Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis

BACKGROUND: Histone acetylation and deacetylation seem processes involved in the pathogenesis of Ewing sarcoma (EwS). Here histone deacetylases (HDAC) class I were investigated. METHODS: Their role was determined using different inhibitors including TSA, Romidepsin, Entinostat and PCI-34051 as well as CRISPR/Cas9 class I HDAC knockouts and HDAC RNAi. To analyze resulting changes microarray analysis, qRT-PCR, western blotting, Co-IP, proliferation, apoptosis, differentiation, invasion assays and xenograft-mouse models were used. RESULTS: Class I HDACs are constitutively expressed in EwS. Patients with high levels of individual class I HDAC expression show decreased overall survival. CRISPR/Cas9 class I HDAC knockout of individual HDACs such as HDAC1 and HDAC2 inhibited invasiveness, and blocked local tumor growth in xenograft mice. Microarray analysis demonstrated that treatment with individual HDAC inhibitors (HDACi) blocked an EWS-FLI1 specific expression profile, while Entinostat in addition suppressed metastasis relevant genes. EwS cells demonstrated increased susceptibility to treatment with chemotherapeutics including Doxorubicin in the presence of HDACi. Furthermore, HDACi treatment mimicked RNAi of EZH2 in EwS. Treated cells showed diminished growth capacity, but an increased endothelial as well as neuronal differentiation ability. HDACi synergizes with EED inhibitor (EEDi) in vitro and together inhibited tumor growth in xenograft mice. Co-IP experiments identified HDAC class I family members as part of a regulatory complex together with PRC2. CONCLUSIONS: Class I HDAC proteins seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in EwS and in combination therapy, co-treatment with HDACi is an interesting new treatment opportunity for this malignant disease

Ten millennia of hepatitis B virus evolution

Hepatitis B virus (HBV) has been infecting humans for millennia and remains a global health problem, but its past diversity and dispersal routes are largely unknown. We generated HBV genomic data from 137 Eurasians and Native Americans dated between similar to 10,500 and similar to 400 years ago. We date the most recent common ancestor of all HBV lineages to between similar to 20,000 and 12,000 years ago, with the virus present in European and South American hunter-gatherers during the early Holocene. After the European Neolithic transition, Mesolithic HBV strains were replaced by a lineage likely disseminated by early farmers that prevailed throughout western Eurasia for similar to 4000 years, declining around the end of the 2nd millennium BCE. The only remnant of this prehistoric HBV diversity is the rare genotype G, which appears to have reemerged during the HIV pandemic.Molecular Technology and Informatics for Personalised Medicine and Healt

Ten millennia of hepatitis B virus evolution

Hepatitis B virus (HBV) has been infecting humans for millennia and remains a global health problem, but its past diversity and dispersal routes are largely unknown. We generated HBV genomic data from 137 Eurasians and Native Americans dated between ~10,500 and ~400 years ago. We date the most recent common ancestor of all HBV lineages to between ~20,000 and 12,000 years ago, with the virus present in European and South American hunter-gatherers during the early Holocene. After the European Neolithic transition, Mesolithic HBV strains were replaced by a lineage likely disseminated by early farmers that prevailed throughout western Eurasia for ~4000 years, declining around the end of the 2nd millennium BCE. The only remnant of this prehistoric HBV diversity is the rare genotype G, which appears to have reemerged during the HIV pandemic

The posterior <em>HOXD</em> locus: Its contribution to phenotype and malignancy of Ewing sarcoma.

Microarray analysis revealed genes of the posterior HOXD locus normally involved in bone formation to be over-expressed in primary Ewing sarcoma (ES). The expression of posterior HOXD genes was not influenced via ES pathognomonic EWS/ETS translocations. However, knock down of the dickkopf WNT signaling pathway inhibitor 2 (DKK2) resulted in a significant suppression of HOXD10, HOXD11 and HOXD13 while over-expression of DKK2 and stimulation with factors of the WNT signaling pathway such as WNT3a, WNT5a or WNT11 increased their expression. RNA interference demonstrated that individual HOXD genes promoted chondrogenic differentiation potential, and enhanced expression of the bone-associated gene RUNX2. Furthermore, HOXD genes increased the level of the osteoblast- and osteoclast-specific genes, osteocalcin (BGLAP) and platelet-derived growth factor beta polypeptide (PDGFB), and may further regulate endochondral bone development via induction of parathyroid hormone-like hormone (PTHLH). Additionally, HOXD11 and HOXD13 promoted contact independent growth of ES, while in vitro invasiveness of ES lines was enhanced by all 3 HOXD genes investigated and seemed mediated via matrix metallopeptidase 1 (MMP1). Consequently, knock down of HOXD11 or HOXD13 significantly suppressed lung metastasis in a xeno-transplant model in immune deficient mice, providing overall evidence that posterior HOXD genes promote clonogenicity and metastatic potential of ES

T cells directed against the metastatic driver chondromodulin-1 in ewing sarcoma: Comparative engineering with CRISPR/Cas9 vs. retroviral gene transfer for adoptive transfer.

Ewing sarcoma (EwS) is a highly malignant sarcoma of bone and soft tissue with early metastatic spread and an age peak in early puberty. The prognosis in advanced stages is still dismal, and the long-term effects of established therapies are severe. Efficacious targeted therapies are urgently needed. Our previous work has provided preliminary safety and efficacy data utilizing T cell receptor (TCR) transgenic T cells, generated by retroviral gene transfer, targeting HLA-restricted peptides on the tumor cell derived from metastatic drivers. Here, we compared T cells engineered with either CRISPR/Cas9 or retroviral gene transfer. Firstly, we confirmed the feasibility of the orthotopic replacement of the endogenous TCR by CRISPR/Cas9 with a TCR targeting our canonical metastatic driver chondromodulin-1 (CHM1). CRISPR/Cas9-engineered T cell products specifically recognized and killed HLA-A*02:01+ EwS cell lines. The efficiency of retroviral transduction was higher compared to CRISPR/Cas9 gene editing. Both engineered T cell products specifically recognized tumor cells and elicited cytotoxicity, with CRISPR/Cas9 engineered T cells providing prolonged cytotoxic activity. In conclusion, T cells engineered with CRISPR/Cas9 could be feasible for immunotherapy of EwS and may have the advantage of more prolonged cytotoxic activity, as compared to T cells engineered with retroviral gene transfer

The endochondral bone protein CHM1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma.

Ewing sarcomas (ES) are highly malignant, osteolytic bone or soft tissue tumors, which are characterized by EWS-ETS translocations and early metastasis to lung and bone. In this study, we investigated the role of the BRICHOS chaperone domain-containing endochondral bone protein chondromodulin I (CHM1) in ES pathogenesis. CHM1 is significantly over-expressed in ES, and chromosome immunoprecipitation (ChIP) data demonstrate CHM1 to be directly bound by an EWS-ETS translocation, EWS-FLI1. Using RNA interference we observed that CHM1 promoted chondrogenic differentiation capacity of ES cells but decreased the expression of osteolytic genes such as HIF1A, IL6, JAG1 and VEGF. This was in line with the induction of the number of tartrate-resistant acid phosphatase (TRAP(+) ) stained osteoclasts in an orthotopic model of local tumor growth after CHM1 knock down, indicating that CHM1-mediated inhibition of osteomimicry might play a role in homing, colonization and invasion into bone tissues. We further demonstrate that CHM1 enhanced the invasive potential of ES cells in vitro. This invasiveness was in part mediated via CHM1-regulated MMP9 expression and correlated with the observation that, in an xenograft mouse model, CHM1 was essential for the establishment of lung metastases. This finding is in line with the observed increase in CHM1 expression in patient specimens with ES lung metastases. Our results suggest that CHM1 seems to have pleiotropic functions in ES, that need to be further investigated, but appears to be essential for the invasive and metastatic capacities of ES