Akihito Otsuki

A Remote GATA2 Hematopoietic Enhancer Drives Leukemogenesis in inv(3)(q21;q26) by Activating EVI1 Expression

Chromosomal inversion between 3q21 and 3q26 results in high-risk acute myeloid leukemia (AML). In this study, we identified a mechanism whereby a GATA2 distal hematopoietic enhancer (G2DHE or -77-kb enhancer) is brought into close proximity to the EVI1 gene in inv(3)(q21;q26) inversions, leading to leukemogenesis. We examined the contribution of G2DHE to leukemogenesis by creating a bacterial artificial chromosome (BAC) transgenic model that recapitulates the inv(3)(q21;q26) allele. Transgenic mice harboring a linked BAC developed leukemia accompanied by EVI1 overexpression-neoplasia that was not detected in mice bearing the same transgene but that was missing the GATA2 enhancer. These results establish the mechanistic basis underlying the pathogenesis of a severe form of leukemia through aberrant expression of the EVI1 proto-oncogene.

Amelioration of inflammation and tissue damage in sickle cell model mice by Nrf2 activation

Significance Sickle cell disease (SCD) is one of the most common inherited disorders. A mutation in the β-globin gene causes deformation of red blood cells into a sickle shape, which in turn causes intravascular hemolysis and vaso-occlusion resulting in damage to multiple organs. Most studies that propose to develop new SCD therapies include the induction of fetal γ-globin expression to inhibit sickle cell formation as their ultimate goal. In contrast, we demonstrate here that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) ameliorates the development of inflammation and tissue damage that strongly affect the morbidity of SCD patients. Notably, several compounds that serve as Nrf2 inducers have been developed or are under development. The data indicate that Nrf2 activation could improve the prognosis for SCD patients. Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene, leading to the production of abnormally shaped red blood cells. Sickle cells are prone to hemolysis and thereby release free heme into plasma, causing oxidative stress and inflammation that in turn result in damage to multiple organs. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a master regulator of the antioxidant cell-defense system. Here we show that constitutive Nrf2 activation by ablation of its negative regulator Keap1 (kelch-like ECH-associated protein 1) significantly improves symptoms in SCD model mice. SCD mice exhibit severe liver damage and lung inflammation associated with high expression levels of proinflammatory cytokines and adhesion molecules compared with normal mice. Importantly, these symptoms subsided after Nrf2 activation. Although hemolysis and stress erythropoiesis did not change substantially in the Nrf2-activated SCD mice, Nrf2 promoted the elimination of plasma heme released by sickle cells’ hemolysis and thereby reduced oxidative stress and inflammation, demonstrating that Nrf2 activation reduces organ damage and segregates inflammation from prevention of hemolysis in SCD mice. Furthermore, administration of the Nrf2 inducer CDDO-Im (2-cyano-3, 12 dioxooleana-1, 9 diene-28-imidazolide) also relieved inflammation and organ failure in SCD mice. These results support the contention that Nrf2 induction may be an important means to protect organs from the pathophysiology of sickle cell-induced damage.

Halofuginone enhances the chemo-sensitivity of cancer cells by suppressing NRF2 accumulation

ABSTRACT The KEAP1‐NRF2 system regulates the cellular defence against oxidative and xenobiotic stresses. NRF2 is a transcription factor that activates the expression of cytoprotective genes encoding antioxidative, detoxifying and metabolic enzymes as well as transporters. Under normal conditions, KEAP1 represses NRF2 activity by degrading the NRF2 protein. When cells are exposed to stresses, KEAP1 stops promoting NRF2 degradation, and NRF2 rapidly accumulates and activates the transcription of target genes. Constitutive accumulation of NRF2 via a variety of mechanisms that disrupt KEAP1‐mediated NRF2 degradation has been observed in various cancer types. Constitutive NRF2 accumulation confers cancer cells with a proliferative advantage as well as resistance to anti‐cancer drugs and radiotherapies. To suppress the chemo‐ and radio‐resistance of cancer cells caused by NRF2 accumulation, we conducted high‐throughput chemical library screening for NRF2 inhibitors and identified febrifugine derivatives. We found that application of the less‐toxic derivative halofuginone in a low dose range rapidly reduced NRF2 protein levels. Halofuginone induced a cellular amino acid starvation response that repressed global protein synthesis and rapidly depleted NRF2. Halofuginone treatment ameliorated the resistance of NRF2‐addicted cancer cells to anti‐cancer drugs both in vitro and in vivo. These results provide preclinical proof‐of‐concept evidence for halofuginone as an NRF2 inhibitor applicable to treatment of chemo‐ and radio‐resistant forms of cancer. Graphical abstract Figure. No Caption Available. HighlightsHigh‐throughput screening identified febrifugine derivatives as NRF2 inhibitors.The febrifugine derivative halofuginone (HF) suppresses NRF2 protein accumulation.HF represses protein synthesis via an amino acid starvation response.HF enhances the sensitivity of NRF2‐addicted cancer cells to anti‐cancer drugs.HF serves as a chemo‐sensitizer of NRF2‐addicted cancer cells.

Gata3 Hypomorphic Mutant Mice Rescued with a Yeast Artificial Chromosome Transgene Suffer a Glomerular Mesangial Cell Defect

ABSTRACT GATA3 is a zinc finger transcription factor that plays a crucial role in embryonic kidney development, while its precise functions in the adult kidney remain largely unexplored. Here, we demonstrate that GATA3 is specifically expressed in glomerular mesangial cells and plays a critical role in the maintenance of renal glomerular function. Newly generated Gata3 hypomorphic mutant mice exhibited neonatal lethality associated with severe renal hypoplasia. Normal kidney size was restored by breeding the hypomorphic mutant with a rescuing transgenic mouse line bearing a 662-kb Gata3 yeast artificial chromosome (YAC), and these animals (termed G3YR mice) survived to adulthood. However, most of the G3YR mice showed degenerative changes in glomerular mesangial cells, which deteriorated progressively during postnatal development. Consequently, the G3YR adult mice suffered severe renal failure. We found that the 662-kb Gata3 YAC transgene recapitulated Gata3 expression in the renal tubules but failed to direct sufficient GATA3 activity to mesangial cells. Renal glomeruli of the G3YR mice had significantly reduced amounts of platelet-derived growth factor receptor (PDGFR), which is known to participate in the development and maintenance of glomerular mesangial cells. These results demonstrate a critical role for GATA3 in the maintenance of mesangial cells and its absolute requirement for prevention of glomerular disease.

Derepression of the DNA Methylation Machinery of the Gata1 Gene Triggers the Differentiation Cue for Erythropoiesis

ABSTRACT GATA1 is a critical regulator of erythropoiesis. While the mechanisms underlying the high-level expression of GATA1 in maturing erythroid cells have been studied extensively, the initial activation of the Gata1 gene in early hematopoietic progenitors remains to be elucidated. We previously identified a hematopoietic stem and progenitor cell (HSPC)-specific silencer element (the Gata1 methylation-determining region [G1MDR]) that recruits DNA methyltransferase 1 (Dnmt1) and provokes methylation of the Gata1 gene enhancer. In the present study, we hypothesized that removal of the G1MDR-mediated silencing machinery is the molecular basis of the initial activation of the Gata1 gene and erythropoiesis. To address this hypothesis, we generated transgenic mouse lines harboring a Gata1 bacterial artificial chromosome in which the G1MDR was deleted. The mice exhibited abundant GATA1 expression in HSPCs, in a GATA2-dependent manner. The ectopic GATA1 expression repressed Gata2 transcription and induced erythropoiesis and apoptosis of HSPCs. Furthermore, genetic deletion of Dnmt1 in HSPCs activated Gata1 expression and depleted HSPCs, thus recapitulating the HSC phenotype associated with GATA1 gain of function. These results demonstrate that the G1MDR holds the key to HSPC maintenance and suggest that release from this suppressive mechanism is a fundamental requirement for subsequent initiation of erythroid differentiation.

GATA2 haploinsufficiency accelerates EVI1-driven leukemogenesis

Chromosomal rearrangements between 3q21 and 3q26 induce inappropriate EVI1 expression by recruiting a GATA2-distal hematopoietic enhancer (G2DHE) to the proximity of the EVI1 gene, leading to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The acquisition of G2DHE by the EVI1 gene reciprocally deprives this enhancer of 1 of the 2 GATA2 alleles, resulting in a loss-of-function genetic reduction in GATA2 abundance. Because GATA2 haploinsufficiency is strongly associated with MDS and AML, we asked whether EVI1 misexpression and GATA2 haploinsufficiency both contributed to the observed leukemogenesis by using a 3q21q26 mouse model that recapitulates the G2DHE-driven EVI1 misexpression, but in this case, it was coupled to a Gata2 heterozygous germ line deletion. Of note, the Gata2 heterozygous deletion promoted the EVI1-provoked leukemic transformation, resulting in early onset of leukemia. The 3q21q26 mice suffered from leukemia in which B220+ cells and/or Gr1+ leukemic cells occupied their bone marrows. We found that the B220+Gr1-c-Kit+ population contained leukemia-initiating cells and supplied Gr1+ leukemia cells in the 3q21q26 leukemia. When Gata2 expression levels in the B220+Gr1-c-Kit+ cells were decreased as a result of Gata2 heterozygous deletion or spontaneous phenomenon, myeloid differentiation of the B220+Gr1-c-Kit+ cells was suppressed, and the cells acquired induced proliferation as well as B-lymphoid-primed characteristics. Competitive transplantation analysis revealed that Gata2 heterozygous deletion confers selective advantage to EVI1-expressing leukemia cell expansion in recipient mice. These results demonstrate that both the inappropriate stimulation of EVI1 and the loss of 1 allele equivalent of Gata2 expression contribute to the acceleration of leukemogenesis.

Establishment of erythroleukemic GAK14 cells and characterization of GATA1 N‐terminal domain

GATA1 is a transcription factor essential for erythropoiesis and megakaryopoiesis. It has been found that Gata1 gene knockdown heterozygous female (Gata1G1.05/+) mice spontaneously develop erythroblastic leukemias. In this study, we have generated a novel Gata1 knockdown erythroblastic cell line, designated GAK14, from the leukemia cells in the Gata1G1.05/+ mice. Although GAK14 cells maintain immature phenotype on OP9 stromal cells in the presence of erythropoietin and stem cell factor, the cells produce Gr‐1‐, Mac1‐, B220‐, CD3e‐ or CD49b‐positive hematopoietic cells when co‐cultured with DAS104‐8 feeder cells. However, GAK14 cells did not produce erythroid and megakaryocytic lineages, perhaps due to the absence of GATA1. Indeed, GAK14 cells became capable of differentiating into mature erythroid cells when complemented with full‐length GATA1 and co‐cultured with fetal liver–derived FLS5 stromal cells. This differentiation potential was impaired when GATA1 lacking the N‐terminal domain was complemented. The N‐terminal domain is known to contribute to the pathogenesis of transient abnormal myelopoiesis and acute megakaryoblastic leukemia related to Down syndrome. These results thus showed that GAK14 cells will serve as a powerful tool for dissecting domain function of GATA1 and that the GATA1 N‐terminal domain is essential for the erythroid differentiation of GAK14 cells.