Fiona Brown

Superiority of Icodextrin Compared with 4.25% Dextrose for Peritoneal Ultrafiltration

Several clinical observations suggest the superiority of icodextrin compared with 4.25% dextrose in optimizing peritoneal ultrafiltration (UF), but no rigorous controlled evaluation has hitherto been performed. For comparing icodextrin and 4.25% dextrose during the long dwell of automated peritoneal dialysis, a multicenter, randomized, double-blind trial was conducted in 92 patients (control, 45; icodextrin, 47) with 4-h dialysate to plasma ratio creatinine >0.70 and D/D(0) glucose <0.34. Long-dwell net UF and the UF efficiency ratio (net UF volume per gram of dialysate carbohydrate absorbed) were determined at baseline, week 1, and week 2. The control and treatment groups were comparable at baseline (all patients using 4.25% dextrose for the long dwell) with regard to mean (+/-SEM) net UF (201.7 +/- 103.1 versus 141.6 +/- 75.4 ml, respectively; P = 0.637) and the percentage of patients with negative net UF (control, 37.8%; treatment, 42.6%; P = 0.641). During the study period, net UF was unchanged from baseline in the control group but increased significantly (P < 0.001) in the icodextrin group from 141.6 +/- 75.4 to 505.8 +/- 46.8 ml at week 1 and 540.2 +/- 46.8 ml at week 2. In the icodextrin group, the incidence of negative net UF was significantly lower (P < 0.0001) than in the control group. Findings were similar for UF efficiency ratio. Rash was reported significantly more often in the icodextrin group. This study showed that in high-average and high transporters, icodextrin is superior to 4.25% dextrose for long-dwell fluid and solute removal.

Loss-of-function mutations of Dynamin 2 promote T-ALL by enhancing IL-7 signalling.

Mutations in the DYNAMIN2 (DNM2) gene are frequently detected in human acute T-cell lymphoblastic leukemia (T-ALL), although the mechanisms linking these mutations to disease pathogenesis remain unknown. Using an ENU-based forward genetic screen for mice with erythroid phenotypes, we identified a heterozygous mouse line carrying a mutation in the GTPase domain of Dnm2 (Dnm2V265G) that induced a microcytic anemia. In vitro assays using the V265G mutant demonstrated loss of GTPase activity and impaired endocytosis that was comparable to other DNM2 mutants identified in human T-ALL. To determine the effects of DNM2 mutations in T-ALL, we bred the Dnm2V265G mice with the Lmo2 transgenic mouse model of T-ALL. Heterozygous Dnm2 mutants lacking the Lmo2 transgene displayed normal T-cell development, and did not develop T-ALL. In contrast, compound heterozygotes displayed an accelerated onset of T-ALL compared with mice carrying the Lmo2 oncogene alone. The leukemias from these mice exhibited a more immature immunophenotype and an expansion in leukemic stem cell numbers. Mechanistically, the Dnm2 mutation impaired clathrin-mediated endocytosis of the interleukin (IL)-7 receptor resulting in increased receptor density on the surface of leukemic stem cells. These findings suggest that DNM2 mutations cooperate with T-cell oncogenes by enhancing IL-7 signalling.

Activation of the erythroid K-Cl cotransporter Kcc1 enhances sickle cell disease pathology in a humanized mouse model

We used an N-ethyl-N-nitrosurea-based forward genetic screen in mice to identify new genes and alleles that regulate erythropoiesis. Here, we describe a mouse line expressing an activated form of the K-Cl cotransporter Slc12a4 (Kcc1), which results in a semi-dominant microcytosis of red cells. A missense mutation from methionine to lysine in the cytoplasmic tail of Kcc1 impairs phosphorylation of adjacent threonines required for inhibiting cotransporter activity. We bred Kcc1(M935K) mutant mice with a humanized mouse model of sickle cell disease to directly explore the relevance of the reported increase in KCC activity in disease pathogenesis. We show that a single mutant allele of Kcc1 induces widespread sickling and tissue damage, leading to premature death. This mouse model reveals important new insights into the regulation of K-Cl cotransporters and provides in vivo evidence that increased KCC activity worsened end-organ damage and diminished survival in sickle cell disease.

ENU mutagenesis identifies the first mouse mutants reproducing human β-thalassemia at the genomic level.

Forward genetic screens have been performed in many species to identify phenotypes in specific organ systems. We have undertaken a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify dominant mutations that perturb erythropoiesis in mice. Mutant mice that displayed an erythrocyte mean cell volume (MCV) greater than three standard deviations from the population mean were identified. Two of these lines, RBC13 and RBC14, displayed a hypochromic, microcytic anemia, accompanied by a marked reticulocytosis, splenomegaly and diminished red cell survival. Timed pregnancies from heterozygous intercrosses revealed that a quarter of the embryos displayed severe anemia and did not survive beyond embryonic day (E) 18.5, consistent with homozygous β-thalassemia. Genetic complementation studies with a β-thalassemia mouse line reproduced the embryonic lethality in compound heterozygotes and a genomic custom capture array and massively parallel sequencing of the β-globin locus identified the causative mutations. The RBC13 line displayed a nonsense mutation at codon 40 in exon 2 of the β-major gene, invoking parallels with the common β(0)39 thalassemia mutation seen in humans. The RBC14 line exhibited a mutation at the polyadenylation signal of the β-major gene, exactly replicating a human β-thalassemia mutation. The RBC13 and RBC14 lines are the first β-thalassemia mouse models that reproduce human β-thalassemia at the genomic level, and as such highlight the power of ENU mutagenesis screens in generating mouse models of human disease.

Genomics of primary chemoresistance and remission induction failure in paediatric and adult acute myeloid leukaemia

Cure rates of children and adults with acute myeloid leukaemia (AML) remain unsatisfactory partly due to chemotherapy resistance. We investigated the genetic basis of AML in 107 primary cases by sequencing 670 genes mutated in haematological malignancies. SETBP1, ASXL1 and RELN mutations were significantly associated with primary chemoresistance. We identified genomic alterations not previously described in AML, together with distinct genes that were significantly overexpressed in therapy‐resistant AML. Defined gene mutations were sufficient to explain primary induction failure in only a minority of cases. Thus, additional genetic or molecular mechanisms must cause primary chemoresistance in paediatric and adult AML.

Induction of human fetal hemoglobin expression by adenosine-2’,3’-dialdehyde

BackgroundPharmacologic reactivation of fetal hemoglobin expression is a promising strategy for treatment of sickle cell disease and β-thalassemia. The objective of this study was to investigate the effect of the methyl transferase inhibitor adenosine-2’,3’-dialdehyde (Adox) on induction of human fetal hemoglobin (HbF) in K562 cells and human hematopoietic progenitor cells.MethodsExpression levels of human fetal hemoglobin were assessed by northern blot analysis and Real-time PCR. HbF and adult hemoglobin (HbA) content were analyzed using high-performance liquid chromatography (HPLC). DNA methylation levels on human gamma-globin gene promoters were determined using Bisulfite sequence analysis. Enrichment of histone marks on genes was assessed by chromosome immunoprecipitation (ChIP).ResultsAdox induced γ-globin gene expression in both K562 cells and in human bone marrow erythroid progenitor cells through a mechanism potentially involving inhibition of protein arginine methyltransferase 5 (PRMT5).ConclusionsThe ability of methyl transferase inhibitors such as Adox to efficiently reactivate fetal hemoglobin expression suggests that these agents may provide a means of reactivating fetal globin expression as a therapeutic option for treating sickle cell disease and β-thalassemia.

MEF2C phosphorylation is required for chemotherapy resistance in acute myeloid leukemia

In acute myeloid leukemia (AML), chemotherapy resistance remains prevalent and poorly understood. Using functional proteomics of patient AML specimens, we identified MEF2C S222 phosphorylation as a specific marker of primary chemoresistance. We found that Mef2cS222A/S222A knock-in mutant mice engineered to block MEF2C phosphorylation exhibited normal hematopoiesis, but were resistant to leukemogenesis induced by MLL-AF9 MEF2C phosphorylation was required for leukemia stem cell maintenance and induced by MARK kinases in cells. Treatment with the selective MARK/SIK inhibitor MRT199665 caused apoptosis and conferred chemosensitivity in MEF2C-activated human AML cell lines and primary patient specimens, but not those lacking MEF2C phosphorylation. These findings identify kinase-dependent dysregulation of transcription factor control as a determinant of therapy response in AML, with immediate potential for improved diagnosis and therapy for this disease.Significance: Functional proteomics identifies phosphorylation of MEF2C in the majority of primary chemotherapy-resistant AML. Kinase-dependent dysregulation of this transcription factor confers susceptibility to MARK/SIK kinase inhibition in preclinical models, substantiating its clinical investigation for improved diagnosis and therapy of AML. Cancer Discov; 8(4); 478-97. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.

Bone marrow transplantation corrects haemolytic anaemia in a novel ENU mutagenesis mouse model of TPI deficiency

ABSTRACT In this study, we performed a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice to identify novel genes or alleles that regulate erythropoiesis. Here, we describe a recessive mouse strain, called RBC19, harbouring a point mutation within the housekeeping gene, Tpi1, which encodes the glycolysis enzyme, triosephosphate isomerase (TPI). A serine in place of a phenylalanine at amino acid 57 severely diminishes enzyme activity in red blood cells and other tissues, resulting in a macrocytic haemolytic phenotype in homozygous mice, which closely resembles human TPI deficiency. A rescue study was performed using bone marrow transplantation of wild-type donor cells, which restored all haematological parameters and increased red blood cell enzyme function to wild-type levels after 7 weeks. This is the first study performed in a mammalian model of TPI deficiency, demonstrating that the haematological phenotype can be rescued. Summary: In a novel ENU mutagenesis mouse model of TPI deficiency, bone marrow transplantation was conducted to demonstrate that haemolytic and red blood cell glycolytic defects can be effectively rescued.

Loss of Dynamin 2 GTPase function results in microcytic anaemia

In a dominant mouse ethylnitrosurea mutagenesis screen for genes regulating erythropoiesis, we identified a pedigree with a novel microcytic hypochromia caused by a V235G missense mutation in Dynamin 2 (Dnm2). Mutations in Dnm2, a GTPase, are highly disease‐specific and have been implicated in four forms of human diseases: centronuclear myopathy, Charcot‐Marie Tooth neuropathy and, more recently, T‐cell leukaemia and Hereditary Spastic Paraplegia, but red cell abnormalities have not been reported to date. The V235G mutation lies within a crucial GTP nucleotide‐binding pocket of Dnm2, and resulted in defective GTPase activity and incompatibility with life in the homozygous state. Dnm2 is an essential mediator of clathrin‐mediated endocytosis, which is required for the uptake of transferrin (Tf) into red cells for incorporation of haem. Accordingly, we observed significantly reduced Tf uptake by Dnm2+/V235G cells, which led to impaired endosome formation. Despite these deficiencies, surprisingly all iron studies were unchanged, suggesting an unexplained alternative mechanism underlies microcytic anaemia in Dnm2+/V235G mice. This study provides the first in vivo evidence for the requirements of Dnm2 in normal erythropoiesis.

A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen

ABSTRACT A genome-wide ethyl-N-nitrosourea (ENU) mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX). A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria. Summary: A mouse mutagenesis model of hereditary coproporphyria has significant genetic and biochemical parallels to that of the human condition.