p53 activation during ribosome biogenesis regulates normal erythroid differentiation.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle-arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Toward RNA Repair of Diamond Blackfan Anemia Hematopoietic Stem Cells.

Diamond blackfan anemia (DBA) is a well-known inherited bone marrow failure syndrome mostly caused by mutations in ribosomal protein (RP) genes but also rarely in the hematopoietic transcription factor gene, GATA1, or TSR2, a ribosomal protein (Rps26) chaperone gene. About 25% of patients have heterozygous mutations in the RPS19 gene, which leads to haploinsufficiency of Rps19 protein in most cases. However, some RPS19 missense mutations appear to act in a dominant negative fashion. DBA typically leads to a hypoplastic anemia that becomes apparent during the first year of life, and standard treatment includes steroids or red blood cell transfusions, each modality having attendant side effects. The only curative therapy is allogeneic stem-cell transplantation, but this option is limited to patients with a histocompatible donor. DBA-mutant embryonic, induced pluripotent, and hematopoietic stem cells all exhibit growth abnormalities that can be corrected by DNA gene transfer, suggesting the possibility of ex vivo autologous gene therapy. The authors have been interested in the application of spliceosome-mediated mRNA trans-splicing (SMaRT) technology to RNA repair of DBA stem cells. Compared with gene replacement or other RNA re-programming approaches, SMaRT has several potential advantages. First, delivery of the entire normal cDNA is unnecessary, thus minimizing the overall size of the construct for packaging into a viral delivery vector. Second, RNA transcription of the corrected gene relies on the cell's endogenous transcriptional, processing, and regulatory machinery, thereby ensuring faithful and contextual expression. Third, RNA trans-splicing employs the endogenous spliceosome enzymatic machinery present in nearly all cells. Fourth, RNA trans-splicing converts mutant transcripts into therapeutically useful mRNA, and thus may be capable of treating disorders caused by dominant negative mutations. This review critically assesses prospects for both gene and RNA repair in DBA stem cells.

Epigenetic control of NF-κB-dependent FAS gene transcription during progression of myelodysplastic syndromes.

The death domain containing TNF receptor 6 (CD95/Fas) is a direct target for the NF-κB transcription factor and is repressed in solid tumors such as colon carcinomas. Previously, we reported that the Fas death receptor, while overexpressed in low-risk myelodysplastic syndromes (MDS), becomes undetectable on CD34(+) progenitors when the disease progresses to secondary acute myeloid leukemia (AML). This study determined the interplay between NF-κB and Fas during MDS progression. We first observed that Fas was induced by TNF-α in the HL60 cell line. In these cells, p65 (RELA) was associated with the FAS promoter, and inhibition of the NF-κB pathway by an IKKα inhibitor (BAY11-7082) or lentiviral expression of a nondegradable mutant of IκBα (IκSR) blocked Fas expression. In contrast, TNF-α failed to induce Fas expression in the colon carcinoma cell line SW480, due to hypermethylation of the FAS promoter. Azacitidine rescued p65 binding on FAS promoter in vitro, and subsequently Fas expression in SW480 cells. Furthermore, inhibition of the NF-κB pathway decreased the expression of Fas in MDS CD45(lo)CD34(+) bone marrow cells. However, despite the nuclear expression of p65, Fas was often low on CD45(lo)CD34(+) AML cells. TNF-α failed to stimulate its expression, while azacitidine efficiently rescued p65 binding and Fas reexpression. Overall, these data suggest that DNA methylation at NF-κB sites is responsible for FAS gene silencing.

Tyrosine kinase inhibitors induce down-regulation of c-Kit by targeting the ATP pocket.

The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-ß-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket.