Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis

MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko). Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (p < 0.01). Competitive BM repopulation experiments showed that donor BM harboring the Mdm2C305F mutation possessed decreased repopulation capacity compared to WT BM, suggesting a functional stem cell deficit. These results suggest that there is a fine tuned balance in the interaction of ribosomal proteins with the MDM2/p53 axis which is important in normal hematopoiesis

Hypercalcemia FOllowing Umbilical Cord Blood Transplantation to Correct Osteopetrosis Associated with the Nemo Mutation

X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency（ XL-EDA-ID） is a congenital developmental and immunologic disorder, which often causes osteopetrosis. These individuals are susceptible to infection with various microorganisms, and most patients die of infection before reaching maturity. Hematopoietic stem cell transplantation （HSCT） is considered the only cure for this disorder. When HSCT is performed in patients with osteopetrosis, on the other hand, transient hypercalcemia often occurs in these patients. We report a XL-EDA-ID patient with osteopetrosis who experienced hypercalcemia and resolution of bone changes after cord blood transplantation

Mice with a Mutation in the <i>Mdm2</i> Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis

<div><p>MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the <i>Mdm2</i> gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in <i>Mdm2</i> causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (<i>Mdm2</i>^<i>C305F</i>), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the <i>Mdm2</i>^<i>C305F</i> mutation we noticed that <i>Mdm2</i>^<i>C305F</i> homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119^hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119^hiCD71^low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (<i>p</i> < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (<i>Trp53</i>^<i>ko/ko</i>). Further investigation revealed that in <i>Mdm2</i>^<i>C305F</i> animals, there was a decrease in Lin^-Sca-1⁺c-Kit⁺ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (<i>p</i> < 0.01). Competitive BM repopulation experiments showed that donor BM harboring the <i>Mdm2</i>^<i>C305F</i> mutation possessed decreased repopulation capacity compared to WT BM, suggesting a functional stem cell deficit. These results suggest that there is a fine tuned balance in the interaction of ribosomal proteins with the MDM2/p53 axis which is important in normal hematopoiesis.</p></div

Mechanism of KIT gene regulation by GATA1 lacking the N-terminal domain in Down syndrome–related myeloid disorders

Abstract Children with Down syndrome (DS) are at high risk of transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). GATA1 mutations are detected in almost all TAM and ML-DS samples, with exclusive expression of short GATA1 protein (GATA1s) lacking the N-terminal domain (NTD). However, it remains to be clarified how GATA1s is involved with both disorders. Here, we established the K562 GATA1s (K562-G1s) clones expressing only GATA1s by CRISPR/Cas9 genome editing. The K562-G1s clones expressed KIT at significantly higher levels compared to the wild type of K562 (K562-WT). Chromatin immunoprecipitation studies identified the GATA1-bound regulatory sites upstream of KIT in K562-WT, K562-G1s clones and two ML-DS cell lines; KPAM1 and CMK11-5. Sonication-based chromosome conformation capture (3C) assay demonstrated that in K562-WT, the − 87 kb enhancer region of KIT was proximal to the − 115 kb, − 109 kb and + 1 kb region, while in a K562-G1s clone, CMK11-5 and primary TAM cells, the − 87 kb region was more proximal to the KIT transcriptional start site. These results suggest that the NTD of GATA1 is essential for proper genomic conformation and regulation of KIT gene expression, and that perturbation of this function might be involved in the pathogenesis of TAM and ML-DS

Prognostic and therapeutic factors influencing the clinical outcome of metastatic Ewing sarcoma family of tumors: A retrospective report from the Japan Ewing Sarcoma Study Group

[Background] The prognosis of patients with metastatic Ewing sarcoma family of tumors (ESFT) remains poor. [Procedure] We retrospectively analyzed 57 patients diagnosed with metastatic ESFT between 2000 and 2018 to identify prognostic and therapeutic factors affecting the clinical outcome. [Results] The 3-year overall survival (OS) rate of the entire cohort was 46.8% (95% confidence interval [CI], 33.0-59.4%). Treatment-related death was not observed. Multivariate analysis identified stem cell transplantation (SCT), response to first-line chemotherapy, and bone metastasis as independent risk factors for OS. Objective response rate to first-line chemotherapy was 65.1% in the 43 evaluable patients. There was no significant difference in the response to different types of first-line chemotherapy. Among patients with lung metastasis alone, the 3-year OS rate was higher in 13 patients who received local treatment than in four who did not, although the difference was not significant. [Conclusions] One possible reason for the high OS rates was the absence of treatment-related mortality even in patients receiving SCT, which could be attributed to advances in the management of post-SCT complications. Novel first-line chemotherapy strategies need to be established to improve the disease status prior to SCT in a higher proportion of patients

Effects of <i>Mdm2</i>^<i>C305F</i> mutant on selected hematopoietic lineage subpopulation in bone marrow.

<p>(A) Representative flow cytometry analysis of the specific precursor populations in control (n = 7), <i>Mdm2</i>^<i>C305F</i> homozygous (n = 6), and <i>Mdm2</i>^{<i>C305F/C305F</i>};<i>Trp53</i>^<i>ko/ko</i> mice (n = 6). (B) <i>Mdm2</i>^<i>C305F</i> mutant animals exhibited a significant paucity in the HSPC population of LSKs (<i>P</i> < 0.01), accompanied by significant decreases of MPP populations (<i>P</i> < 0.01). HSPC indicates hematopoietic stem and progenitor cell; LSK, Lin^-Sca-1⁺c-Kit⁺; LT-HSC, long-term hematopoietic stem cells; ST-HSC, short-term hematopoietic stem cells; MPP, multipotent progenitor; CLP, common lymphoid progenitor; and CMP, common myeloid progenitor.</p

Effects of <i>Mdm2</i>^<i>C305F</i> mutant on peripheral blood, bone marrow, and spleen histology.

<p>(A) Hematoxylin and eosin stained splenic sections (Magnification: 40x). (B) Ter119-stained (brown) splenic sections. Blue is counterstained lymphoid tissue.</p

Effects of <i>Mdm2</i>^<i>C305F</i> mutant on blood counts, lactate dehydrogenase, bilirubin, erythropoietein level, and eADA activity.

<p>Effects of <i>Mdm2</i>^<i>C305F</i> mutant on blood counts, lactate dehydrogenase, bilirubin, erythropoietein level, and eADA activity.</p

Diagram of the hematopoietic lineage and differences observed in <i>Mdm2</i>^<i>C305F</i> mutant mice.

<p>(A) <i>Mdm2</i>^<i>C305F</i> phenotype corresponded to blocks at MPP and orthochromatic erythroblast. LSK indicates Lin^-Sca-1⁺c-Kit⁺; LT-HSC, long-term hematopoietic stem cells; ST-HSC, short-term hematopoietic stem cells; MPP, multipotent progenitor; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte/macrophage progenitor; PreMegE, megakaryocyte–erythroid progenitor; and MKP, megakaryocyte progenitor. (B) RP/MDM2 interaction may be important for the mature hematopoiesis rather than for the HSC proliferation.</p

Effects of <i>Mdm2</i>^<i>C305F</i> mutant on erythroid maturation in bone marrow and spleen.

<p>Flow cytometric assessment of cell-surface Ter119 and CD71 expression in bone marrow and spleen, used to define population of proerythroblasts (I; Ter119^medCD71^hi), basophilic erythroblasts (II; Ter119^hiCD71^hi), chromatophilic erythroblasts (III; Ter119^hiCD71^med), and orthochromatic erythroblasts (IV; Ter119^hiCD71^low). Specific populations in control (n = 25), <i>Mdm2</i>^<i>C305F</i> heterozygous (n = 9), <i>Mdm2</i>^<i>C305F</i> homozygous (n = 8), and <i>Mdm2</i>^{<i>C305F/C305F</i>}<i>;Trp53</i>^<i>ko/ko</i> mice (n = 8). (A) Representative flow cytometry analysis of bone marrow. (B) <i>Mdm2</i>^<i>C305F</i> homozygous mice showed a significant decrease in Ter119^high cells compared to wild type littermates in the bone marrow. (C) Representative flow cytometry analysis of spleen. (D) <i>Mdm2</i>^<i>C305F</i> homozygous mice showed no decrease in the number of mature erythroid cells (Ter119^hiCD71^low) compared to wild type littermates in the spleen.</p