Project description:Early B-cell development is primarily regulated at the transcriptional level and comprises the consecutive differentiation stages B-cell progenitor, pro-B-cell and pre-B-cell. These entities provide the cells of origin in B-cell precursor acute lymphoid leukemia (BCP-ALL) which show deregulation of developmental transcription factors (TFs), representing major oncogenic drivers. Analysis of TF activities in these developmental entities helps understanding both their normal and aberrant expression and regulatory connections. Here, we focused on NKL-subclass homeodomain TF NKX6-3 which is active in normal B-cell progenitors and TCF3::PBX1-positive BCP-ALL cases. Performing siRNA-mediated knockdown and forced expression experiments in BCP-ALL model cell lines, we established a gene regulatory network for NKX6-3 together with NKL-TFs HLX, MSX1 and HHEX, TALE-class homeodomain TFs MEIS1 and IRX1, ETS-TFs ERG, ETS2 and SPIB, and additionally with PAX5, EBF1 and IRF4. NKX6-3 and TCF3::PBX1 were found to be mutual activators, underlying their co-expression. Furthermore, comparative expression profiling analysis of BCP-ALL patients revealed TGFb-pathway inhibitor CD109 as downregulated target gene of NKX6-3. TGFb in turn enhanced NKX6-3 expression, indicating mutual activation. Finally, RNA-seq analysis of BCP-ALL cell line RCH-ACV after NKX6-3 knockdown revealed MPP7 as upregulated target gene of both, NKX6-3 and TCF3::PBX1. Elevated MPP7 expression indicated an oncogenic role of the HIPPO-pathway in TCF3::PBX1 positive BCP-ALL. Collectively, our data add novel players and regulatory connections to normal and aberrant TF-networks in B-cell progenitors which may serve as diagnostic markers or therapeutic targets.
Project description:RCH-ACV cells was depleted for SETDB2 by shRNA lentivirus construct co-expressing mCherry fluorescent. After sorting of mCherry positive cells, the cells were cultured and expanded and total RNA was extracted for Microarray experiment.
Project description:Study of BLaER1 cell line epigenetic changes induced throughout transdifferentiation. The Illumina Infinium MethylationEPIC Beadchip was used to obtain genomewide methylation profiles of BLaER1 cells at 7 different times throughout transdifferentiation treatment (0h, 3h, 12h, 24h, 48h, 72h and 168h). As a reference, the parental RCH-ACV cell line at 168h of treatment and anonymous donor blood derived macrophages were also profiled.
Project description:The NSD2 p.E1099K (EK) mutation has been shown to be enriched in patients with relapsed ALL indicating a role in clonal evolution and drug resistance. We previously demonstrated that the impact of EK is highly cell context dependent. To uncover 3D chromatin architecture-related mechanisms underlying drug resistance, we performed Hi-C on three B-ALL cell lines heterozygous for NSD2 EK (RS4;11, RCH-ACV, SEM) and assessed changes upon knockdown.
Project description:The NSD2 p.E1099K (EK) mutation increases methyltransferase activity and is observed in 10% of acute lymphoblastic leukemia (ALL) samples with enrichment at relapse indicating a role in clonal evolution and drug resistance. To discover mechanisms that mediate the clonal expansion, we engineered B-ALL cell lines (Reh, 697) to overexpress wildtype (WT) and EK NSD2, but observed no differences in proliferation, clonal growth, or chemosensitivity. To address whether NSD2 EK acts collaboratively with other pathways, we used shRNAs to knockdown expression of NSD2 in B-ALL cell lines heterozygous for NSD2 EK (RS4;11, RCH-ACV, SEM). Knockdown resulted in decreased proliferation in all three lines, decreased clonal growth in RCH-ACV, and increased sensitivity to chemotherapeutic agents routinely used in ALL therapy, although the pattern of drug sensitivity varied among cell lines implying that the oncogenic properties of NSD2 mutations are likely cell context specific and rely on cooperative pathways to exert its effect. Knockdown of both Type II and REIIBP EK isoforms had a greater impact than knockdown of Type II alone, suggesting that both SET containing EK isoforms contribute to phenotypic changes driving relapse. Furthermore, in vivo models using both cell lines and patient samples revealed dramatically enhanced proliferation of NSD2 EK compared to WT and reduced sensitivity to 6-mercaptopurine in the relapse sample relative to diagnosis. Finally, EK-mediated changes in chromatin state and transcriptional output differed dramatically among cell lines further supporting a cell context specific role of NSD2 EK. These results demonstrate a unique role of NSD2 EK in mediating clonal fitness through pleiotropic mechanisms dependent on the genetic and epigenetic landscape.