Search

The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity., Competing Interests: Declaration of interests R.D.H. is a Chief Scientific Advisor of Pimera, Inc. (San Diego, CA). All other authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Diamond-Blackfan anemia is a congenital erythroid hypoplasia and is associated with physical malformations and a predisposition to cancer. Twenty-five percent of patients with Diamond-Blackfan anemia have mutations in a gene encoding ribosomal protein S19 (RPS19). Through overexpression of RPS19 using a lentiviral vector with the spleen focus-forming virus promoter, we demonstrated that the Diamond-Blackfan anemia phenotype can be successfully treated in Rps19-deficient mice. In our present study, we assessed the efficacy of a clinically relevant promoter, the human elongation factor 1α short promoter, with or without the locus control region of the β-globin gene for treatment of RPS19-deficient Diamond-Blackfan anemia. The findings demonstrate that these vectors rescue the proliferation defect and improve erythroid development of transduced RPS19-deficient bone marrow cells. Remarkably, bone marrow failure and severe anemia in Rps19-deficient mice was cured with enforced expression of RPS19 driven by the elongation factor 1α short promoter. We also demonstrate that RPS19-deficient bone marrow cells can be transduced and these cells have the capacity to repopulate bone marrow in long-term reconstituted mice. Our results collectively demonstrate the feasibility to cure RPS19-deficient Diamond-Blackfan anemia using lentiviral vectors with cellular promoters that possess a reduced risk of insertional mutagenesis., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Current sources of platelets for transfusion are insufficient and associated with risk of alloimmunization and blood-borne infection. These limitations could be addressed by the generation of autologous megakaryocytes (MKs) derived in vitro from somatic cells with the ability to engraft and differentiate in vivo. Here, we show that overexpression of a defined set of six transcription factors efficiently converts mouse and human fibroblasts into MK-like progenitors. The transdifferentiated cells are CD41 + , display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. No models exist for RPS19-deficient DBA and the molecular pathogenesis is unknown. To establish an in vitro inducible model for DBA, human erythroid leukemic cell lines, TF-1 and UT-7 cells, were cotransduced with a lentiviral vector expressing the green fluorescent protein (GFP) gene and small interfering RNA (siRNA) against RPS19 controlled by a tet operator regulatory element and another transactivator vector containing the red fluorescent protein (RFP) gene and the cDNA encoding a tetracycline-controllable transcriptional repressor. Following transduction, the RFP-positive and GFP-negative cell population was sorted by flow cytometry. Upon incubation with doxycycline (0.5 mug/ml), more than 98% of cells expressed GFP and the siRNA. Significant suppression of erythroid differentiation, cell growth, and colony formation was observed in cells treated with siRNA against RPS19 but not in cells treated with a control vector. These findings show that RPS19 plays an important role in the regulation of hematopoietic cell proliferation and erythroid differentiation. These novel cell lines represent models for RPS19-deficient DBA and can be used to identify the molecular mechanisms in RPS19-deficient DBA.

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Since some patients with DBA develop a reduction in thrombocytes and granulocytes with age, we asked whether multipotent hematopoietic progenitors from DBA patients had normal proliferative capacity in liquid expansion cultures. CD34(+) cells derived from DBA patients showed deficient proliferation in liquid culture containing IL-3, IL-6, and SCF. Single CD34(+) CD38(-) cells from DBA patients exhibited deficient proliferation recruitment in a limiting dilution assay containing IL-3, IL-6, SCF, Tpo, FL, and G-CSF or containing IL-3, IL-6, and SCF. Our findings suggest that the underlying hematopoietic defect in DBA may not be limited to the erythroid lineage. Since a fraction of DBA patients have a deficiency in ribosomal protein S19 (RPS19), we constructed lentiviral vectors containing the RPS19 gene for overexpression in hematopoietic progenitors from RPS19-deficient DBA patients. Enforced expression of the RPS19 transgene improved the proliferation of CD34(+) cells from DBA patients with RPS19 mutation. Similarly, enforced expression of RPS19 improved erythroid development of RPS19-deficient hematopoietic progenitors as determined by colony assays and erythroid differentiation cultures. These findings suggest that gene therapy for RPS19-deficient DBA is feasible.