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Induction of fetal hemoglobin (HbF) has been shown to be a viable therapeutic approach to treating sickle cell disease and potentially other β-hemoglobinopathies. To identify targets and target-modulating small molecules that enhance HbF expression, we engineered a human umbilical-derived erythroid progenitor reporter cell line (HUDEP2_HBG1_HiBiT) by genetically tagging a HiBiT peptide to the carboxyl (C)-terminus of the endogenous HBG1 gene locus, which codes for γ-globin protein, a component of HbF. Employing this reporter cell line, we performed a chemogenomic screen of approximately 5000 compounds annotated with known targets or mechanisms that have achieved clinical stage or approval by the US Food and Drug Administration (FDA). Among them, 10 compounds were confirmed for their ability to induce HbF in the HUDEP2 cell line. These include several known HbF inducers, such as pomalidomide, lenalidomide, decitabine, idoxuridine, and azacytidine, which validate the translational nature of this screening platform. We identified avadomide, autophinib, triciribine, and R574 as novel HbF inducers from these screens. We orthogonally confirmed HbF induction activities of the top hits in both parental HUDEP2 cells as well as in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs). Further, we demonstrated that pomalidomide and avadomide, but not idoxuridine, induced HbF expression through downregulation of several transcriptional repressors such as BCL11A, ZBTB7A, and IKZF1. These studies demonstrate a robust phenotypic screening workflow that can be applied to large-scale small molecule profiling campaigns for the discovery of targets and pathways, as well as novel therapeutics for sickle cell disease and other β-hemoglobinopathies. [ABSTRACT FROM AUTHOR]

Alport syndrome (AS) is a genetic disease characterized by haematuric glomerulopathy variably associated with hearing loss and anterior lenticonus. It is caused by mutations in the COL4A3, COL4A4 or COL4A5 genes encoding the α3α4α5(IV) collagen heterotrimer. AS is rare, but it accounts for >1% of patients receiving renal replacement therapy. Angiotensin-converting enzyme inhibition slows, but does not stop, the progression to renal failure; therefore, there is an urgent requirement to expand and intensify research towards discovering new therapeutic targets and new therapies. The 2015 International Workshop on Alport Syndrome targeted unmet needs in basic science, genetics and diagnosis, clinical research and current clinical care. In three intensive days, more than 100 international experts including physicians, geneticists, researchers from academia and industry, and patient representatives from all over the world participated in panel discussions and breakout groups. This report summarizes the most important priority areas including (i) understanding the crucial role of podocyte protection and regeneration, (ii) targeting mutations by new molecular techniques for new animal models and potential gene therapy, (iii) creating optimal interaction between nephrologists and geneticists for early diagnosis, (iv) establishing standards for mutation screening and databases, (v) improving widespread accessibility to current standards of clinical care, (vi) improving collaboration with the pharmaceutical/biotech industry to investigate new therapies, (vii) research in hearing loss as a huge unmet need in Alport patients and (viii) the need to evaluate the risk and benefit of novel (including 'repurposing') therapies on an international basis., (© The Author 2016. Published by Oxford University Press on behalf of ERAEDTA.)

As described for a long time, carcinoma-derived Caco-2 cells form a polarized epithelium in culture, whereas HT29-D4 cells are nonpolarized and undifferentiated but can form a polarized monolayer when cultured in a galactose-supplemented medium. Using NF-kappaB translocation and IL-8 and ICAM-1 gene activation as an index, we have studied the relationship between the differentiation state and the cell response to cytokines. We found that differentiated Caco-2 and HT29-D4 cells were responsive to both cytokines TNFalpha- and IL-1beta-mediated activation of NF-kappaB but that undifferentiated HT29-D4 cells were unresponsive to IL-1beta. However, the expression of endogenous ICAM-1 and IL-8 genes was upregulated by these cytokines in either cell lines differentiated or not. Upregulation of ICAM-1 gene occurred when IL-1beta or TNFalpha was added to the basal, but not apical surface of the differentiated epithelia. Finally, it appeared that in polarized HT29-D4 cells, the IL-1beta-induced translocation of NF-kappaB was connected to PKCdelta translocation.

IkappaBbeta, one of the major IkappaB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IkappaBbeta exists in at least two different forms: one that is bound to the NF-kappaB dimer and the other bound to both NF-kappaB and kappaB-Ras, a Ras-like small G protein. Removal of cellular kappaB-Ras enhances whereas excess kappaB-Ras blocks induced IkappaBbeta degradation. Remarkably, kappaB-Ras functions in both GDP- and GTP-bound states, and mutations of the conserved guanine-binding residues of kappaB-Ras abrogate its ability to block degradation of IkappaBbeta. kappaB-Ras also directly blocks the in vitro phosphorylation of IkappaBbeta by IKKbeta. These observations suggest that IkappaBbeta in the ternary complex is resistant to degradation by most signals. We suggest that specific signals, in addition to those that activate only IKK, are essential for the complete degradation of IkappaBbeta.