Landscape of driver mutations and their clinical effects on Down syndrome-related myeloid neoplasms.

Transient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse, and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group AML-D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival [28.6% vs. 90.5%, P < 0.001; 25.0% vs. 89.5%, P < 0.001] than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.

Exploring Large MAF Transcription Factors: Functions, Pathology, and Mouse Models with Point Mutations.

Large musculoaponeurotic fibrosarcoma (MAF) transcription factors contain acidic, basic, and leucine zipper regions. Four types of MAF have been elucidated in mice and humans, namely c-MAF, MAFA, MAFB, and NRL. This review aimed to elaborate on the functions of MAF transcription factors that have been studied in vivo so far, as well as describe the pathology of human patients and corresponding mouse models with c-MAF, MAFA, and MAFB point mutations. To identify the functions of MAF transcription factors in vivo, we generated genetically modified mice lacking c-MAF, MAFA, and MAFB and analyzed their phenotypes. Further, in recent years, c-MAF, MAFA, and MAFB have been identified as causative genes underpinning many rare diseases. Careful observation of human patients and animal models is important to examine the pathophysiological mechanisms underlying these conditions for targeted therapies. Murine models exhibit phenotypes similar to those of human patients with c-MAF, MAFA, and MAFB mutations. Therefore, generating these animal models emphasizes their usefulness for research uncovering the pathophysiology of point mutations in MAF transcription factors and the development of etiology-based therapies.

Generation and mutational analysis of a transgenic murine model of the human MAF mutation.

Aymé-Gripp syndrome is an autosomal dominant multisystem disorder. The major clinical features of this syndrome include congenital cataracts, sensorineural hearing loss, intellectual disability, and a distinctive flat facial appearance. MAF has been identified as a causative gene of the syndrome, and heterozygous variants owing to impairment in glycogen synthase kinase 3 (GSK3)-mediated MAF phosphorylation shows related disorders. However, the underlying mechanisms of these types of disorders in affected individuals remain poorly understood. To explore the underlying mechanisms and discover new phenotypes, a murine model with a Maf mutation on a GSK3 phosphorylation motif, p.Thr58Ile, was generated using CRISPR-Cas9 gene editing. This is a homologous mutation to that in human patients. Our murine model exhibited similar phenotypes to those in humans, such as lens abnormalities, short stature, growth retardation, and abnormal skull morphology. The murine model showed decreased brain volume and malocclusion. Considering the sequencing and genotyping data, our models were successfully generated for the first time (to the best of our knowledge). Therefore, this study offers new and unique functional insights into human and murine MAF and novel clinical values of MAF pathogenic variants associated with changes in the functions of several organs based on a viable murine model.

Transcription factor c-Maf deletion improves streptozotocin-induced diabetic nephropathy by directly regulating Sglt2 and Glut2.

The transcription factor c-Maf has been widely studied and has been reported to play a critical role in embryonic kidney development; however, the postnatal functions of c-Maf in adult kidneys remain unknown as c-Maf-null C57BL/6J mice exhibit embryonic lethality. In this study, we investigated the role of c-Maf in adult mouse kidneys by comparing the phenotypes of tamoxifen-inducible (TAM-inducible) c-Maf-knockout mice (c-Maffl/fl; CAG-Cre-ERTM mice named "c-MafΔTAM") with those of c-Maffl/fl control mice, 10 days after TAM injection [TAM(10d)]. In addition, we examined the effects of c-Maf deletion on diabetic conditions by injecting the mice with streptozotocin, 4 weeks before TAM injection. c-MafΔTAM mice displayed primary glycosuria caused by sodium-glucose cotransporter 2 (Sglt2) and glucose transporter 2 (Glut2) downregulation in the kidneys without diabetes, as well as morphological changes and life-threatening injuries in the kidneys on TAM(10d). Under diabetic conditions, c-Maf deletion promoted recovery from hyperglycemia and suppressed albuminuria and diabetic nephropathy by causing similar effects as did Sglt2 knockout and SGLT2 inhibitors. In addition to demonstrating the potentially unique gene regulation of c-Maf, these findings highlight the renoprotective effects of c-Maf deficiency under diabetic conditions and suggest that c-Maf could be a novel therapeutic target gene for treating diabetic nephropathy.

MafB Maintains ß-Cell Identity under MafA-Deficient Conditions.

The transcription factor MafB plays an essential role in ß-cell differentiation during the embryonic stage in rodents. Although MafB disappears from ß-cells after birth, it has been reported that MafB can be evoked in ß-cells and is involved in insulin+ß-cell number and islet architecture maintenance in adult mice under diabetic conditions. However, the underlying mechanism by which MafB protects ß-cells remains unknown. To elucidate this, we performed RNA sequencing using an inducible diabetes model (A0BΔpanc mice) that we previously generated. We found that the deletion of Mafb can induce ß-cell dedifferentiation, characterized by the upregulation of dedifferentiation markers, Slc5a10 and Cck, as well as several ß-cell-disallowed genes, and by the downregulation of mature ß-cell markers, Slc2a2 and Ucn3. However, there is no re-expression of well-known progenitor cell markers, Foxo1 and Neurog3. Further, the appearance of ALDH1A3+ cells and the disappearance of UCN3+ cells also verify the ß-cell dedifferentiation state. Collectively, our results suggest that MafB can maintain ß-cell identity under certain pathological conditions in adult mice, providing novel insight into the role of MafB in ß-cell identity maintenance.

An Inducible Diabetes Mellitus Murine Model Based on MafB Conditional Knockout under MafA-Deficient Condition.

Diabetes mellitus is an increasingly severe chronic metabolic disease that is occurring at an alarming rate worldwide. Various diabetic models, including non-obese diabetic mice and chemically induced diabetic models, are used to characterize and explore the mechanism of the disease's pathophysiology, in hopes of detecting and identifying novel potential therapeutic targets. However, this is accompanied by disadvantages, such as specific conditions for maintaining the incidence, nonstable hyperglycemia induction, and potential toxicity to other organs. Murine MAFA and MAFB, two closely-linked islet-enriched transcription factors, play fundamental roles in glucose sensing and insulin secretion, and maintenance of pancreatic ß-cell, respectively, which are highly homologous to human protein orthologs. Herein, to induce the diabetes mellitus model at a specific time point, we generated Pdx1-dependent Mafb-deletion mice under Mafa knockout condition (A0BΔpanc), via tamoxifen-inducible Cre-loxP system. After 16 weeks, metabolic phenotypes were characterized by intraperitoneal glucose tolerance test (IPGTT), urine glucose test, and metabolic parameters analysis. The results indicated that male A0BΔpanc mice had obvious impaired glucose tolerance, and high urine glucose level. Furthermore, obvious renal lesions, impaired islet structure and decreased proportion of insulin positive cells were observed. Collectively, our results indicate that A0BΔpanc mice can be an efficient inducible model for diabetes research.

c-MAF deletion in adult C57BL/6J mice induces cataract formation and abnormal differentiation of lens fiber cells.

The transcription factor c-MAF is a member of the large MAF family, members of which possess transactivation and bZIP domains. c-MAF plays an important role in lens formation, T-lymphocyte differentiation, hypertrophic chondrocyte differentiation, and kidney development in mouse embryos. However, because homozygous deletion of c-Maf in C57BL/6J mice causes embryonic lethality, the functions of c-MAF in adult mice remain largely uninvestigated. To address this issue, we generated c-Maf floxed (c-Maffl/fl) C57BL/6J mice and established tamoxifen-inducible c-Maf knockout mice (c-Maffl/fl; CAG-Cre-ERTM mice, c-MafΔTAM). After tamoxifen injection, adult c-MafΔTAM mice showed successful deletion of c-Maf protein and developed severe cataracts; cataracts are also seen in human patients who have mutations in the c-MAF DNA binding domain. Furthermore, adult c-MafΔTAM mice exhibited abnormal lens structure and impaired differentiation of lens fiber cells. In summary, we established c-Maffl/fl and c-MafΔTAM C57BL/6J mice, which can be useful animal models for the investigation of c-MAF function in various developmental stages and can also be used as a disease model for cataracts.

Phenotypic analysis of mice carrying human-type MAFB p.Leu239Pro mutation.

The transcription factor, MafB, plays important role in the differentiation and functional maintenance of various cells and tissues, such as the inner ear, kidney podocyte, parathyroid gland, pancreatic islet, and macrophages. The rare heterozygous substitution (p.Leu239Pro) of the DNA binding domain in MAFB is the cause of Focal Segmental Glomerulosclerosis associated with Duane Retraction Syndrome, which is characterized by impaired horizontal eye movement due to cranial nerve maldevelopment in humans. In this research, we generated mice carrying MafB p.Leu239Pro (Mafbmt/mt) and retrieved their tissues for analysis. As a result, we found that the phenotype of Mafbmt/mt mouse was similar to that of the conventional Mafb deficient mouse. This finding suggests that the Leucine residue at 239 in the DNA binding domain plays a key role in MafB function and could contribute to the diagnosis or development of treatment for patients carrying the MafB p.Leu239Pro missense variant.

Uncovering the role of MAFB in glucagon production and secretion in pancreatic α-cells using a new α-cell-specific Mafb conditional knockout mouse model.

Cre/loxP is a site-specific recombination system extensively used to enable the conditional deletion or activation of target genes in a spatial- and/or temporal-specific manner. A number of pancreatic-specific Cre driver mouse lines have been broadly established for studying the development, function and pathology of pancreatic cells. However, only a few models are currently available for glucagon-producing α-cells. Disagreement exists over the role of the MAFB transcription factor in glucagon expression during postnatal life, which might be due to the lack of α-cell-specific Cre driver mice. In the present study, we established a novel Gcg-Cre knock-in mouse line with the Cre transgene expressed under the control of the preproglucagon (Gcg) promoter without disrupting the endogenous Gcg gene expression. Then, we applied this newly developed Gcg-Cre mouse line to generate a new α-cell-specific Mafb conditional knockout mouse model (MafbΔGcg). Not only α-cell number but also glucagon production were significantly decreased in MafbΔGcg mice compared to control littermates, suggesting an indispensable role of MAFB in both α-cell development and function. Taken together, our newly developed Gcg-Cre mouse line, which was successfully utilized to uncover the role of MAFB in α-cells, is a useful tool for genetic manipulation in pancreatic α-cells, providing a new platform for future studies in this field.

Neuron-specific Mafb knockout causes growth retardation accompanied by an impaired growth hormone/insulin-like growth factor I axis.

Mammalian postnatal growth is regulated primarily by the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. MafB is a basic leucine zipper (bZip) transcription factor that has pleiotropic functions. Although MafB plays a critical role in fetal brain development, such as in guidance for hindbrain segmentation, its postnatal role in neurons remains to be elucidated. To investigate this, we used neuron-specific Mafb conditional knockout (cKO) mice. In addition to an approximately 50% neonatal viability, the Mafb cKO mice exhibited growth retardation without apparent signs of low energy intake. Notably, serum IGF-I levels of these mice in the postnatal stage were lower than those of control mice. They seemed to have a neuroendocrine dysregulation, as shown by the upregulation of serum GH levels in the resting state and an inconsistent secretory response of GH upon administration of growth hormone-releasing hormone. These findings reveal that neuronal MafB plays an important role in postnatal development regulated by the GH/IGF-I axis.

Transcription factor MafB may play an important role in secondary hyperparathyroidism.

The transcription factor MafB is essential for development of the parathyroid glands, the expression of which persists after morphogenesis and in adult parathyroid glands. However, the function of MafB in adult parathyroid tissue is unclear. To investigate this, we induced chronic kidney disease (CKD) in wild-type and MafB heterozygote (MafB+/-) mice by feeding them an adenine-supplemented diet, leading to secondary hyperparathyroidism. The elevated serum creatinine and blood urea nitrogen levels in heterozygous and wild-type mice fed the adenine-supplemented diet were similar. Interestingly, secondary hyperparathyroidism, characterized by serum parathyroid hormone elevation and enlargement of parathyroid glands, was suppressed in MafB+/- mice fed the adenine-supplemented diet compared to similarly fed wild-type littermates. Quantitative RT-PCR and immunohistochemical analyses showed that the increased expression of parathyroid hormone and cyclin D2 in mice with CKD was suppressed in the parathyroid glands of heterozygous CKD mice. A reporter assay indicated that MafB directly regulated parathyroid hormone and cyclin D2 expression. To exclude an effect of a developmental anomaly in MafB+/- mice, we analyzed MafB tamoxifen-induced global knockout mice. Hypocalcemia-stimulated parathyroid hormone secretion was significantly impaired in MafB knockout mice. RNA-sequencing analysis indicated PTH, Gata3 and Gcm2 depletion in the parathyroid glands of MafB knockout mice. Thus, MafB appears to play an important role in secondary hyperparathyroidism by regulation of parathyroid hormone and cyclin D2 expression. Hence, MafB may represent a new therapeutic target in secondary hyperparathyroidism.

Th2-biased GATA-3 transgenic mice developed severe experimental peritoneal fibrosis compared with Th1-biased T-bet and Th17-biased RORγt transgenic mice.

Encapsulating peritoneal sclerosis is one of the most serious complications of long-term peritoneal dialysis. The pathogenesis of encapsulating peritoneal sclerosis has not been elucidated, but several putative factors necessary for the development of peritoneum fibrosis (PF) have been reported. However, the roles of T helper (Th) cells in the progression of PF are unknown. The purpose of this study was to clarify the roles of Th1, Th2, and Th17 cells in the progression of PF. T-bet, GATA-3, and RORγt are Th1, Th2, and Th17 lineage commitment transcription factors, respectively. We previously generated Th1-biased (T-bet transgenic (Tg)) mice, Th2-biased (GATA-3 Tg) mice, and Th17-biased (RORγt Tg) mice. In this study, Th1, Th2, Th17-biased, and wild-type mice were administered chlorhexidine gluconate (CG) intraperitoneally and analyzed on day 21. CG-injected GATA-3 Tg mice showed a distended intestinal tract and developed marked thickening of the submesothelial space compared with the other groups. CG-injected GATA-3 Tg mice also showed significant expression of α-SMA positive cells, macrophages, and collagen III in the submesothelium. In contrast, CG-injected T-bet Tg mice only developed mild peritoneal fibrosis. Cytokines analysis in peritoneal fluid showed that IFN-γ was significantly increased in CG-injected T-bet Tg mice and that IL-13 was significantly increased in CG-injected GATA-3 Tg mice. Moreover, intraperitoneal administration of IFN-γ improved PF in GC-injected wild-type mice. Our results suggest that Th2 cells may play roles in the development of experimental PF and that Th1 cells may alleviate the severity of experimental PF.

Overexpression of GATA-3 in T cells accelerates dextran sulfate sodium-induced colitis.

Ulcerative colitis (UC) is an inflammatory bowel disease, and its pathogenesis includes genetic, environmental, and immunological factors, such as T helper cells and their secreted cytokines. T helper cells are classified as Th1, Th2, and Th17 cells. However, it is unclear which T helper cells are important in UC. Dextran sulfate sodium (DSS)-induced colitis is a commonly used model of UC. In this study, we induced DSS colitis in Th1 dominant (T-bet transgenic (Tg)) mice, Th2 dominant (GATA-3 Tg) mice, and Th17 dominant (RORγt Tg) mice to elucidate the roles of T helper cell in DSS colitis. The results showed that GATA-3 Tg mice developed the most severe DSS colitis compared with the other groups. GATA-3 Tg mice showed a significant decreased in weight from day 1 to day 7, and an increased high score for the disease activity index compared with the other groups. Furthermore, GATA-3 Tg mice developed many ulcers in the colon, and many neutrophils and macrophages were detected on day 4 after DSS treatment. Measurement of GATA-3-induced cytokines demonstrated that IL-13 was highly expressed in the colon from DSS-induced GATA-3 Tg mice. In conclusion, GATA-3 overexpression in T-cells and IL-13 might play important roles in the development of DSS colitis.

Overexpression of Mafb in podocytes protects against diabetic nephropathy.

We previously showed that the transcription factor Mafb is essential for podocyte differentiation and foot process formation. Podocytes are susceptible to injury in diabetes, and this injury leads to progression of diabetic nephropathy. In this study, we generated transgenic mice that overexpress Mafb in podocytes using the nephrin promoter/enhancer. To examine a potential pathogenetic role for Mafb in diabetic nephropathy, Mafb transgenic mice were treated with either streptozotocin or saline solution. Diabetic nephropathy was assessed by renal histology and biochemical analyses of urine and serum. Podocyte-specific overexpression of Mafb had no effect on body weight or blood glucose levels in either diabetic or control mice. Notably, albuminuria and changes in BUN levels and renal histology observed in diabetic wild-type animals were ameliorated in diabetic Mafb transgenic mice. Moreover, hyperglycemia-induced downregulation of Nephrin was mitigated in diabetic Mafb transgenic mice, and reporter assay results suggested that Mafb regulates Nephrin directly. Mafb transgenic glomeruli also overexpressed glutathione peroxidase, an antioxidative stress enzyme, and levels of the oxidative stress marker 8-hydroxydeoxyguanosine decreased in the urine of diabetic Mafb transgenic mice. Finally, Notch2 expression increased in diabetic glomeruli, and this effect was enhanced in diabetic Mafb transgenic glomeruli. These data indicate Mafb has a protective role in diabetic nephropathy through regulation of slit diaphragm proteins, antioxidative enzymes, and Notch pathways in podocytes and suggest that Mafb could be a therapeutic target.

Overexpression of RORγt under control of the CD2 promoter induces polyclonal plasmacytosis and autoantibody production in transgenic mice.

Retinoic acid related orphan receptor gamma-t (RORγt) is known to be a master regulator of Th17-cell development. In this study, we generated RORγt-overexpressing transgenic (RORγt Tg) mice in which transgene expression was driven by the CD2 promoter, and found that these mice developed polyclonal plasmacytosis and autoantibody production. RORγt Tg mice were generated on a C57BL/6 background, and also were intercrossed with BALB/c mice. BALB/c F1 (BALB/F1) RORγt Tg mice developed massive polyclonal plasma-cytosis, and had shorter life spans. Splenomegaly and infiltration of plasma cells into the lung were observed. Hyperglobulinemia, anti-double-stranded DNA antibodies, anti-erythrocyte antibodies, and anti-platelet antibodies were detected in BALB/F1 RORγt Tg mice. In the present study, polyclonal plasmacytosis in BALB/F1 RORγt Tg mice appeared to be due to the induction of excessive IL-6 production by IL-17. We detected increased numbers of CD11b(+) cells that produced IL-6. We also generatedIL-6-deficient RORγt Tg BALB/F1 background mice, which displayed high levels of serum IL-17, but did not develop severe hyperglobulinemia. Excessive IL-6 production by several cell types, including macrophages, in BALB/F1 RORγt Tg mice, might effect the development of plasma-cytosis. These results suggest that RORγt plays important roles in the development of plasmacytosis and autoantibody production.

A novel diabetes mellitus mouse model, MAFA-deficient and beta cell-specific MAFK-overexpressing hybrid transgenic mice, developed severe diabetic nephropathy and improved with TCV-116 (candesartan cilexetil) treatment.

Many models of diabetic nephropathy have been reported. However, it is rare that the characteristic findings of severe human diabetic nephropathy, such as diffuse, nodular, and exudative lesions, are all detected in one model mouse. Previously, we reported that MAFA-deficient and beta cell-specific MAFK-overexpressing hybrid transgenic (Mafa(-/-)Mafk (+)) mice develop diabetes mellitus and, after uninephrectomy, demonstrate these characteristic lesions. In this study, we administered TCV-116 (candesartan cilexetil) to Mafa(-/-)Mafk (+) mice after uninephrectomy and examined whether TCV-116 ameliorated the diabetic nephropathy. We also evaluated the utility of these mice as a model for developing treatments for diabetic nephropathy. We performed uninephrectomy of the Mafa(-/-)Mafk (+) mice at 8 weeks old. We then divided these mice into two groups as follows: 1) an untreated group and 2) a group treated with TCV-116 at 5 µg/g/day from 10 to 20 weeks. TCV-116 treatment did not affect serum glucose levels. However, in the treated group, urinary protein excretion, mesangial matrix expansion, enlargement of the kidney, and glomerular surface area were all improved relative to untreated mice. Oxidative stress is known to be increased in diabetic nephropathy and to be suppressed by TCV-116. The urinary level of 8-OHdG, an oxidative stress marker, at 20 weeks was lower in the TCV-116-treated group than in the untreated group. From these results, we concluded that the Mafa(-/-)Mafk (+) mouse is a useful model to analyze diabetic nephropathy and a useful tool for the development of new drugs to treat diabetic nephropathy.

MafA-deficient and beta cell-specific MafK-overexpressing hybrid transgenic mice develop human-like severe diabetic nephropathy.

Transcription factor MafA is a key molecule in insulin secretion and the development of pancreatic islets. Previously, we demonstrated that some of the MafA-deficient mice develop overt diabetes mellitus, and the phenotype of these mice seems to be mild probably because of redundant functions of other Maf proteins. In this study, we generated hybrid transgenic mice that were MafA-deficient and also over-expressed MafK specifically in beta cells (MafA(-/-)MafK(+)). MafA(-/-)MafK(+) mice developed severe overt diabetes mellitus within 5weeks old, and showed higher levels of proteinuria and serum creatinine. Histological analysis revealed that embryonic development of beta cells in the MafA(-/-)MafK(+) mice was significantly suppressed and the reduced number of beta cells was responsible for the early onset of diabetes. Furthermore, after uninephrectomy, these mice demonstrated three characteristics of human diabetic nephropathy: diffuse, nodular, and exudative lesions. MafA(-/-)MafK(+) mice might be a useful model for the analysis of human diabetic nephropathy.

Hyperglycemia induces oxidative and nitrosative stress and increases renal functional impairment in Nrf2-deficient mice.

The transcription factor Nrf2 regulates the expression of antioxidant genes. Hyperglycemia-induced oxidative stress is involved in the pathogenesis of diabetes and its complications. However, little is known about the protective role of Nrf2 in diabetes. To gain insight into the protective role of Nrf2 in diabetes we treated Nrf2 knockout (Nrf2 KO) mice with streptozotocin (STZ). The STZ Nrf2 KO mice did not develop renal hyperfiltration, which was observed in the STZ-treated wild-type (STZ WT) mice, but renal function gradually deteriorated over the 10-week observation period. Urinary excretion of nitric oxide metabolites and the occurrence of 8-nitroguanosine, which was detected in glomerular lesions, were increased in STZ Nrf2 KO mice during the early stages after treatment. In vivo electron paramagnetic resonance analysis revealed an accelerated rate of decay of the 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl spin probe signal in STZ Nrf2 KO mice. The addition of superoxide dismutase prolonged the half-life of the signal, which suggested that increased oxygen radical formation occurred in the STZ Nrf2 KO mice. These results suggested that hyperglycemia increased oxidative and nitrosative stress and accelerated renal injury in the Nrf2 KO mice and that Nrf2 serves as a defense factor against some diabetic complications.