Cre recombinase expression cooperates with homozygous FLT3 internal tandem duplication knockin mouse model to induce acute myeloid leukemia

Murine models offer a valuable tool to recapitulate genetically defined subtypes of AML, and to assess the potential of compound mutations and clonal evolution during disease progression. This is of particular importance for difficult to treat leukemias such as FLT3 internal tandem duplication (ITD) positive AML. While conditional gene targeting by Cre recombinase is a powerful technology that has revolutionized biomedical research, consequences of Cre expression such as lack of fidelity, toxicity or off-target effects need to be taken into consideration. We report on a transgenic murine model of FLT3-ITD induced disease, where Cre recombinase expression alone, and in the absence of a conditional allele, gives rise to an aggressive leukemia phenotype. Here, expression of various Cre recombinases leads to polyclonal expansion of FLT3(ITD/ITD) progenitor cells, induction of a differentiation block and activation of Myc-dependent gene expression programs. Our report is intended to alert the scientific community of potential risks associated with using this specific mouse model and of unexpected effects of Cre expression when investigating cooperative oncogenic mutations in murine models of cancer

A Subset of Osteoblasts Expressing High Endogenous Levels of PPARγ Switches Fate to Adipocytes in the Rat Calvaria Cell Culture Model

Understanding fate choice and fate switching between the osteoblast lineage (ObL) and adipocyte lineage (AdL) is important to understand both the developmental inter-relationships between osteoblasts and adipocytes and the impact of changes in fate allocation between the two lineages in normal aging and certain diseases. The goal of this study was to determine when during lineage progression ObL cells are susceptible to an AdL fate switch by activation of endogenous peroxisome proliferator-activated receptor (PPAR)gamma.Multiple rat calvaria cells within the ObL developmental hierarchy were isolated by either fractionation on the basis of expression of alkaline phosphatase or retrospective identification of single cell-derived colonies, and treated with BRL-49653 (BRL), a synthetic ligand for PPARgamma. About 30% of the total single cell-derived colonies expressed adipogenic potential (defined cytochemically) when BRL was present. Profiling of ObL and AdL markers by qRT-PCR on amplified cRNA from over 160 colonies revealed that BRL-dependent adipogenic potential correlated with endogenous PPARgamma mRNA levels. Unexpectedly, a significant subset of relatively mature ObL cells exhibited osteo-adipogenic bipotentiality. Western blotting and immunocytochemistry confirmed that ObL cells co-expressed multiple mesenchymal lineage determinants (runt-related transcription factor 2 (Runx2), PPARgamma, Sox9 and MyoD which localized in the cytoplasm initially, and only Runx2 translocated to the nucleus during ObL progression. Notably, however, some cells exhibited both PPARgamma and Runx2 nuclear labeling with concomitant upregulation of expression of their target genes with BRL treatment.We conclude that not only immature but a subset of relatively mature ObL cells characterized by relatively high levels of endogenous PPARgamma expression can be switched to the AdL. The fact that some ObL cells maintain capacity for adipogenic fate selection even at relatively mature developmental stages implies an unexpected plasticity with important implications in normal and pathological bone development

Imetelstat-mediated alterations in fatty acid metabolism to induce ferroptosis as a therapeutic strategy for acute myeloid leukemia

Telomerase enables replicative immortality in most cancers including acute myeloid leukemia (AML). Imetelstat is a first-in-class telomerase inhibitor with clinical efficacy in myelofibrosis and myelodysplastic syndromes. Here, we develop an AML patient-derived xenograft resource and perform integrated genomics, transcriptomics and lipidomics analyses combined with functional genetics to identify key mediators of imetelstat efficacy. In a randomized phase II-like preclinical trial in patient-derived xenografts, imetelstat effectively diminishes AML burden and preferentially targets subgroups containing mutant NRAS and oxidative stress-associated gene expression signatures. Unbiased, genome-wide CRISPR/Cas9 editing identifies ferroptosis regulators as key mediators of imetelstat efficacy. Imetelstat promotes the formation of polyunsaturated fatty acid-containing phospholipids, causing excessive levels of lipid peroxidation and oxidative stress. Pharmacological inhibition of ferroptosis diminishes imetelstat efficacy. We leverage these mechanistic insights to develop an optimized therapeutic strategy using oxidative stress-inducing chemotherapy to sensitize patient samples to imetelstat causing substantial disease control in AML

Evidence for multiple peroxisome proliferator-activated receptor γ transcripts in bone: fine-tuning by hormonal regulation and mRNA stability

The expression, regulation and functional significance of multiple Peroxisome proliferator-activated receptor γ transcript variants in bone were studied. PPARG transcripts giving rise to PPARg-1 protein were expressed in human osteoblasts, whereas PPARG-2 transcript and protein remained virtually absent. PPARG expression underwent homologous regulation, was upregulated during differentiation and directly induced by the osteogenic hormone dexamethasone, suggesting a role of PPARg-1 for osteogenesis. Differences between the stabilities of PPARG-1, -3 and -4 were observed. We hypothesize that cell-specific expression patterns of multiple PPARG transcript variants encoding for the same protein but differing in mRNA stabilities enable a fine-tuning of PPARG action, which eventually supports a well-adjusted signal transduction between the cell and its environment

New insights into peroxisome proliferatoractivated receptor gamma action: Stimulation of human osteoblast differentiation

Peroxisome proliferator-activated receptor γ (PPARG) is a member of the nuclear receptor superfamily, which orchestrates responses to environmental factors by sensing diverse nutrients and xenobiotics. Imbalances in nuclear receptor signalling have been associated with various aging-related diseases, e.g. osteoporosis, metabolic syndromes and cancer. Two PPARG proteins exist derived from one single gene by alternative promoter usage and splicing: PPARγ1, which is translated from three alternative transcripts PPARG1, 3 and 4, and PPARγ2, which is translated from PPARG2 transcript. PPARG2 is known to control adipogenesis. Here we report our recent findings concerning the presence, regulation and functional significance of PPARG signalling during differentiation and mineralization of our human osteoblast cell model SV-HFO. The latter can be induced by dexamethasone (dex) to differentiate and to mineralize the extracellular matrix formed in a three-week period. Realtime-PCR analysis of the four PPARG transcripts revealed a at least 250, 30 and 150-fold higher expression of PPARG1, 3 and 4, respectively, compared to PPARG2 at all time points during culture. This markedly contrasts the expression pattern in adipocytes in which PPARG2 is strongly expressed. Expression of the four PPARG transcripts increased during differentiation, with PPARG3 showing strongest induction. Short-term dex-treatment for three hours increased expression of all four PPARG transcripts suggesting direct regulation by dex. We furthermore studied the effect of PPARG ligands on human osteoblast differentiation and mineralization. Treatment with rosiglitazone, a PPARG-specific agonist, resulted in significant increases in alkaline phosphatase (ALP) activity and mineralization at all three time points during culture. The PPARG antagonist GW9662, however, revealed a significant decrease in ALP activity and did not show any effect on mineralization. In conclusion, PPARG expression is controlled by cell-type specific promoter usage and splicing, leading to PPARG1, 3 and 4 (= PPARγ1) expression in osteoblasts and PPARG2 (= PPARγ2) expression in adipocytes. The PPARG ligand rosiglitazone does not exclusively control adipocyte differentiation but also stimulates human osteoblast differentiation. Our data implicate an alternative view on PPARG in the balance between adipocyte and osteoblast differentiation in relation to the development of osteoporosis

1 alpha,25-Dihydroxyvitamin D3 and rosiglitazone synergistically enhance osteoblast-mediated mineralization

Both vitamin D receptor (VDR) and peroxisome proliferator-activated receptor gamma (PPAR-gamma) are ligand-activated nuclear transcription factors that are instrumental for bone health. While 1 alpha,25-dihydroxyvitamin D3 (1,25D3), the ligand for VDR, is essential for the development and maintenance of healthy bone, PPAR-gamma agonists cause detrimental skeletal effects. Recent studies have revealed evidence for a cross-talk between 1,25D3- and PPAR-alpha/-delta ligand-mediated signaling but there is a current lack of knowledge regarding cross-talk between signaling of 1,25D3 and the PPAR-gamma ligand mediated signaling. In this study, we investigated the cross-talk between 1,25D3- and PPAR-gamma agonist rosiglitazone-mediated signaling in human osteoblasts. 1,25D3 slightly but significantly induced expression of the primary PPAR-gamma target gene ANGPTL4 but did not influence FABP4 1,25D3 did not change rosiglitazone regulation of ANGPTL4 and FABP4. The other way around, rosiglitazone reduced CYP24A1 gene expression but this did not change CYP24A1 induction by 1,25D3. The findings regarding CYP24A1 gene expression are in line with the observation that 1,25D3 levels in medium were not affected by rosiglitazone. Furthermore, rosiglitazone significantly inhibited 1,25D3-induction of BGLAP while rosiglitazone alone did not change BGLAP. Additionally, 1,25D3 and rosiglitazone increase osteoblast alkaline phosphatase activity and synergistically stimulated extracellular matrix mineralization. In conclusion, these data provide evidence for a cross-talk between rosiglitazone- and 1,25D3-mediated signaling leading to an acceleration of extracellular matrix mineralization. The data suggest that the reduction of the mineralization inhibitor BGLAP and the increased differentiation status underlie the increased mineralization. (c) 2012 Elsevier B.V. All rights reserved