Establishment of the Myeloid TBX-Code Reveals Aberrant Expression of T-Box Gene TBX1 in Chronic Myeloid Leukemia.

T-box genes encode transcription factors, which control developmental processes and promote cancer if deregulated. Recently, we described the lymphoid TBX-code, which collates T-box gene activities in normal lymphopoiesis, enabling identification of members deregulated in lymphoid malignancies. Here, we have extended this analysis to cover myelopoiesis, compiling the myeloid TBX-code and, thus, highlighting which of these genes might be deregulated in myeloid tumor types. We analyzed public T-box gene expression datasets bioinformatically for normal and malignant cells. Candidate T-box-gene-expressing model cell lines were identified and examined by RQ-PCR, Western Blotting, genomic profiling, and siRNA-mediated knockdown combined with RNA-seq analysis and live-cell imaging. The established myeloid TBX-code comprised 10 T-box genes, including progenitor-cell-restricted TBX1. Accordingly, we detected aberrant expression of TBX1 in 10% of stem/progenitor-cell-derived chronic myeloid leukemia (CML) patients. The classic CML cell line K-562 expressed TBX1 at high levels and served as a model to identify TBX1 activators, including transcription factor GATA1 and genomic amplification of the TBX1 locus at 22q11; inhibitors, including BCR::ABL1 fusion and downregulated GNAI2, as well as BMP, FGF2, and WNT signaling; and the target genes CDKN1A, MIR17HG, NAV1, and TMEM38A. The establishment of the myeloid TBX-code permitted identification of aberrant TBX1 expression in subsets of CML patients and cell lines. TBX1 forms an integral part of an oncogenic regulatory network impacting proliferation, survival, and differentiation. Thus, the data spotlight novel diagnostic markers and potential therapeutic targets for this malignancy.

The Hematopoietic TALE-Code Shows Normal Activity of IRX1 in Myeloid Progenitors and Reveals Ectopic Expression of IRX3 and IRX5 in Acute Myeloid Leukemia.

Homeobox genes encode transcription factors that control basic developmental decisions. Knowledge of their hematopoietic activities casts light on normal and malignant immune cell development. Recently, we constructed the so-called lymphoid TALE-code that codifies expression patterns of all active TALE class homeobox genes in early hematopoiesis and lymphopoiesis. Here, we present the corresponding myeloid TALE-code to extend this gene signature, covering the entire hematopoietic system. The collective data showed expression patterns for eleven TALE homeobox genes and highlighted the exclusive expression of IRX1 in megakaryocyte-erythroid progenitors (MEPs), implicating this TALE class member in a specific myeloid differentiation process. Analysis of public profiling data from acute myeloid leukemia (AML) patients revealed aberrant activity of IRX1 in addition to IRX3 and IRX5, indicating an oncogenic role for these TALE homeobox genes when deregulated. Screening of RNA-seq data from 100 leukemia/lymphoma cell lines showed overexpression of IRX1, IRX3, and IRX5 in megakaryoblastic and myelomonocytic AML cell lines, chosen as suitable models for studying the regulation and function of these homeo-oncogenes. Genomic copy number analysis of IRX-positive cell lines demonstrated chromosomal amplification of the neighboring IRX3 and IRX5 genes at position 16q12 in MEGAL, underlying their overexpression in this cell line model. Comparative gene expression analysis of these cell lines revealed candidate upstream factors and target genes, namely the co-expression of GATA1 and GATA2 together with IRX1, and of BMP2 and HOXA10 with IRX3/IRX5. Subsequent knockdown and stimulation experiments in AML cell lines confirmed their activating impact in the corresponding IRX gene expression. Furthermore, we demonstrated that IRX1 activated KLF1 and TAL1, while IRX3 inhibited GATA1, GATA2, and FST. Accordingly, we propose that these regulatory relationships may represent major physiological and oncogenic activities of IRX factors in normal and malignant myeloid differentiation, respectively. Finally, the established myeloid TALE-code is a useful tool for evaluating TALE homeobox gene activities in AML.

NKL Homeobox Genes NKX2-3 and NKX2-4 Deregulate Megakaryocytic-Erythroid Cell Differentiation in AML.

NKL homeobox genes encode transcription factors that impact normal development and hematopoietic malignancies if deregulated. Recently, we established an NKL-code that describes the physiological expression pattern of eleven NKL homeobox genes in the course of hematopoiesis, allowing evaluation of aberrantly activated NKL genes in leukemia/lymphoma. Here, we identify ectopic expression of NKL homeobox gene NKX2-4 in an erythroblastic acute myeloid leukemia (AML) cell line OCI-M2 and describe investigation of its activating factors and target genes. Comparative expression profiling data of AML cell lines revealed in OCI-M2 an aberrantly activated program for endothelial development including master factor ETV2 and the additional endothelial signature genes HEY1, IRF6, and SOX7. Corresponding siRNA-mediated knockdown experiments showed their role in activating NKX2-4 expression. Furthermore, the ETV2 locus at 19p13 was genomically amplified, possibly underlying its aberrant expression. Target gene analyses of NKX2-4 revealed activated ETV2, HEY1, and SIX5 and suppressed FLI1. Comparative expression profiling analysis of public datasets for AML patients and primary megakaryocyte-erythroid progenitor cells showed conspicuous similarities to NKX2-4 activating factors and the target genes we identified, supporting the clinical relevance of our findings and developmental disturbance by NKX2-4. Finally, identification and target gene analysis of aberrantly expressed NKX2-3 in AML patients and a megakaryoblastic AML cell line ELF-153 showed activation of FLI1, contrasting with OCI-M2. FLI1 encodes a master factor for myelopoiesis, driving megakaryocytic differentiation and suppressing erythroid differentiation, thus representing a basic developmental target of these homeo-oncogenes. Taken together, we have identified aberrantly activated NKL homeobox genes NKX2-3 and NKX2-4 in AML, deregulating genes involved in megakaryocytic and erythroid differentiation processes, and thereby contributing to the formation of specific AML subtypes.

False and mycoplasma-contaminated leukemia-lymphoma cell lines: time for a reappraisal.

Leukemia-lymphoma cell lines are important research tools in a variety of fields. To represent adequate model systems it is of utmost importance that cell lines faithfully model the primary tumor material and are not cross-contaminated with unrelated cell material (or contaminated with mycoplasma). As it has been previously reported that cross-contaminated cell lines represent a significant problem, it is of interest to know whether any improvement in the prevalence of such "false cell lines" had occurred since we called the alert in 1999. A retrospective review of our data archives covered 848 cell lines received from 1990 to 2014 from 290 laboratories in 23 countries spanning the spectrum of leukemia-lymphoma entities. Two variables were considered: authenticity and freedom from mycoplasma infection. Regarding provenance, we separately considered primary sources (original investigators having established the cell lines or reference repositories) and secondary sources. The percentages of mycoplasma-contaminated cell lines decreased significantly over the 25-year timespan. Among primary sourced material: mycoplasma-contamination fell from 23% to 0%; among secondary sourced: from 48% to 21%. The corresponding figures for cross-contamination declined from 15% to 6%, while among material obtained from secondary sources prevalence remained remarkably high, throughout the time periods at 14-18%. Taken together, our data indicate that using non-authenticated cell lines from secondary sources carries a risk of about 1:6 for obtaining a false cell line. The use of authentic leukemia-lymphoma cell lines holds important translational value for their model character and the reproducibility of the laboratory data in the clinical arena.

Mechanisms underlying epigenetic and transcriptional heterogeneity in Chinese hamster ovary (CHO) cell lines.

BACKGROUND: Recombinant cell lines developed for therapeutic antibody production often suffer instability or lose recombinant protein expression during long-term culture. Heterogeneous gene expression among cell line subclones may result from epigenetic modifications of DNA or histones, the protein component of chromatin. We thus investigated in such cell lines, DNA methylation and the chromatin environment along the human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter in an antibody protein-expression vector which was integrated into the Chinese hamster ovary (CHO) cell line genome. RESULTS: We analyzed four PT1-CHO cell lines which exhibited losses of protein expression at advanced passage number (>P35) growing in adherent conditions and in culture medium with 10 % FCS. These cell lines exhibited different integration sites and transgene copy numbers as determined by fluorescence in situ hybridization (FISH) and quantitative PCR (qPCR), respectively. By qRT-PCR, we analyzed the recombinant mRNA expression and correlated it with DNA methylation and with results from various approaches interrogating the chromatin landscape along the EEF1A1 promoter region. Each PT1-CHO cell line displayed specific epigenetic signatures or chromatin marks correlating with recombinant mRNA expression. The cell line with the lowest recombinant mRNA expression (PT1-1) was characterized by the highest nucleosome occupancy and displayed the lowest enrichment for histone marks associated with active transcription. In contrast, the cell line with the highest recombinant mRNA expression (PT1-55) exhibited the highest numbers of formaldehyde-assisted isolation of regulatory elements (FAIRE)-enriched regions, and was marked by enrichment for histone modifications H3K9ac and H3K9me3. Another cell line with the second highest recombinant mRNA transcription and the most stable protein expression (PT1-7) had the highest enrichments of the histone variants H3.3 and H2A.Z, and the histone modification H3K9ac. A further cell line (PT1-30) scored the highest enrichments for the bivalent marks H3K4me3 and H3K27me3. Finally, DNA methylation made a contribution, but only in the culture medium with reduced FCS or in a different expression vector. CONCLUSIONS: Our results suggest that the chromatin state along the EEF1A1 promoter region can help predict recombinant mRNA expression, and thus may assist in selecting desirable clones during cell line development for protein production.

A recurrent 11q aberration pattern characterizes a subset of MYC-negative high-grade B-cell lymphomas resembling Burkitt lymphoma.

The genetic hallmark of Burkitt lymphoma (BL) is the t(8;14)(q24;q32) and its variants leading to activation of the MYC oncogene. It is a matter of debate whether true BL without MYC translocation exists. Here, we identified 59 lymphomas concordantly called BL by 2 gene expression classifiers among 753 B-cell lymphomas. Only 2 (3%) of these 59 molecular BL lacked a MYC translocation, which both shared a peculiar pattern of chromosome 11q aberration characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter. We extended our analysis to 17 MYC-negative high-grade B-cell lymphomas with a similar 11q aberration and showed this aberration to be recurrently associated with morphologic and clinical features of BL. The minimal region of gain was defined by high-level amplifications in 11q23.3 and associated with overexpression of genes including PAFAH1B2 on a transcriptional and protein level. The recurrent region of loss contained a focal homozygous deletion in 11q24.2-q24.3 including the ETS1 gene, which was shown to be mutated in 4 of 16 investigated cases. These findings indicate the existence of a molecularly distinct subset of B-cell lymphomas reminiscent of BL, which is characterized by deregulation of genes in 11q.

NFkB is activated by multiple mechanisms in hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare chronic B-cell lymphoproliferative disorder of unclear pathogenesis. Recent studies have identified BRAF(V600E) mutations in most HCL patients, highlighting this abnormality as a molecular hallmark for this disease. Cell lines originating from HCL patients lack BRAF mutations but retain the typical piliferous morphology and the distinctive HCL immunophenotype, thus, constituting suitable tools for identifying alternative tumor genes and leukemic mechanisms in this malignancy. To this end, we integrated genomic and transcriptional profiling of the HCL cell line MLMA. The expression levels of genomically targeted genes were compared to four HCL control cell lines, thus, identifying 91 chromosomally deregulated genes. Gene set enrichment analysis of these indicted apoptosis, proliferation, and DNA damage response as altered processes. Accordingly, prominent target genes overexpressed in this cell line include ATM, BRAF, CDK6, CUTL1/CUX1, H2AFX, and REL. Treatment of MLMA with selective pharmacological inhibitors and specific siRNA-mediated gene knockdowns highlighted a central role for NFkB in their deregulation in HCL. Moreover, relevant expression profiling data from HCL and ABC-DLBCL cell lines display elevated NFkB-pathway activity when compared to GC-DLBCL equivalents. Finally, analysis of HCL patient samples in silico collectively supported the clinical significance of NFkB activation in this disease. In conclusion, we identified deregulated genes and multiple mechanisms underlying aberrantly activated NFkB-pathway in HCL. Therefore, NFkB may represent a B-cell specific hallmark of HCL and a promising novel therapeutic target, most notably in patients lacking BRAF mutations in this entity including variant HCL.

Biomarkers Provide Clues to Early Events in the Pathogenesis of Breast Implant-Associated Anaplastic Large Cell Lymphoma.

Almost 200 women worldwide have been diagnosed with breast implant-associated anaplastic large cell lymphoma (BIA-ALCL). The unique location and specific lymphoma type strongly suggest an etio-pathologic link between breast implants and BIA-ALCL. It is postulated that chronic inflammation via bacterial infection may be an etiological factor. BIA-ALCL resembles primary cutaneous ALCL (pcALCL) in morphology, activated T-cell phenotype, and indolent clinical course. Gene expression array analysis, flow cytometry, and immunohistochemistry were used to study pcALCL and BIA-ALCL cell lines. Clinical samples were also studied to characterize transcription factor and cytokine profiles of tumor cells and surrounding lymphocytes. BIA-ALCL and pcALCL were found to have common expression of transcription factors SOCS3, JunB, SATB1, and a cytokine profile suggestive of a Th1 phenotype. Similar patterns were observed in a CD30+ cutaneous lymphoproliferative disorder (LPD). The patterns of cytokine and transcription factor expression suggest that BIA-ALCL is likely to arise from chronic bacterial antigen stimulation of T-cells. Further analysis of cytokine and transcription factor profiles may allow early detection and treatment of BIA-ALCL leading to better prognosis and survival. LEVEL OF EVIDENCE 5: Risk.

BCL6--regulated by AhR/ARNT and wild-type MEF2B--drives expression of germinal center markers MYBL1 and LMO2.

Genetic heterogeneity is widespread in tumors, but poorly documented in cell lines. According to immunoglobulin hypermutation analysis, the diffuse large B-cell lymphoma cell line U-2932 comprises two subpopulations faithfully representing original tumor subclones. We set out to identify molecular causes underlying subclone-specific expression affecting 221 genes including surface markers and the germinal center oncogenes BCL6 and MYC. Genomic copy number variations explained 58/221 genes differentially expressed in the two U-2932 clones. Subclone-specific expression of the aryl-hydrocarbon receptor (AhR) and the resulting activity of the AhR/ARNT complex underlaid differential regulation of 11 genes including MEF2B. Knock-down and inhibitor experiments confirmed that AhR/ARNT regulates MEF2B, a key transcription factor for BCL6. AhR, MEF2B and BCL6 levels correlated not only in the U-2932 subclones but in the majority of 23 cell lines tested, indicting overexpression of AhR as a novel mechanism behind BCL6 diffuse large B-cell lymphoma. Enforced modulation of BCL6 affected 48/221 signature genes. Although BCL6 is known as a transcriptional repressor, 28 genes were up-regulated, including LMO2 and MYBL1 which, like BCL6, signify germinal center diffuse large B-cell lymphoma. Supporting the notion that BCL6 can induce gene expression, BCL6 and the majority of potential targets were co-regulated in a series of B-cell lines. In conclusion, genomic copy number aberrations, activation of AhR/ARNT, and overexpression of BCL6 are collectively responsible for differential expression of more than 100 genes in subclones of the U-2932 cell line. It is particularly interesting that BCL6 - regulated by AhR/ARNT and wild-type MEF2B - may drive expression of germinal center markers in diffuse large B-cell lymphoma.

Deregulated FOX genes in Hodgkin lymphoma.

FOX genes encode transcription factors which regulate basic developmental processes during embryogenesis and in the adult. Several FOX genes show deregulated expression in particular malignancies, representing oncogenes or tumor suppressors. Here, we screened six Hodgkin lymphoma (HL) cell lines for FOX gene activity by comparative microarray profiling, revealing overexpression of FOXC1 and FOXD1, and reduced transcription of FOXN3, FOXO1, and FOXP1. In silico expression analyses of these FOX gene candidates in HL patient samples supported the cell line data. Chromosomal analyses demonstrated an amplification of the FOXC1 locus at 6p25 and a gain of the FOXR2 locus at Xp11, indicting genomic aberrations for their upregulation. Comparative expression profiling and ensuing stimulation experiments revealed implementation of the TGFß- and WNT-signaling pathways in deregulation of FOXD1 and FOXN3. Functional analysis of FOXP1 implicated miR9 and miR34a as upstream regulators and PAX5, TCF3, and RAG2 as downstream targets. A similar exercise for FOXC1 revealed repression of MSX1 and activation of IPO7, both mediating inhibition of the B-cell specific homeobox gene ZHX2. Taken together, our data show that aberrantly expressed FOX genes and their downstream targets are involved in the pathogenesis of HL via deregulation of B-cell differentiation and may represent useful diagnostic markers and/or therapeutic targets.

Chromothripsis in Hodgkin lymphoma.

Chromosomal rearrangements are common features of most cancers, where they contribute to deregulated gene expression. Chromothripsis is a recently described oncogenic mechanism whereby small genomic pieces originating from one chromosomal region undergo massive rearrangements in a single step. Here, we document chromothripsis in Hodgkin lymphoma (HL) cell lines by genomic profiling, showing alternating amplicons of defined chromosomal regions. In L-1236 cells, fluorescent in situ hybridization analyses identified aberrations affecting amplified chromosomal segments that derived from the long arm regions of chromosomes 3 and 9 and that colocalized to a derivative chromosome 6, indicating the cataclysmic origin of this mutation. The ABL1 gene at 9q34 was targeted by these rearrangements leading to its overexpression in L-1236 cells, correlating with pharmacological resistance to treatment with the kinase inhibitor dasatinib. Collectively, we identified and characterized chromothriptic rearrangements in HL cell lines to serve as models for analyzing this novel oncogenomic mechanism.

Match criteria for human cell line authentication: where do we draw the line?

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.

Neoplastic MiR-17~92 deregulation at a DNA fragility motif (SIDD).

Chromosomal or mutational activation of BCL6 (at 3q27) typifies diffuse large B-cell lymphoma (DLBCL) which in the germinal center subtype may be accompanied by focal amplification of chromosome band 13q31 effecting upregulation of miR-17~92. Using long distance inverse-polymerase chain reaction, we mapped and sequenced six breakpoints of a complex BCL6 rearrangement t(3;13)(q27;q31)t(12;13)(p11;q31) in DLBCL cells, which places miR-17~92 antisense within the resulting ITPR2-BCL6 chimeric fusion gene rearrangement. MiR-17~92 members were upregulated ~15-fold over controls in a copy number independent manner consistent with structural deregulation. MIR17HG and ITPR2-BCL6 were, despite their close configuration, independently expressed, discounting antisense regulation. MIR17HG in t(3;13)t(12;13) cells proved highly responsive to treatment with histone deacetylase inhibitors implicating epigenetic deregulation, consistent with which increased histone-H3 acetylation was detected by chromatin immunoprecipitation near the upstream MIR17HG breakpoint. Remarkably, 5/6 DNA breaks in the t(3;13)t(12;13) precisely cut at stress-induced DNA duplex destabilization (SIDD) peaks reminiscent of chromosomal fragile sites, while the sixth lay 150 bp distant. Extended SIDD profiling showed that additional oncomiRs also map to SIDD peaks. Fluorescence in situ hybridization analysis showed that 11 of 52 (21%) leukemia-lymphoma (L-L) cell lines with 13q31 involvement bore structural rearrangements at/near MIR17HG associated with upregulation. As well as fueling genome instability, SIDD peaks mark regulatory nuclear-scaffold matrix attachment regions open to nucleosomal acetylation. Collectively, our data indict a specific DNA instability motif (SIDD) in chromosome rearrangement, specifically alterations activating miR-17~92 epigenetically via promoter hyperacetylation, and supply a model for the clustering of oncomiRs near cancer breakpoints.

Chromosomal Aberration t(14;17)(q32;q21) Simultaneously Activates HOXB5 and miR10a in Triple-Hit B-Cell Lymphoma.

BCL2, BCL6 and MYC are major oncogenes in B-cell lymphoma. Their aberrant activation frequently occurs via chromosomal translocations which juxtapose light or heavy chain immunoglobulin (IG) genes to BCL2 and MYC or fuse diverse partner genes with BCL6. So-called double-hit lymphomas usually carry BCL2 and MYC rearrangements, while triple-hit lymphomas additionally bear BCL6-fusions. All these translocations are of diagnostic relevance and usually denote poor prognosis. Here, we genomically characterized classic follicular lymphoma (FL) cell line SC-1, thereby identifying t(14;18)(q32;q21) juxtaposing IGH and BCL2, t(8;14)(q24;q32) juxtaposing IGH and MYC, and t(3;3)(q25;q27) fusing MBNL1 to BCL6. In addition, we found that SC-1 carries a novel chromosomal rearrangement, t(14;17)(q32;q21), which, though present at establishment, has remained unreported until now. We further show that t(14;17)(q32;q21) juxtaposes IGH with the HOXB gene cluster at 17q21 and affect the oncogenic activation of both homeobox gene HOXB5 and neighboring micro-RNA gene miR10a. Moreover, we detected aberrant overexpression of HOXB5 in subsets of Burkitt lymphoma, FL, and multiple myeloma patients, confirming the clinical relevance of its deregulation. In SC-1, HOXB5 activation was additionally supported by co-expression of hematopoietic stem cell factor ZNF521, indicating an aberrant impact in cell differentiation. Functional investigations showed that HOXB5 represses the apoptotic driver BCL2L11 and promotes survival in the presence of etoposide, and that miR10a inhibits BCL6 and may thus play an oncogenic role in later stages of lymphomagenesis. Collectively, we characterize triple-hit B-cell line SC-1 and identify the aberrant expression of HOXB5 and miR10a, both novel oncogenes in B-cell lymphoma.

Clinical, immunophenotypic, cytogenetic, and molecular genetic features in 117 adult patients with mixed-phenotype acute leukemia defined by WHO-2008 classification.

Among 4,780 consecutive adult acute lymphoblastic/myeloblastic leukemia patients, we identified 117 (2.4%) patients with mixed-phenotype acute leukemia fulfilling WHO 2008 criteria; these were classified as: Blymphoid+ myeloid (n=64), T-lymphoid+myeloid (n=38), B+T-lymphoid (n=14) and trilineage (n=1). Of 92 patients karyotyped, 59 were abnormal and were classified as: complex (22 of 92), t(9;22)(q34;q11) (14 of 92), monosomy 7 (7 of 92), polysomy 21 (7 of 92), t(v;11q23) (4 of 92), t(10;11)(p15;q21) (3 of 92), while STIL-TAL1 fusion was detected in one (T+My) patient. After investigating common acute leukemia-related mutations in 17 genes, 12 of 31 (39%) patients were found to have at least one mutation, classified with: IKZF1 deletion (4 of 31), and EZH2 (3 of 31), ASXL1 (3 of 31), ETV6 (2 of 31), NOTCH1 (1 of 31), and TET2 (1 of 31) mutations. Array-CGH revealed genomic deletions of CDKN2A (4 of 12), IKZF1 (3 of 12), MEF2C (2 of 12), BTG1 (2 of 12), together with BCOR, EBF1, K-RAS, LEF1, MBNL1, PBX3, and RUNX1 (one of 12 each). Our results indicate that mixed-phenotype acute leukemia is a complex entity with heterogeneous clinical, immunophenotypic, cytogenetic, and molecular genetic features.

t(4;8)(q27;q24) in Hodgkin lymphoma cells targets phosphodiesterase PDE5A and homeobox gene ZHX2.

Hodgkin/Reed-Sternberg (HRS) cells represent the malignant fraction of infiltrated lymph nodes in Hodgkin lymphoma (HL). Although HRS cells display multiple chromosomal aberrations, few are recurrent and the targeted genes unknown. However, understanding the pathology of HL and developing rational therapies may well require identifying putative deregulated genes. Here, we analyzed the karyotype of the well-defined HL cell line L-1236 by spectral karyotyping and identified multiple abnormalities, therein, notably t(4;8)(q27;q24) which includes two breakpoint regions previously highlighted in HL. Target genes at 4q27 and 8q24 were shortlisted by high density genomic arrays and fluorescence in situ hybridization. Expression analysis of candidate target genes revealed conspicuous activation of phosphodiesterase PDE5A at 4q27 and inhibition of homeobox gene ZHX2 at 8q24. Treatment of L-1236 with PDE5A-inhibitor sildenafil or with siRNA directed against PDE5A and concomitant stimulation with cyclic guanosine monophosphate (cGMP) resulted in enhanced apoptosis, indicating PDE5A as an oncogene. Expression profiling of L-1236 cells following siRNA-mediated knockdown of ZHX2 showed inhibition of genes regulating differentiation and apoptosis, suggesting tumor suppressor activity of ZHX2. Downstream genes included STAT1 and several STAT1-target genes, indicating activation of STAT1-signaling by ZHX2 as analyzed by RQ-PCR and western blot. Taken together, we have identified a novel aberration with recurrent breakpoints in HL, t(4;8)(q27;q24), which activate PDE5A and repress ZHX2, deregulating apoptosis, differentiation, and STAT1-signaling in HL cells.

Genomic Aberrations Generate Fusion Gene FOXK2::TP63 and Activate NFKB1 in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a severe lymphoid malignancy with a worse prognosis lacking curative treatment regimens. Several gene mutations and deregulated pathways, including NFkB signaling, have been implicated in its pathogenesis. Accordingly, CTCL cell line HUT-78 reportedly contains mutated NFKB2, which is constitutively activated via partial gene deletion, also demonstrating that genomic rearrangements cause driving mutations in this malignancy. Here, along with HUT-78, we analyzed CTCL cell line HH to identify additional aberrations underlying gene deregulation. Karyotyping and genomic profiling of HH showed several rearrangements worthy of detailed investigation. Corresponding to the established karyotype, RNA-seq data and PCR analysis confirmed the presence of t(3;17)(q28;q25), generating a novel fusion gene, FOXK2::TP63. Furthermore, chromosomal rearrangement t(1;4)(p32;q25) was connected to amplification at 4q24-26, affecting aberrant NFKB1 overexpression thereat. Transcription factor binding-site analysis and knockdown experiments demonstrated that IRF4 contributed to NFKB1 expression. Within the same amplicon, we identified amplification and overexpression of NFkB signaling activator CAMK2D (4q26) and p53-inhibitor UBE2D3 (4q24). Genomic profiling data for HUT-78 detailed a deletion at 10q25 underlying reported NFKB2 activation. Moreover, amplifications of ID1 (20q11) and IKZF2 (2q34) in this cell line drove overexpression of these NK cell differentiation factors and possibly thus formed corresponding lineage characteristics. Target gene analysis for NFKB1 via siRNA-mediated knockdown in HH revealed activation of TP63, MIR155, and NOTCH pathway component RBPJ. Finally, treatment of HH with NFkB inhibitor demonstrated a role for NFkB in supporting proliferation, while usage of inhibitor DAPT showed significant survival effects via the NOTCH pathway. Collectively, our data suggest that NFkB and/or NOTCH inhibitors may represent reasonable treatment options for subsets of CTCL patients.

New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling.

Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype-specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell-like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.