Publications

Mechanisms regulating thrombus stabilization remain largely unknown. Here, we report that loss of any 1 of the Gas6 receptors (Gas6-Rs), i.e., Tyro3, Axl, or Mer, or delivery of a soluble extracellular domain of Axl that traps Gas6 protects mice against life-threatening thrombosis. Loss of a Gas6-R does not prevent initial platelet aggregation but impairs subsequent stabilization of platelet aggregates, at least in part by reducing “outside-in” signaling and platelet granule secretion. Gas6, through its receptors, activates PI3K and Akt and stimulates tyrosine phosphorylation of the β(3) integrin, thereby amplifying outside-in signaling via α(IIb)β(3). Blocking the Gas6-R–α(IIb)β(3) integrin cross-talk might be a novel approach to the reduction of thrombosis.

Abstract Background: About 40% of IDH1 mutated (IDH1mut) acute myeloid leukemia (AML) patients respond to IDH1 inhibitors with a median duration of response of 8.2 months. A better understanding of the biology of IDH1mut leukemia may further improve the treatment of these patients. IDH1mut produces R-2-hydroxyglutarate (R-2HG), which activates PHD1 and PHD2 but have negligible effects on PHD3. In the present study we assessed whether PHD3 plays a role in the pathogenesis of IDH1 mutated leukemia and can be targeted in a patient-derived xenograft (PDX) model of IDH1 mutated AML. Methods: Bone marrow cells from Phdwt and Phd3ko mice were immortalized with HoxA9, and IDH1wildtype (IDH1wt) and IDH1mut respectively, were constitutively expressed. The effects on cell proliferation, apoptosis and colony formation were evaluated in vitro, whereas the leukemic potential was evaluated in vivo by transplantation in syngeneic mice. To show that PHD3 is a therapeutic target, either IDH1mut cells from AML patients were transduced with shRNA against PHD3 and transplanted in immunocompromised mice, or leukemic cells from an AML patient with mutated IDH1 were xenografted in immunocompromised mice and treated with the PHD inhibitor molidustat. Results: In in-vitro functional assays loss of Phd3 specifically impaired proliferation, apoptosis and clonogenic capacity of HoxA9-IDH1mut but not HoxA9-IDH1wt cells. Likewise, in mouse transplantation assays, loss of Phd3 eliminated HoxA9-IDH1mut induced leukemia. However, Phd3 was dispensable to the engraftment and proliferation of HoxA9-IDH1wt cells. Additionally, the IDH1-independent model of MN1-induced leukemia remained unaltered in the absence of Phd3, indicating the specificity of the role of Phd3 in mutant IDH1-induced transformation. To identify molecular pathways that might explain in vitro and in vivo phenotypes gene expression profiling was performed. Immune and stress-response pathways as well as metabolism-related genes were most prominently dysregulated in Phd3ko IDH1-mutant cells. Analysis of dysregulated transcription factors by gene set enrichment analysis revealed a depletion of key oncogenic transcription factors (Myc, Rb, Stk33, and Rps14) in Phd3ko IDH1mut cells compared to Phd3ko IDH1wt cells. To study if IDH1mut signals to Phd3 through R-2HG, we transduced Phd3kocells, with a splice variant of mutant IDH1, which does not produce R-2HG but causes leukemia in mice with similar kinetics as in mice with the full-length IDH1 mutant protein. Interestingly, loss of Phd3 also eliminated leukemia in these mice, which demonstrates that mutant IDH1 signals through Phd3 independently of R-2HG. To study the functional relevance of PHD3 inhibition in patients, cells from an IDH1 mutated AML patient were transduced with an shRNA against PHD3 and were transplanted in immunodeficient NSG mice. Inhibition of PHD3 depleted human AML cells in the IDH1-mutated PDX model. Moreover, the PHD inhibitor molidustat was 50-fold more active in IDH1mut (80 nM) compared to IDH1wt AML patient cells (4000 nM) in colony-forming unit assays. In a xenograft model of IDH1 mutated AML, molidustat significantly prolonged survival compared to control-treated mice (P<.001). Conclusion: We demonstrate that the leukemogenic activity of the mutant IDH1 protein depends on PHD3 independently of R-2HG. We identified inhibition of PHD3 as a novel therapeutic strategy in IDH1 mutated AML. Since PHD3 can be targeted pharmacologically, combinatorial treatment of PHD3 and IDH1 inhibitors is warranted to improve eradication of leukemic stem cells in IDH1 mutated AML. #AC and MMAC share first authorship Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Karyopharm: Research Funding; Daiichi Sankyo: Research Funding; Sunesis: Research Funding; Tetralogic: Research Funding; Bayer Pharma AG: Consultancy, Research Funding; StemLine Therapeutics: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; BergenBio: Research Funding; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

<p>Supplementary Figure 5 - Effects of VEGFR-1 signaling ablation on PAN02 tumor progression in body-weight matched mice.</p>

Our results provide evidence that immaturity of plaque vessels is associated with the degradation of angiogenic factors by haemorrhage-conveyed leucocytes and proteases.

Neutrophils are unusual in their reliance on glycolysis to maintain their energy requirements 1 despite the presence of mitochondria and TCA cycle intermediaries 2 .This metabolic adaptation is thought in part to underpin their survival and anti-microbial function in tissues that are typically hypoxic [3][4][5] .Despite their unique metabolism, little is known about the importance of flux between metabolic pathways in determining neutrophil survival responses.Recent work has demonstrated the importance of the HIF/PHD oxygen-sensing pathway in this regard, identifying both HIF-1α and PHD3 as critical regulators of neutrophil survival in hypoxia 6,7 , with the extended survival of neutrophils in hypoxia being dependent upon HIF-1α expression.In parallel, an expanding body of work has addressed the role of HIF-1α in coordinating macrophage functional responses to pro-inflammatory mediators [8][9][10][11] .This work led to the observation that, in macrophages, LPS causes an intracellular increase in succinate levels resulting in HIF-1α stabilization and enhanced IL-1β signaling 11 .Subsequently, the metabolic re-wiring of anti-microbial (M1) and tissue repair (M2) macrophages has been elucidated, with important consequences of TCA cycle activity and integrity for regulation of NO and N-glycosylation signaling respectively 12 .Whether TCA cycle activity and succinate accumulation regulates HIF-1α activity and hypoxic survival in neutrophils is unknown.

Introduction Angiogenesis is genetically pre‐determined Mutations causing vascular anomalies Translocations reveal novel angiogenic genes Single nucleotide polymorphisms shape the angio‐genome Copy number variability affects angiogenesis Epigenetic regulation of angiogenesis Micromanagers of angiogenesis Perspectives Abstract Angiogenesis is controlled by a balance between pro‐ and anti‐angiogenic factors. Studies in mice and human beings have shown that this balance, as well as the general sensitivity of the endothelium to these factors, is genetically pre‐determined. In an effort to dissect this genetic basis, different types of genetic variability have emerged: mutations and translocations in angiogenic factors have been linked to several vascular malformations and haemangiomas, whereas SNPs have been associated with complex genetic disorders, such as cancer, neurodegeneration and diabetes. In addition, copy number alterations of angiogenic factors have been reported in several tumours. More recently, epigenetic changes caused by aberrant DNA methylation or histone acetylation of anti‐angiogenic molecules have been shown to determine angiogenesis as well. Initial studies also revealed a crucial role for microRNAs in stimulating or reducing angiogenesis. So far, most of these genetic studies have focused on tumour angiogenesis, but future research is expected to improve our understanding of how genetic variants determine angiogenesis in other diseases. Importantly, these genetic insights might also be of important clinical relevance for the use of anti‐angiogenic strategies in cancer or macular degeneration.

Supplementary Data from A Phase I Trial of TB-403 in Relapsed Medulloblastoma, Neuroblastoma, Ewing Sarcoma, and Alveolar Rhabdomyosarcoma

Summary The cDNA encoding murine coagulation factor VII (mfVII) was isolated and reconstructed from a Λ Zap cDNA library generated from murine liver mRNA. The cDNA contains 1903 nucleotides spanning 15 bases upstream of the 5’-translation initiation codon, an open reading frame of 1338 nucleotides, 550 nucleotides downstream of the first stop codon and a 3’ poly(A) tail. The translation product is composed of a 41-amino acid signal/propeptide region followed by a 405-residue mature protein. The latter is highly homologous to that of human, rabbit, bovine, Rhesus monkey, and canine fVII. All protein domains of hfVII are strictly conserved in mfVII.

The Bmx gene, a member of the Tec tyrosine kinase gene family, is known to be expressed in subsets of hematopoietic and endothelial cells. In this study, mice were generated in which the first coding exon of the Bmx gene was replaced with the lacZ reporter gene by a knock-in strategy. The homozygous mice lacking Bmx activity were fertile and had a normal life span without an obvious phenotype. Staining of their tissues using beta-galactosidase substrate to assess the sites of Bmx expression revealed strong signals in the endothelial cells of large arteries and in the endocardium starting between days 10.5 and 12.5 of embryogenesis and continuing in adult mice, while the venular endothelium showed a weak signal only in the superior and inferior venae cavae. Of the five known endothelial receptor tyrosine kinases tested, activated Tie-2 induced tyrosyl phosphorylation of the Bmx protein and both Tie-2 and vascular endothelial growth factor receptor 1 (VEGFR-1) stimulated Bmx tyrosine kinase activity. Thus, the Bmx tyrosine kinase has a redundant role in arterial endothelial signal transduction downstream of the Tie-2 and VEGFR-1 growth factor receptors.