A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis—and, in particular, the genes that they regulate—however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation

The biodistribution and clearance of AlbudAb, a novel biopharmaceutical medicine platform, assessed via PET imaging in humans

Abstract: Conjugation or fusion to AlbudAbs™ (albumin-binding domain antibodies) is a novel approach to extend the half-life and alter the tissue distribution of biological and small molecule therapeutics. To understand extravasation kinetics and extravascular organ concentrations of AlbudAbs in humans, we studied tissue distribution and elimination of a non-conjugated 89Zr-labeled AlbudAb in healthy volunteers using positron emission tomography/computed tomography (PET/CT). Methods: A non-conjugated AlbudAb (GSK3128349) was radiolabeled with 89Zr and a single 1 mg (~ 15 MBq) dose intravenously administered to eight healthy males. 89Zr-AlbudAb tissue distribution was followed for up to 7 days with four whole-body PET/CT scans. 89Zr-AlbudAb tissue concentrations were quantified in organs of therapeutic significance, measuring standardized uptake value and tissue/plasma ratios. Plasma pharmacokinetics were assessed by gamma counting and LC-MS/MS of blood samples. Results: 89Zr-AlbudAb administration and PET/CT procedures were well tolerated, with no drug-related immunogenicity or adverse events. 89Zr-AlbudAb rapidly distributed throughout the vasculature, with tissue/plasma ratios in the liver, lungs, and heart relatively stable over 7 days post-dose, ranging between 0.1 and 0.5. The brain tissue/plasma ratio of 0.025 suggested minimal AlbudAb blood-brain barrier penetration. Slowly increasing ratios in muscle, testis, pancreas, and spleen reflected either slow AlbudAb penetration and/or 89Zr residualization in these organs. Across all tissues evaluated, the kidney tissue/plasma ratio was highest (0.5–1.5 range) with highest concentration in the renal cortex. The terminal half-life of the 89Zr-AlbudAb was 18 days. Conclusion: Evaluating the biodistribution of 89Zr-AlbudAb in healthy volunteers using a low radioactivity dose was successful (total subject exposure ~ 10 mSv). Results indicated rapid formation of reversible, but stable, complexes between AlbudAb and albumin upon dosing. 89Zr-AlbudAb demonstrated albumin-like pharmacokinetics, including limited renal elimination. This novel organ-specific distribution data for AlbudAbs in humans will facilitate a better selection of drug targets to prosecute using the AlbudAb platform and significantly contribute to modeling work optimizing dosing of therapeutic AlbudAbs in the clinic

The biodistribution and clearance of AlbudAb, a novel biopharmaceutical medicine platform, assessed via PET imaging in humans

Conjugation or fusion to AlbudAbs (albumin-binding domain antibodies) is a novel approach to extend the half-life and alter the tissue distribution of biological and small molecule therapeutics. To understand extravasation kinetics and extravascular organ concentrations of AlbudAbs in humans, we studied tissue distribution and elimination of a non-conjugated Zr-89-labeled AlbudAb in healthy volunteers using positron emission tomography/computed tomography (PET/CT).MethodsA non-conjugated AlbudAb (GSK3128349) was radiolabeled with Zr-89 and a single 1mg (similar to 15MBq) dose intravenously administered to eight healthy males. Zr-89-AlbudAb tissue distribution was followed for up to 7days with four whole-body PET/CT scans. Zr-89-AlbudAb tissue concentrations were quantified in organs of therapeutic significance, measuring standardized uptake value and tissue/plasma ratios. Plasma pharmacokinetics were assessed by gamma counting and LC-MS/MS of blood samples.Results(89)Zr-AlbudAb administration and PET/CT procedures were well tolerated, with no drug-related immunogenicity or adverse events. Zr-89-AlbudAb rapidly distributed throughout the vasculature, with tissue/plasma ratios in the liver, lungs, and heart relatively stable over 7days post-dose, ranging between 0.1 and 0.5. The brain tissue/plasma ratio of 0.025 suggested minimal AlbudAb blood-brain barrier penetration. Slowly increasing ratios in muscle, testis, pancreas, and spleen reflected either slow AlbudAb penetration and/or Zr-89 residualization in these organs. Across all tissues evaluated, the kidney tissue/plasma ratio was highest (0.5-1.5 range) with highest concentration in the renal cortex. The terminal half-life of the Zr-89-AlbudAb was 18days.ConclusionEvaluating the biodistribution of Zr-89-AlbudAb in healthy volunteers using a low radioactivity dose was successful (total subject exposure similar to 10mSv). Results indicated rapid formation of reversible, but stable, complexes between AlbudAb and albumin upon dosing. Zr-89-AlbudAb demonstrated albumin-like pharmacokinetics, including limited renal elimination. This novel organ-specific distribution data for AlbudAbs in humans will facilitate a better selection of drug targets to prosecute using the AlbudAb platform and significantly contribute to modeling work optimizing dosing of therapeutic AlbudAbs in the clinic

Transforming activity of Fbxo7 is mediated specifically through regulation of cyclin D/cdk6

D cyclins (D1, D2 and D3) and their catalytic subunits (cyclin-dependent kinases cdk4 and cdk6) have a facilitating, but nonessential, role in cell cycle entry. Tissue-specific functions for D-type cyclins and cdks have been reported; however, the biochemical properties of these kinases are indistinguishable. We report that an F box protein, Fbxo7, interacted with cellular and viral D cyclins and distinguished among the cdks that bind D-type cyclins, specifically binding cdk6, in vitro and in vivo. Fbxo7 specifically regulated D cyclin/cdk6 complexes: Fbxo7 knockdown decreased cdk6 association with cyclin and its overexpression increased D cyclin/cdk6 activity and E2F activity. Fbxo7 interacted with p27, but its enhancement of cyclin D/cdk6 activity was p21/p27 independent. Fbxo7 overexpression transformed murine fibroblasts, rendering them tumorigenic in athymic nude mice. Transformed phenotypes were dependent on cdk6, as knockdown of cdk6 reversed them. Fbxo7 was highly expressed in epithelial tumors, but not in normal tissues, suggesting that it may have a proto-oncogenic role in human cancers