Maciej Giefing

Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma

Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell–derived Hodgkins lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkins lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberrantly. We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications.

Detection of genomic imbalances in microdissected Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma by array-based comparative genomic hybridization

Cytogenetic analysis of classical Hodgkin’s lymphoma is limited by the low content of the neoplastic Reed-Sternberg cells in the affected tissues. The present study demonstrates that array comparative genomic hybridization of microdissected Hodgkin-Reed-Sternberg cells is suitable for identifying and characterizing chromosomal imbalances. Se related perspective article on page 1292. Background Cytogenetic analysis of classical Hodgkin’s lymphoma is limited by the low content of the neoplastic Hodgkin-Reed-Sternberg cells in the affected tissues. However, available cytogenetic data point to an extreme karyotype complexity. To obtain insights into chromosomal imbalances in classical Hodgkin’s lymphoma, we applied array-based comparative genomic hybridization (array comparative genomic hybridization) using DNA from microdissected Hodgkin-Reed-Sternberg cells. Design and Methods To avoid biases introduced by DNA amplification for array comparative genomic hybridization, cHL cases rich in Hodgkin-Reed-Sternberg cells were selected. DNA obtained from approximately 100,000 microdissected Hodgkin-Reed-Sternberg cells of each of ten classical Hodgkin’s lymphoma cases was hybridized onto commercial 105 K oligonucleotide comparative genomic hybridization microarrays. Selected imbalances were confirmed by interphase cytogenetics and quantitative polymerase chain reaction analysis and further studied in an independent series of classical Hodgkin’s lymphoma. Results Gains identified in at least five cHL affected 2p12-16, 5q15-23, 6p22, 8q13, 8q24, 9p21-24, 9q34, 12q13-14, 17q12, 19p13, 19q13 and 20q11 whereas losses recurrent in at least five cases involved Xp21, 6q23-24 and 13q22. Copy number changes of selected genes and a small deletion (156 kb) of the CDKN2B (p15) gene were confirmed by interphase cytogenetics and polymerase chain reaction analysis, respectively. Several gained regions included genes constitutively expressed in cHL. Among these, gains of STAT6 (12q13), NOTCH1 (9q34) and JUNB (19p13) were present in additional cHL with the usual low Hodgkin-Reed-Sternberg cell content. Conclusions The present study demonstrates that array comparative genomic hybridization of microdissected Hodgkin-Reed-Sternberg cells is suitable for identifying and characterizing chromosomal imbalances. Regions affected by genomic changes in Hodgkin-Reed-Sternberg cells recurrently include genes constitutively expressed in cHL.

Genetic lesions of the TRAF3 and MAP3K14 genes in classical Hodgkin lymphoma.

Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) show constitutive activation of nuclear factor (NF)‐κB. Several genetic lesions contribute to this deregulated NF‐κB activity. Here, we analysed two further NF‐κB regulators for genetic lesions, the inhibitory factor TRAF3 and the key signalling component of the alternative NF‐κB pathway, MAP3K14 (NIK). Single nucleotide polymorphism (SNP) array analysis of cHL cell lines revealed a uniparental disomy of the long arm of chromosome 14 associated with a biallelic deletion of TRAF3 located on this chromosome in cell line U‐HO1. Cloning of the deletion breakpoint showed a 123 371 bp deletion. No inactivating mutations of TRAF3 were found in six other cHL cell lines or in microdissected HRS cells from seven cHL. However, in primary cHL samples interphase cytogenetic analyses revealed signal patterns indicating monoallelic deletion of TRAF3 in 3/20 other cases. SNP array analysis revealed a gain of copy number for MAP3K14 in three cHL cell lines. Gains of MAP3K14 were detected in 5/16 cases of primary cHL. In conclusion, in rare instances, HRS cells harbour inactivating mutations of the TRAF3 gene and recurrently show gains of MAP3K14, indicating that more components of NF‐κB signalling show genetic lesions in HRS cells than previously known.

High Resolution ArrayCGH and Expression Profiling Identifies PTPRD and PCDH17/PCH68 as Tumor Suppressor Gene Candidates in Laryngeal Squamous Cell Carcinoma

Many classical tumor suppressor genes (TSG) were identified by delineation of bi‐allelic losses called homozygous deletions. To identify systematically homozygous deletions in laryngeal squamous cell carcinoma (LSCC) and to unravel novel putative tumor suppressor genes, we screened 10 LSCC cell lines using high resolution array comparative genomic hybridization (arrayCGH) and array based expression analysis. ArrayCGH identified altogether 113 regions harboring protein coding genes that showed strong reduction in copy number indicating a potential homozygous deletion. Out of the 113 candidate regions, 22 novel homozygous deletions that affected the coding sequences of 15 genes were confirmed by multiplexPCR. Three genes were homozygously lost in two cell lines: PCDH17/PCH68, PRR20, and PTPRD. For the 15 homozygously deleted genes, four showed statistically significant downregulation of expression in LSCC cell lines as compared with normal human laryngeal controls. These were ATG7 (1/10 cell line), ZMYND11 (BS69) (1/10 cell line), PCDH17/PCH68 (9/10 cell lines), and PTPRD (7/10 cell lines). Quantitative real‐time PCR was used to confirm the downregulation of the candidate genes in 10 expression array‐studied cell lines and an additional cohort of cell lines; statistical significant downregulation of PCDH17/PCH68 and PTPRD was observed. In line with this also Western blot analyses demonstrated a complete absence of the PCDH17 and PTPRD proteins. Thus, expression profiling confirmed recurrent alterations of two genes identified primarily by delineation of homozygous deletions. These were PCDH17/PCH68, the protocadherin gene, and the STAT3 inhibiting receptor protein tyrosine phosphatase gene PTPRD. These genes are good candidates for novel TSG in LSCC.

Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma

Significance Human lymphomas and leukemias are characterized by molecular and structural alterations of transcription factors (TFs). The identification of such deregulated TFs is therefore central to the understanding of lymphomagenesis. We addressed this question in classical Hodgkin lymphoma (HL), a common B-cell–derived malignancy that is one of the most prominent examples for complex patterns of deregulated TFs including the activation of NF-κB or AP-1 and a profound deregulation of lineage-specific TFs. We found that IRF5 together with NF-κB induces a number of HL characteristic features in non-Hodgkin cells, such as expression of cytokines and chemokines or AP-1 activation. Our work exemplifies how the global lymphoma type-specific characterization of TF activities can improve the understanding of tumor biology. Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TF-binding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell–derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-κB to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5.

Loss of expression of LyGDI (ARHGDIB), a rho GDP‐dissociation inhibitor, in Hodgkin lymphoma*

The guanosine triphosphatase (GTPase) inhibitor LyGDI (ARHGDIB, Ly/D4‐GDI, RhoGDIb or RhoGDI 2) is abundantly expressed in haematopoetic cells and possibly plays a role in the onset of apoptosis. Gene expression profiling of Hodgkin cell lines revealed that LyGDI expression was downregulated in these cell lines. The present study evaluated the expression of LyGDI in Hodgkin cells in vivo and studied the function of LyGDI in Hodgkin cell lines in vitro. Our results showed that virtually all Hodgkin and Reed‐Sternberg cells in classical Hodgkin lymphoma lacked LyGDI protein expression. On the other hand, almost all non‐Hodgkin lymphomas, except for anaplastic large cell lymphomas, expressed LyGDI protein. Transfection of the classical Hodgkin cell line L428 with a vector containing full‐length LyGDI‐induced apoptosis in a subset of cells. However, the majority of Hodgkin cells with transgenic expression of LyGDI escaped apoptosis. Our data show that lack of LyGDI expression is a frequent feature of cHL but that it is not of vital importance for the growth and survival of these cells.

Identification of candidate tumour suppressor gene loci for Hodgkin and Reed-Sternberg cells by characterisation of homozygous deletions in classical Hodgkin lymphoma cell lines.

Several tumour suppressor genes (TSG) have been identified as a result of mapping homozygous deletions in cancer cells. To identify putative TSG involved in the pathogenesis of classical Hodgkin lymphoma (cHL), we investigated four cHL cell lines (L428, HDLM2, KMH2, L1236) using four different array‐Comparative Genomic Hybridisation (array‐CGH) platforms and focused on high resolution identification of homozygous deletions. Out of 79 candidate regions of bi‐allelic loss identified by array‐CGH, besides previously described regions, 28 novel regions of homozygous deletions could be verified by polymerase chain reaction. These regions ranged from 13 kb to 619 kb in size. Eleven of the 28 novel bi‐allelic losses were putative copy number polymorphisms. This left 17 regions that might harbour novel tumour suppressors involved in Hodgkin lymphoma. Expression profiling with two different platforms confirmed lack of expression of the majority of the genes located in the homozygous deletions. Furthermore, analysis of ontology annotations of genes located in the homozygously deleted regions indicated an enrichment of genes involved in apoptosis and cell death. In summary, through the mapping of homozygous deletions in cell lines this study identified a series of genes, such as SEPT9, GNG7 and CYBB, which might encode candidate tumour suppressors involved in the pathogenesis of cHL.

Pyrosequencing-based DNA methylation profiling of Fanconi anemia/BRCA pathway genes in laryngeal squamous cell carcinoma

Fanconi anemia (FA) associated genes [FANCA, -B, -C, FANCD1(BRCA2), -D2, -E, -F, -G, -I, -L, -M, FANCN (PALB2), FANCJ(BRIP1) and FA-linked BRCA1] encode proteins of DNA damage response pathways mutated in FA patients. FA is characterized by congenital malformations, chromosomal instability and high cancer susceptibility. FA patients have a 500-700 times higher risk of head and neck squamous cell carcinoma (HNSCC) compared to the non-FA population. As DNA methylation comprises one of the known gene inactivation mechanisms in cancer we have investigated the methylation status of 13 FA and one FA-linked gene in order to assess the role of FA in sporadic laryngeal squamous cell carcinoma (LSCC) tumor samples. Thirteen laryngeal squamous carcinoma cell lines (UT-SCC) and 64 primary laryngeal carcinoma cases were analyzed by bisulfite pyrosequencing. DNA from buccal swabs of 10 healthy volunteers was used as a control group. Promoter regions of FANCA, BRCA1 and BRCA2 displayed recurrent alterations in the methylation levels in cancer samples as compared to buccal swabs controls. For FANCA, hypomethylation was observed in 11/13 cell lines (p<0.0003) and all 64 primary larynx samples (p<0.001) compared to buccal swabs. For BRCA1, 4/13 cell lines (p=0.04) and 3/58 primary laryngeal cases (p=0.22) showed hypomethylation. In BRCA2, all 13 cell lines (p<0.0001) 4/63 primary LSCC (p<0.01) showed hypermethylation as compared to controls. In conclusion, we show recurrent alterations of DNA methylation levels in three Fanconi anemia genes which might contribute to the pathogenesis of LSCC.

Rare occurrence of biallelic CYLD gene mutations in classical Hodgkin lymphoma

Survival of the malignant Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) is dependent on constitutive activation of the nuclear factor κB (NF‐κB) transcription factor. The deubiquitinating enzyme CYLD is a negative regulator of NF‐κB and known to function as a tumor suppressor. To determine whether CYLD mutations play a role in cHL pathogenesis, we sequenced the gene in cHL cell lines and microdissected HRS cells obtained from lymph‐node biopsies. A biallelic inactivation by mutations was found in the cHL cell‐line KM‐H2. However, the other seven cHL cell lines analyzed and HRS cells of 10 primary cHL cases did not show any mutations. By interphase cytogenetics, a (sub)clonal biallelic CYLD deletion was observed by interphase cytogenetics in 1 of 29 primary cHL, whereas signal patterns indicating decreased CYLD copy numbers were observed in a total of 10 of 29 primary cases. Our results suggest that biallelic CYLD mutations are rarely involved in cHL pathogenesis. Nevertheless, it is remarkable that KM‐H2 cells, besides the CYLD mutations, also carry inactivating mutations in the genes of two other NF‐κB inhibitors, that is, NFKBIA and TNFAIP3, exemplifying that multiple lesions in regulators of this signaling pathway can likely cooperatively contribute to the strong NF‐κB activity of these cells.

Combining array-based approaches for the identification of candidate tumor suppressor loci in mature lymphoid neoplasms†

Tumor suppressor gene (TSG) inactivation by chromosomal deletions and/or mutations is a well‐characterized genetic alteration in lymphomas. Array‐based technologies have greatly increased the detection and characterization of chromosomal imbalances and regions with loss of heterozygosity (LOH), leading to the identification of a number of novel candidate TSG loci. In addition, microarray platforms for studying DNA methylation and histone modifications enable identifying epigenetic changes affecting gene expression of TSG. Combining these microarray technologies with gene expression profiling is a promising strategy to discover novel TSG in regions targeted by genetic or epigenetic alterations. In this review we present an outline of methodological aspects of the various microarray technologies, and discuss their potentials and restrictions. Furthermore, we survey research findings derived from these high‐throughput techniques, which are allowing a deeper insight into the mechanisms of lymphomagenesis.