Transforming activities of the NUP98-KMT2A fusion gene associated with myelodysplasia and acute myeloid leukemia

Inv(11)(p15q23), found in myelodysplastic syndromes and acute myeloid leukemia, leads to expression of a fusion protein consisting of the N-terminal of nucleoporin 98 (NUP98) and the majority of the lysine methyltransferase 2A (KMT2A). To explore the transforming potential of this fusion we established inducible iNUP98-KMT2A transgenic mice. After a median latency of 80 weeks, over 90% of these mice developed signs of disease, with anemia and reduced bone marrow cellularity, increased white blood cell numbers, extramedullary hematopoiesis, and multilineage dysplasia. Additionally, induction of iNUP98-KMT2A led to elevated lineage marker-negative Sca-1+ c-Kit+ cell numbers in the bone marrow, which outcompeted wildtype cells in repopulation assays. Six iNUP98-KMT2A mice developed transplantable acute myeloid leukemia with leukemic blasts infiltrating multiple organs. Notably, as reported for patients, iNUP98-KMT2A leukemic blasts did not express increased levels of the HoxA-B-C gene cluster, and in contrast to KMT2A-AF9 leukemic cells, the cells were resistant to pharmacological targeting of menin and BET family proteins by MI-2-2 or JQ1, respectively. Expression of iNUP98-KMT2A in mouse embryonic fibroblasts led to an accumulation of cells in G1 phase, and abrogated replicative senescence. In bone marrow-derived hematopoietic progenitors, iNUP98-KMT2A expression similarly resulted in increased cell numbers in the G1 phase of the cell cycle, with aberrant gene expression of Sirt1, Tert, Rbl2, Twist1, Vim, and Prkcd, mimicking that seen in mouse embryonic fibroblasts. In summary, we demonstrate that iNUP98-KMT2A has in vivo transforming activity and interferes with cell cycle progression rather than primarily blocking differentiation

Accelerating functional gene discovery in osteoarthritis

Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease

Potent co-operation between the NUP98-NSD1 fusion and the FLT3-ITD mutation in acute myeloid leukemia induction

The NUP98-NSD1 fusion, product of the t(5;11)(q35;p15.5) chromosomal translocation, is one of the most prevalent genetic alterations in cytogenetically normal pediatric acute myeloid leukemias and is associated with poor prognosis. Co-existence of an FLT3-ITD activating mutation has been found in more than 70% of NUP98-NSD1-positive patients. To address functional synergism, we determined the transforming potential of retrovirally expressed NUP98-NSD1 and FLT3-ITD in the mouse. Expression of NUP98-NSD1 provided mouse strain-dependent, aberrant self-renewal potential to bone marrow progenitor cells. Co-expression of FLT3-ITD increased proliferation and maintained self-renewal in vitro. Transplantation of immortalized progenitors co-expressing NUP98-NSD1 and FLT3-ITD into mice resulted in acute myeloid leukemia after a short latency. In contrast, neither NUP98-NSD1 nor FLT3-ITD single transduced cells were able to initiate leukemia. Interestingly, as reported for patients carrying NUP98-NSD1, an increased Flt3-ITD to wild-type Flt3 mRNA expression ratio with increased FLT3-signaling was associated with rapidly induced disease. In contrast, there was no difference in the expression levels of the NUP98-NSD1 fusion or its proposed targets HoxA5, HoxA7, HoxA9 or HoxA10 between animals with different latencies to develop disease. Finally, leukemic cells co-expressing NUP98-NSD1 and FLT3-ITD were very sensitive to a small molecule FLT3 inhibitor, which underlines the significance of aberrant FLT3 signaling for NUP98-NSD1-positive leukemias and suggests new therapeutic approaches that could potentially improve patient outcome

Loss of CCDC6 affects cell cycle through impaired intra-S-phase checkpoint control

In most cancers harboring Ccdc6 gene rearrangements, like papillary thyroid tumors or myeloproliferative disorders, the product of the normal allele is supposed to be functionally impaired or absent. To address the consequence of the loss of CCDC6 expression, we applied lentiviral shRNA in several cell lines. Loss of CCDC6 resulted in increased cell death with clear shortening of the S phase transition of the cell cycle. Upon exposure to etoposide, the cells lacking CCDC6 did not achieve S-phase accumulation. In the absence of CCDC6 and in the presence of genotoxic stress, like etoposide treatment or UV irradiation, increased accumulation of DNA damage was observed, as indicated by a significant increase of pH2Ax Ser139. 14-3-3σ, a major cell cycle regulator, was down-regulated in CCDC6 lacking cells, regardless of genotoxic stress. Interestingly, in the absence of CCDC6, the well-known genotoxic stress-induced cytoplasmic sequestration of the S-phase checkpoint CDC25C phosphatase did not occur. These observations suggest that CCDC6 plays a key role in cell cycle control, maintenance of genomic stability and cell survival and provide a rational of how disruption of CCDC6 normal function contributes to malignancy. © 2012 Thanasopoulou et al

Thymidylate synthase inhibition induces p53-dependent and p53-independent apoptotic responses in human urinary bladder cancer cells

Purpose: In search for more effective clinical protocols, the antimetabolite drug 5-fluorouracil (5-FU) has been successfully included in new regimens of bladder cancer combination chemotherapy. In the present study, we have investigated the effects of 5-FU treatment on apoptosis induction in wild-type and mutant p53 urinary bladder cancer cells. Methods: We have used MTT-based assays, FACS analysis, Western blotting and semi-quantitative RT-PCR in RT4 and RT112 (grade I, wild-type p53), as well as in T24 (grade III, mutant p53) and TCCSUP (grade IV, mutant p53) human urinary bladder cancer cell lines. Results: In the urothelial bladder cancer cell lines RT4 and T24, 5-FU-induced TS inhibition proved to be associated with cell type-dependent (a) sensitivity to the drug, (b) Caspase-mediated apoptosis, (c) p53 stabilization and activation, as well as Rb phosphorylation and E2F1 expression and (d) transcriptional regulation of p53 target genes and their cognate proteins, while an E2F-dependent transcriptional network did not seem to be critically engaged in such type of responses. Conclusions: We have shown that in the wild-type p53 context of RT4 cells, 5-FU-triggered apoptosis was prominently efficient and mainly regulated by p53-dependent mechanisms, whereas the mutant p53 environment of T24 cells was able to provide notable levels of resistance to apoptosis, basically ascribed to E2F-independent, and still unidentified, pathways. Nevertheless, the differential vulnerability of RT4 and T24 cells to 5-FU administration could also be associated with cell-type-specific transcriptional expression patterns of certain genes critically involved in 5-FU metabolism. © 2010 Springer-Verlag

Gelsolin expression is necessary for the development of modelled pulmonary inflammation and fibrosis

Background: Despite intense research efforts, the aetiology and pathogenesis of idiopathic pulmonary fibrosis remain poorly understood. Gelsolin, an actin-binding protein that modulates cytoskeletal dynamics, was recently highlighted as a likely disease modifier through comparative expression profiling and target prioritisation. Methods: To decipher the possible role of gelsolin in pulmonary inflammation and fibrosis, immunocytochemistry on tissue microarrays of human patient samples was performed followed by computerised image analysis. The results were validated in the bleomycin-induced animal model of pulmonary inflammation and fibrosis using genetically-modified mice lacking gelsolin expression. Moreover, to gain mechanistic insights into the mode of gelsolin activity, a series of biochemical analyses was performed ex vivo in mouse embryonic fibroblasts. Results: Increased gelsolin expression was detected in lung samples of patients with idiopathic interstitial pneumonia as well as in modelled pulmonary inflammation and fibrosis. Genetic ablation of gelsolin protected mice from the development of modelled pulmonary inflammation and fibrosis attributed to attenuated epithelial apoptosis. Conclusions: Gelsolin expression is necessary for the development of modelled pulmonary inflammation and fibrosis, while the caspase-3-mediated gelsolin fragmentation was shown to be an apoptotic effector mechanism in disease pathogenesis and a marker of lung injury

PFI-1, a highly selective protein interaction inhibitor, targeting BET Bromodomains.

Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and antiapoptotic genes as well as for cell-cycle progression. BET proteins are recruited on transcriptionally active chromatin via their two N-terminal bromodomains (BRD), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional downregulation of a number of oncogenes, providing a novel pharmacologic strategy for the treatment of cancer. Here, we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1, which efficiently blocks the interaction of BET BRDs with acetylated histone tails. Cocrystal structures showed that PFI-1 acts as an acetyl-lysine (Kac) mimetic inhibitor efficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell-cycle arrest, downregulation of MYC expression, as well as induction of apoptosis and induces differentiation of primary leukemic blasts. Intriguingly, cells exposed to PFI-1 showed significant downregulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10, providing an alternative strategy for the specific inhibition of this well-established oncology target