Wanda Y. Li

IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the ‘oncometabolite’ R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.

Fas Receptor Expression in Germinal-Center B Cells Is Essential for T and B Lymphocyte Homeostasis

Fas is highly expressed in activated and germinal center (GC) B cells but can potentially be inactivated by misguided somatic hypermutation. We employed conditional Fas-deficient mice to investigate the physiological functions of Fas in various B cell subsets. B cell-specific Fas-deficient mice developed fatal lymphoproliferation due to activation of B cells and T cells. Ablation of Fas specifically in GC B cells reproduced the phenotype, indicating that the lymphoproliferation initiates in the GC environment. B cell-specific Fas-deficient mice also showed an accumulation of IgG1(+) memory B cells expressing high amounts of CD80 and the expansion of CD28-expressing CD4(+) Th cells. Blocking T cell-B cell interaction and GC formation completely prevented the fatal lymphoproliferation. Thus, Fas-mediated selection of GC B cells and the resulting memory B cell compartment is essential for maintaining the homeostasis of both T and B lymphocytes.

Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15

Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.

Estrogen controls the survival of BRCA1-deficient cells via a PI3K–NRF2-regulated pathway

Significance Our establishment of a connection between the phosphatidylinositol 3-kinase (PI3K) and NRF2 pathways provides the basis for the tissue specificity of BRCA1-related cancers. Because BRCA1 is a vital component of the intracellular machinery maintaining genomic stability, BRCA1 functions as a major tumor suppressor in cells of all types. However, BRCA1-related cancers occur overwhelmingly in breasts and ovaries. Our work demonstrates that estrogen (E2) acts via the PI3K–AKT pathway to regulate NRF2 activation in BRCA1-deficient cells, resulting in the induction of antioxidant genes that protect the cell from reactive oxygen species-induced death. BRCA1-deficient cells are thus allowed to survive and may accumulate mutations, such as loss of PTEN, that perpetuate NRF2 activation. Our work supports the emerging clinical strategy of treating BRCA1-related cancers with PI3K inhibitors. Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1’s tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase–AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.

Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2.

Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.

Mutant IDH is sufficient to initiate enchondromatosis in mice.

Significance Current genomic and biochemical analysis revealed mutations in isocitrate dehydrogenase (IDH) genes associated with several neoplasms and a novel enzymatic activity of IDH mutations to catalyze α-ketoglutarate to d-2-hydroxyglutarate, contributing to tumorigenesis. We identified a broad range of IDH1 mutations, including a previously unidentified IDH1-R132Q mutation, in cartilage tumors. Cartilage-specific Col2a1-Cre/ERT2;Idh1-R132 mutant knock-in mice developed multiple enchondroma-like lesions. These data show that mutant Idh in growth-plate cells causes persistence of chondrocytes, giving rise to enchondromas adjacent to the growth cartilage in bone. Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

TAp73 is required for spermatogenesis and the maintenance of male fertility

Significance Defects in spermatogenesis, many of which are unexplained, underlie the infertility problems of ∼20% of couples. Although specific roles for the p53 family members in female fertility have been described, their involvement in spermatogenesis is largely unexpected. Using gene-targeted mice, we have demonstrated that deficiency of TAp73, but not p53 or ∆Np73, leads to male infertility caused by severely impaired germ cell differentiation and maturation to viable sperms in the testes. Importantly, our work has established that TAp73, but not p53, regulates many genes involved in spermatogenesis. Thus, our results provide previously unidentified in vivo evidence that TAp73 is a “guardian” of male germ cells and may point toward a novel avenue for the diagnosis and management of male infertility. The generation of viable sperm proceeds through a series of coordinated steps, including germ cell self-renewal, meiotic recombination, and terminal differentiation into functional spermatozoa. The p53 family of transcription factors, including p53, p63, and p73, are critical for many physiological processes, including female fertility, but little is known about their functions in spermatogenesis. Here, we report that deficiency of the TAp73 isoform, but not p53 or ΔNp73, results in male infertility because of severe impairment of spermatogenesis. Mice lacking TAp73 exhibited increased DNA damage and cell death in spermatogonia, disorganized apical ectoplasmic specialization, malformed spermatids, and marked hyperspermia. We demonstrated that TAp73 regulates the mRNA levels of crucial genes involved in germ stem/progenitor cells (CDKN2B), spermatid maturation/spermiogenesis (metalloproteinase and serine proteinase inhibitors), and steroidogenesis (CYP21A2 and progesterone receptor). These alterations of testicular histology and gene expression patterns were specific to TAp73 null mice and not features of mice lacking p53. Our work provides previously unidentified in vivo evidence that TAp73 has a unique role in spermatogenesis that ensures the maintenance of mitotic cells and normal spermiogenesis. These results may have implications for the diagnosis and management of human male infertility.

The E3 ubiquitin ligase Mule acts through the ATM–p53 axis to maintain B lymphocyte homeostasis

Genetic manipulation reveals that Mule is vital for B cell development, proliferation, and homeostasis as a result of its ability to regulate p53 and ATM.

Crucial role for TNF receptor-associated factor 2 (TRAF2) in regulating NFκB2 signaling that contributes to autoimmunity

TNF receptor-associated factor 2 (TRAF2) is a key intracellular signaling mediator that acts downstream of not only TNFα but also various members of the TNFα superfamily. Here, we report that, despite their lack of TNFα signaling, TRAF2−/−TNFα−/− mice develop an inflammatory disorder characterized by autoantibody accumulation and organ infiltration by T cells with the phenotypes of activated, effector, and memory cells. RAG1−/− mice reconstituted with TRAF2−/−TNFα−/− bone marrow cells showed increased numbers of hyperactive T cells and rapidly developed progressive and eventually lethal inflammation. No inflammation was observed in RAG1−/− mice reconstituted with TRAF2−/−TNFα−/−T-cell receptor β−/− or TRAF2−/−TNFα−/−NFκB-induced kinase+/− bone marrow cells. The pathogenic TRAF2−/−TNFα−/− T cells showed constitutive NFκB2p52 activation and produced elevated levels of T-helper 1 and T-helper 17 cytokines. Our results suggest that a regulatory circuit consisting of TRAF2–NFκB-induced kinase–NFκB2p52 is essential for the proper control of effector T-cell polarization and that loss of T-cell TRAF2 function induces constitutive NFκB2p52 activity that drives fatal autoimmune inflammation independently of TNFα signaling. The involvement of this regulatory circuit in controlling autoimmune responses highlights the delicate balance required to avoid paradoxical adverse events when implementing new targeted anti-inflammatory therapies.

Idh1 protects murine hepatocytes from endotoxin-induced oxidative stress by regulating the intracellular NADP+/NADPH ratio

Isocitrate dehydrogenase-1 (Idh1) is an important metabolic enzyme that produces NADPH by converting isocitrate to α-ketoglutarate. Idh1 is known to reduce reactive oxygen species (ROS) induced in cells by treatment with lipopolysaccharide (LPS) in vitro. Here, we used Idh1-deficient knockout (Idh1 KO) mice to investigate the role of Idh1 in antioxidant defense in vivo. Idh1 KO mice showed heightened susceptibility to death induced by LPS and exhibited increased serum levels of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The serum of LPS-injected Idh1 KO mice also contained elevated levels of AST, a marker of inflammatory liver damage. Furthermore, after LPS injection, livers of Idh1 KO mice showed histological evidence of elevated oxidative DNA damage compared with livers of wild-type (WT) mice. Idh1 KO livers showed a faster and more pronounced oxidative stress than WT livers. In line with that, Idh1 KO hepatocytes showed higher ROS levels and an increase in the NADP+/NADPH ratio when compared with hepatocytes isolated from WT mice. These results suggest that Idh1 has a physiological function in protecting cells from oxidative stress by regulating the intracellular NADP+/NADPH ratio. Our findings suggest that stimulation of Idh1 activity may be an effective therapeutic strategy for reducing oxidative stress during inflammatory responses, including the early stages of septic shock.