Generation of three iPSC lines with inducible systems to be used in Angelman syndrome research.

The neurodevelopmental disorder Angelman syndrome (AS) has an incidence of 1:15.000 live births and is caused by absence of UBE3A protein, showing imprinted gene expression in the brain. Imprinted genes are controlled by differentially methylated regions resulting in parent-of-origin dependent gene expression. Two iPS cell lines derived from patients with AS and one healthy control iPSC line were used to integrate a 3rd generation reverse tetracycline transactivator protein (rtTA3) into the AAVS1 locus on chromosome 19. The rtTA allows tetracycline-dependent activation of an inducible promoter that can be introduced at a position of interest in the cell lines described here.

Molecular Characterization and Subtyping of Breast Cancer Cell Lines Provide Novel Insights into Cancer Relevant Genes.

Continuous cell lines are important and commonly used in vitro models in breast cancer (BC) research. Selection of the appropriate model cell line is crucial and requires consideration of their molecular characteristics. To characterize BC cell line models in depth, we profiled a panel of 29 authenticated and publicly available BC cell lines by mRNA-sequencing, mutation analysis, and immunoblotting. Gene expression profiles separated BC cell lines in two major clusters that represent basal-like (mainly triple-negative BC) and luminal BC subtypes, respectively. HER2-positive cell lines were located within the luminal cluster. Mutation calling highlighted the frequent aberration of TP53 and BRCA2 in BC cell lines, which, therefore, share relevant characteristics with primary BC. Furthermore, we showed that the data can be used to find novel, potential oncogenic fusion transcripts, e.g., FGFR2::CRYBG1 and RTN4IP1::CRYBG1 in cell line MFM-223, and to elucidate the regulatory circuit of IRX genes and KLF15 as novel candidate tumor suppressor genes in BC. Our data indicated that KLF15 was activated by IRX1 and inhibited by IRX3. Moreover, KLF15 inhibited IRX1 in cell line HCC-1599. Each BC cell line carries unique molecular features. Therefore, the molecular characteristics of BC cell lines described here might serve as a valuable resource to improve the selection of appropriate models for BC research.

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.

Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

STAT3 is a transcription factor which is activated via various signaling transduction pathways or Epstein-Barr virus (EBV) infection and plays an oncogenic role in lymphoid malignancies including Hodgkin lymphoma (HL). The tumor cells of HL are derived from germinal center B-cells and transformed by chromosomal rearrangements, aberrant signal transduction, deregulation of developmental transcription factors, and EBV activity. HL cell lines represent useful models to investigate molecular principles and deduced treatment options of this malignancy. Using cell line L-540, we have recently shown that constitutively activated STAT3 drives aberrant expression of hematopoietic NKL homeobox gene HLX. Here, we analyzed HL cell line AM-HLH which is EBV-positive but, nevertheless, HLX-negative. Consistently, AM-HLH expressed decreased levels of STAT3 proteins which were additionally inactivated and located in the cytoplasm. Combined genomic and expression profiling data revealed several amplified and overexpressed gene candidates involved in opposed regulation of STAT3 and EBV. Corresponding knockdown studies demonstrated that IRF4 and NFATC2 inhibited STAT3 expression. MIR155 (activated by STAT3) and SPIB (repressed by HLX) showed reduced and elevated expression levels in AM-HLH, respectively. However, treatment with IL6 or IL27 activated STAT3, elevated expression of HLX and MIR155, and inhibited IRF4. Taken together, this cell line deals with two conflicting oncogenic drivers, namely, JAK2-STAT3 signaling and EBV infection, but is sensitive to switch after cytokine stimulation. Thus, AM-HLH represents a unique cell line model to study the pathogenic roles of STAT3 and EBV and their therapeutic implications in HL.