NKX2-2 (NK2 homeobox 2)

Review on NKX2-2 (NK2 homeobox 2), with data on DNA, on the protein encoded, and where the gene is implicated

EWS/FLI Mediates Transcriptional Repression via NKX2.2 during Oncogenic Transformation in Ewing's Sarcoma

EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip) revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease

IGF1 Is a Common Target Gene of Ewing's Sarcoma Fusion Proteins in Mesenchymal Progenitor Cells

The EWS-FLI-1 fusion protein is associated with 85-90% of Ewing's sarcoma family tumors (ESFT), the remaining 10-15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1) for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin. To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC) permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression. Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis

Comparison of MAPIE versus MAP in patients with a poor response to preoperative chemotherapy for newly diagnosed high-grade osteosarcoma (EURAMOS-1): an open-label, international, randomised controlled trial

Background We designed the EURAMOS-1 trial to investigate whether intensified postoperative chemotherapy for patients whose tumour showed a poor response to preoperative chemotherapy (≥10% viable tumour) improved event-free survival in patients with high-grade osteosarcoma. Methods EURAMOS-1 was an open-label, international, phase 3 randomised, controlled trial. Consenting patients with newly diagnosed, resectable, high-grade osteosarcoma aged 40 years or younger were eligible for randomisation. Patients were randomly assigned (1:1) to receive either postoperative cisplatin, doxorubicin, and methotrexate (MAP) or MAP plus ifosfamide and etoposide (MAPIE) using concealed permuted blocks with three stratification factors: trial group; location of tumour (proximal femur or proximal humerus vs other limb vs axial skeleton); and presence of metastases (no vs yes or possible). The MAP regimen consisted of cisplatin 120 mg/m2, doxorubicin 37·5 mg/m2 per day on days 1 and 2 (on weeks 1 and 6) followed 3 weeks later by high-dose methotrexate 12 g/m2 over 4 h. The MAPIE regimen consisted of MAP as a base regimen, with the addition of high-dose ifosfamide (14 g/m2) at 2·8 g/m2 per day with equidose mesna uroprotection, followed by etoposide 100 mg/m2 per day over 1 h on days 1–5. The primary outcome measure was event-free survival measured in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT00134030. Findings Between April 14, 2005, and June 30, 2011, 2260 patients were registered from 325 sites in 17 countries. 618 patients with poor response were randomly assigned; 310 to receive MAP and 308 to receive MAPIE. Median follow-up was 62·1 months (IQR 46·6–76·6); 62·3 months (IQR 46·9–77·1) for the MAP group and 61·1 months (IQR 46·5–75·3) for the MAPIE group. 307 event-free survival events were reported (153 in the MAP group vs 154 in the MAPIE group). 193 deaths were reported (101 in the MAP group vs 92 in the MAPIE group). Event-free survival did not differ between treatment groups (hazard ratio [HR] 0·98 [95% CI 0·78–1·23]); hazards were non-proportional (p=0·0003). The most common grade 3–4 adverse events were neutropenia (268 [89%] patients in MAP vs 268 [90%] in MAPIE), thrombocytopenia (231 [78% in MAP vs 248 [83%] in MAPIE), and febrile neutropenia without documented infection (149 [50%] in MAP vs 217 [73%] in MAPIE). MAPIE was associated with more frequent grade 4 non-haematological toxicity than MAP (35 [12%] of 301 in the MAP group vs 71 [24%] of 298 in the MAPIE group). Two patients died during postoperative therapy, one from infection (although their absolute neutrophil count was normal), which was definitely related to their MAP treatment (specifically doxorubicin and cisplatin), and one from left ventricular systolic dysfunction, which was probably related to MAPIE treatment (specifically doxorubicin). One suspected unexpected serious adverse reaction was reported in the MAP group: bone marrow infarction due to methotrexate. Interpretation EURAMOS-1 results do not support the addition of ifosfamide and etoposide to postoperative chemotherapy in patients with poorly responding osteosarcoma because its administration was associated with increased toxicity without improving event-free survival. The results define standard of care for this population. New strategies are required to improve outcomes in this setting. Funding UK Medical Research Council, National Cancer Institute, European Science Foundation, St Anna Kinderkrebsforschung, Fonds National de la Recherche Scientifique, Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, Parents Organization, Danish Medical Research Council, Academy of Finland, Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, Federal Ministry of Education and Research, Semmelweis Foundation, ZonMw (Council for Medical Research), Research Council of Norway, Scandinavian Sarcoma Group, Swiss Paediatric Oncology Group, Cancer Research UK, National Institute for Health Research, University College London Hospitals, and Biomedical Research Centre

Transformation induced by Ewing's sarcoma associated EWS/FLI-1 is suppressed by KRAB/FLI-1

Ewing's sarcoma is a childhood bone tumour with poor prognosis, most commonly associated with a t(11;22)(q24;q12) reciprocal translocation that fuses the EWS and FLI-1 genes, resulting in the production of an aberrant chimeric transcription factor EWS/FLI-1. To erucidate the mechanisms by which EWS/FLI-1 mediates transformation in mouse models, we have generated a murine Ews/Fli-1 fusion protein. We demonstrate that this protein transforms fibroblast celrs in vitro similar to human EWS/FLI-1 as demonstrated by serum and anchorage-independent growth, the formation of tumours in nude mice and elevation of the oncogenic marker c-myc. Furthermore, transformation of these cells was inhibited by a specific represser, KRAB/FLI-1. The KRAB/FLI-1 repressor also suppressed the tumorigenic phenotype of a human Ewing's sarcoma cell line. These findings suggest that the transformed phenotype of Ewing's sarcoma cells can be reversed by using the sequence-specific FLI-1-DNA-binding domain to target a gone repressor domain. The inhibition of EWS/FLI-1 is the first demonstration of the KRAB domain suppressing the action of an ETS factor. This approach provides potential avenues for the elucidation of the biological mechanisms of EWS/FLI-1 oncogenesis and the development of novel therapeutic strategies. © 2003 Cancer Research UK.link_to_subscribed_fulltex

Modeling Initiation of Ewing Sarcoma in Human Neural Crest Cells

Ewing sarcoma family tumors (ESFT) are aggressive bone and soft tissue tumors that express EWS-ETS fusion genes as driver mutations. Although the histogenesis of ESFT is controversial, mesenchymal (MSC) and/or neural crest (NCSC) stem cells have been implicated as cells of origin. For the current study we evaluated the consequences of EWS-FLI1 expression in human embryonic stem cell-derived NCSC (hNCSC). Ectopic expression of EWS-FLI1 in undifferentiated hNCSC and their neuro-mesenchymal stem cell (hNC-MSC) progeny was readily tolerated and led to altered expression of both well established as well as novel EWS-FLI1 target genes. Importantly, whole genome expression profiling studies revealed that the molecular signature of established ESFT is more similar to hNCSC than any other normal tissue, including MSC, indicating that maintenance or reactivation of the NCSC program is a feature of ESFT pathogenesis. Consistent with this hypothesis, EWS-FLI1 induced hNCSC genes as well as the polycomb proteins BMI-1 and EZH2 in hNC-MSC. In addition, up-regulation of BMI-1 was associated with avoidance of cellular senescence and reversible silencing of p16. Together these studies confirm that, unlike terminally differentiated cells but consistent with bone marrow-derived MSC, NCSC tolerate expression of EWS-FLI1 and ectopic expression of the oncogene initiates transition to an ESFT-like state. In addition, to our knowledge this is the first demonstration that EWS-FLI1-mediated induction of BMI-1 and epigenetic silencing of p16 might be critical early initiating events in ESFT tumorigenesis

Stable interference of EWS–FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target

BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS–FLI1 target genes still remain unknown and many have been identified in heterologous model systems

Network potential identifies therapeutic miRNA cocktails in Ewing sarcoma.

MicroRNA (miRNA)-based therapies are an emerging class of targeted therapeutics with many potential applications. Ewing Sarcoma patients could benefit dramatically from personalized miRNA therapy due to inter-patient heterogeneity and a lack of druggable (to this point) targets. However, because of the broad effects miRNAs may have on different cells and tissues, trials of miRNA therapies have struggled due to severe toxicity and unanticipated immune response. In order to overcome this hurdle, a network science-based approach is well-equipped to evaluate and identify miRNA candidates and combinations of candidates for the repression of key oncogenic targets while avoiding repression of essential housekeeping genes. We first characterized 6 Ewing sarcoma cell lines using mRNA sequencing. We then estimated a measure of tumor state, which we term network potential, based on both the mRNA gene expression and the underlying protein-protein interaction network in the tumor. Next, we ranked mRNA targets based on their contribution to network potential. We then identified miRNAs and combinations of miRNAs that preferentially act to repress mRNA targets with the greatest influence on network potential. Our analysis identified TRIM25, APP, ELAV1, RNF4, and HNRNPL as ideal mRNA targets for Ewing sarcoma therapy. Using predicted miRNA-mRNA target mappings, we identified miR-3613-3p, let-7a-3p, miR-300, miR-424-5p, and let-7b-3p as candidate optimal miRNAs for preferential repression of these targets. Ultimately, our work, as exemplified in the case of Ewing sarcoma, describes a novel pipeline by which personalized miRNA cocktails can be designed to maximally perturb gene networks contributing to cancer progression