Agent Activity and Synergizes with Chemotherapy in Diffuse Large B-Cell Lymphoma

Abstract 2732The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the signal transduction mechanims of the DNA damage response pathway. Once activated by upstream kinases [Ataxia-Telangiectasia mutated (ATM) and Ataxia-Telangiectasia and Rad3-related (ATR) kinases] following DNA damage, they phosphorylate downstream targets such as CDC25 phosphatases and p53, promoting G2/M cell cycle arrest, in order to facilitate DNA repair. Furthermore is now clear that the efficacy of conventional DNA-damaging anticancer drugs is limited by the activity of these protective cell cycle checkpoints. The tumor suppressor p53 is activated in normal cells following extensive DNA damage and promotes G1 cell cycle arrest and apoptosis. Cells lacking p53 activity are more resistant to genotoxic agents. It has been shown that CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, by inhibiting the response to DNA damage, preferentially in p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL harboring p53 mutations and/or CDKN2A loss have been recently shown to have a dismal outcome, being refractory to conventional antracyclin-based chemotherapy. Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a large panel of B cell lymphoma cell lines, and explore its mechanisms of action. Nine cell lines were used for in vitro viability assays: 3 Germinal center (GCB) Diffuse Large B-cell lymphoma (DLBCL) derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2. All the cell lines were screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but an homozygous deletion of CDKN2A was detected. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were first incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs of incubation, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with PF-0477736 10nM for 24 hours. The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 250–500 nM induced cell death by apoptosis in a time and dose dependent manner after 24 and 48 hours of incubation. Lower concentrations of PF-0477736 (25–50 nM) promoted a statistically significant increase in cell death only in the BJAB cells. For functional studies we characterized the two most sensitive cell lines (BJAB and U2932) and the two resistant cell lines (SUDHL-4 and KM-H2). Inhibition of cdc25c ser216 phosphorylation was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25–250 nM). A marked increase in levels of the DNA damage marker γH2AX, was detected in the BJAB, U2932, SUDHL-4 cell lines after 24 hrs. KM-H2 did not show any increase of γH2AX following treatment. All the cell lines demonstrated baseline CHK1 activation but there was no correlation with outcome. Interestingly levels of baseline pcdc25c ser216 were higher in the sensitive BJAB and U2932 cells. PF-0477736 at the fixed dose of 50 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) in the BJAB and U2932 cells at 24 hrs.These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The integrity of the p53 axis seems to be the major determinant of efficacy of PF-0477736. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome. Our study provides the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy.PF-0477736 was provided by Pfizer. Disclosures:No relevant conflicts of interest to declare.

Constitutively overexpressed 21 kDa protein in Hodgkin lymphoma and aggressive non-Hodgkin lymphomas identified as cytochrome B5b (CYB5B)

BackgroundWe have previously reported a novel constitutively overexpressed 21 kDa protein in Hodgkin Lymphoma (HL) and aggressive Non-Hodgkin Lymphomas (NHL). The objective of the current study was to 1) identify this protein using two independent methods, 2) study the expression of the protein and its encoding mRNA in reactive lymph nodes, normal lymphocytes and CD34+ bone marrow precursor cells, 3) analyse patterns of expression of the protein in tissue microarrays assembled from a large number of diagnostic clinical biopsies from patients with HL, and 4) determine the copy number variation and mutation status of the encoding gene in HL cell lines.ResultsPeptide sequencing by LC-MS/MS and protein identification by protein array screening identified a single protein, CYB5B. No mutations were detected in the CYB5B gene in HL cell lines. Quantitative PCR showed CYB5B gene expression was increased in HL and NHL cell lines. Array CGH using a submegabase resolution tiling array revealed gains in the CYB5B locus in HL cell lines KMH2 and L428. Membrane expression was seen in Reed-Sternberg cells in clinical biopsies from patients with HL but not in reactive lymph nodes. Bone marrow CD34+ precursor cells were CYB5B negative on the cell surface. RT-PCR assays of RNA extracted from T and B cell enriched fractions obtained from normal peripheral blood mononuclear cells, reactive lymph nodes, tonsils and normal bone marrow samples showed no evidence of increased mRNA levels of CYB5B in comparison to housekeeping gene GAPDH.ConclusionsThe 21 kDa protein overexpressed in HL and aggressive NHL is identical to CYB5B. CYB5B gene expression is increased in a subset of HL and NHL cell lines tested. This is associated with CYB5B gene amplification in HL cell lines KMH2 and L428. CYB5B may be a potential target for antibody-based therapy of HL and aggressive NHL as although cytoplasmic expression is present in reactive lymphocytes, it is not expressed on the cell surface of non-neoplastic lymphocytes or bone marrow precursor cells.

Transactivation of CCL20 Gene by CD30 in Hodgkin's Lymphoma.

Hodgkin's lymphoma (HL) is characterized by a minority of neoplastic cells, the so-called Hodgkin/Reed-Sternberg (H/RS) cells, and an admixture of reactive cells including lymphocytes, plasma cells, eosinophils, and histiocytes. Chemokines produced by H/RS cells play a major role in leukocyte trafficking. These molecules with specific receptor affinities contribute to the maintenance of a favorable environment for survival of H/RS cells. The C-C chemokine CCL20 is expected to play a crucial role in the initiation of immune responses and tumor growth. However, expression of CCL20 in HL has not been studied. We found that in lymph nodes from patients with HL, CCL20 was expressed in H/RS cells by immunohistochemistry. CCL20 was expressed in HL cell lines KM-H2 and L428 established from B-cell type HL patients, but not in cell lines L540 and HDLM-2 established from T-cell type HL patients. CD30 was identified in H/RS cells of HL and has attracted much interest as a molecular marker of HL. All four H/RS cell lines expressed CD30, but not CD30 ligand. CD30 induced CCL20 mRNA expression. Analysis of the CCL20 promoter showed the importance of NF-κB binding site for CD30-induced CCL20 expression. Coexpression of IκB, IKK, NIK, and TRAF dominant-negative constructs with CD30 inhibited CD30-induced activation of CCL20 promoter, suggesting that CD30 induces CCL20 via NF-κB signaling. Electrophoretic mobility shift assay showed the NF-κB complexes that consisted of p50, p65, and c-Rel in KM-H2 and L428 cells. In contrast, the NF-κB complexes contained p50 homodimer that was transcriptionally repressive in L540 and HDLM-2 cells. The CCL20 promoter was activated by the C-terminal region of CD30, which activated NF-κB signaling. A decoy CD30 lacking the cytoplasmic region inhibited CCL20 promoter activity in KM-H2 and L428 cells. Thus, in B-cell derived H-RS cells, ligand-independent activation of CD30 signaling activates NF-κB and this leads to constitutive CCL20 expression.