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The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/- mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/- mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/- mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear.

Purpose Patients with reciprocal balanced translocations (RBT) have a risk for recurrent pregnancy losses (RPL), affected child, and infertility. Currently, genetic counseling is based on karyotypes found among the products of conception (POC), although factors influencing the success of assisted reproductive technologies (ART) in RBT couples are not established. Methods Cytogenetic results from 261 POC and offspring of the parents (113 women and 90 men) with RBT were evaluated. Chromosome segregation modes and number of euploid embryos were assessed in couples undergoing in vitro fertilization. Results Patients with translocations involving an acrocentric chromosome have a higher risk of unbalanced gametes caused by a 3:1 segregation. Female RBT patients have a statistically higher risk of aneuploidy due to an interchromosomal effect. The rate of euploid embryos is low due to meiosis I malsegregation of RBT, meiosis II nondisjunction, additional whole chromosome or segmental aneusomies. RBT patients with RPL have a higher rate of miscarriage of euploid fetuses with RBT. Conclusion Chromosome-specific factors, female gender, age, and history of RPL are the risk elements influencing pregnancy and in vitro fertilization success in RBT patients. Chromosomal microarray analysis of POC is necessary to provide an accurate and timely diagnosis for patients with adverse reproductive outcomes.

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ~80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine DNMT3a −/− bone marrow cells. To determine whether the hypomethylation phenotype of DNMT3a −/− hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from DNMT3a −/− mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic “state” created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

The DNA methyltransferases DNMT3A and DNMT3B are primarily responsible for de novo methylation of specific cytosine residues in CpG dinucleotides during mammalian development. While loss-of-function mutations in DNMT3A are highly recurrent in acute myeloid leukemia (AML), DNMT3A mutations are almost never found in AML patients with translocations that create oncogenic fusion genes such as PML-RARA, RUNX1-RUNX1T1, and MLL-AF9. Here, we explored how DNMT3A is involved in the function of these fusion genes. We used retroviral vectors to express PML-RARA, RUNX1-RUNX1T1, or MLL-AF9 in bone marrow cells derived from WT or DNMT3A-deficient mice. Additionally, we examined the phenotypes of hematopoietic cells from Ctsg-PML-RARA mice, which express PML-RARA in early hematopoietic progenitors and myeloid precursors, with or without DNMT3A. We determined that the methyltransferase activity of DNMT3A, but not DNMT3B, is required for aberrant PML-RARA-driven self-renewal ex vivo and that DNMT3A is dispensable for RUNX1-RUNX1T1- and MLL-AF9-driven self-renewal. Furthermore, both the PML-RARA-driven competitive transplantation advantage and development of acute promyelocytic leukemia (APL) required DNMT3A. Together, these findings suggest that PML-RARA requires DNMT3A to initiate APL in mice.

Acute promyelocytic leukemia (APL) is initiated by the PML-RARA ( PR ) fusion oncogene and has a characteristic expression profile that includes high levels of the Notch ligand Jagged-1 ( JAG1 ). In this study, we used a series of bioinformatic, in vitro , and in vivo assays to assess the role of Notch signaling in human APL samples, and in a PML-RARA knock-in mouse model of APL (Ctsg-PML-RARA) . We identified a Notch expression signature in both human primary APL cells and in Kit+Lin−Sca1+ cells from pre-leukemic Ctsg-PML-RARA mice. Both genetic and pharmacologic inhibition of Notch signaling abrogated the enhanced self-renewal seen in hematopoietic stem/progenitor cells from pre-leukemic Ctsg-PML-RARA mice, but had no influence on cells from age-matched wild-type mice. In addition, six of nine murine APL tumors tested displayed diminished growth in vitro when Notch signaling was inhibited pharmacologically. Finally, we found that genetic inhibition of Notch signaling with a dominant-negative Mastermind-like protein reduced APL growth in vivo in a subset of tumors. These findings expand the role of Notch signaling in hematopoietic diseases, and further define the mechanistic events important for PML-RARA -mediated leukemogenesis.

Mammalian epidermis is a stratified epithelium that serves as a barrier protecting the organism from mechanical stress and dehydration. Previous studies have demonstrated the importance of the actin cytoskeleton in the establishment of a functional skin epithelium. Despite what is known about the actin cytoskeleton in epithelial sheet formation, the molecules important for controlling the actin cytoskeleton during epidermal development have not been determined. Serum response factor (SRF) is a transcription factor that is considered to be an important regulator of the actin cytoskeleton. To examine the role of SRF in the developing mouse epidermis, we have employed gene targeting to ablate Srf in keratinocytes. Conditional inactivation of Srf during the embryonic timepoint leads to a defect in the organization of the epidermis. Immunohistochemical analyses demonstrated a marked loss of the filamentous actin cytoskeleton and E-cadherin localization in epidermis, as well as an aberration in the localization of tight junction proteins. Moreover, impairment of the “inside-out” epidermal barrier was shown. Srf conditional knockout keratinocytes are unable to establish proper intercellular connections or form an epithelial sheet as shown by histological examination and induced keratinocyte differentiation experiments. Our results demonstrate that Srf is essential for the actin-mediated sealing of epithelial cell-cell contacts and the development of functional stratified skin epithelium in vivo.

Destrin (DSTN) is a member of the ADF/cofilin family of proteins and is an important regulator of actin dynamics. The primary function of destrin is to depolymerize filamentous actin into its monomeric form and promote filament severing. While progress has been made in understanding the biochemical functions of the ADF/cofilin proteins, the study of an animal model for cells deficient for DSTN provides an opportunity to investigate the physiological processes regulated by proper actin dynamics in vivo. A spontaneous mouse mutant, corneal disease 1(corn1), is deficient for DSTN, which causes epithelial hyperproliferation and neovascularization in the cornea. Dstn(corn1) mice exhibit an actin dynamics defect in the cornea as evidenced by the formation of actin stress fibers in the epithelial cells. Previously, we observed a significant infiltration of leukocytes into the cornea of Dstn(corn1) mice as well as the upregulation of proinflammatory molecules. In this study, we sought to characterize this inflammatory condition and explore the physiological mechanism through which a loss of Dstn function leads to inflammation. Through immunofluorescent analyses, we observed a significant recruitment of neutrophils and macrophages to the Dstn(corn1) cornea, demonstrating that the innate immune system is spontaneously activated in this mutant. The inflammatory chemokine, CXCL5, was ectopically expressed in the corneal epithelial cells of Dstn(corn1) mice, and targeting of the receptor for this chemokine inhibited neutrophil recruitment. An inflammatory reaction was not observed in the cornea of allelic mutant strain, Dstn(corn1-2J), which has a milder defect in actin dynamics in the corneal epithelial cells. This study shows that severe defects in actin dynamics lead to an autoinflammatory condition that is mediated by the expression of CXC chemokines.

Cell hyperproliferation, inflammation, and angiogenesis are biological processes central to the pathogenesis of corneal disease, as well as other conditions including tumorigenesis and chronic inflammatory disorders. Due to the number of disease conditions that arise as a result of these abnormalities, identifying the molecular mechanisms underlying these processes is critical. The avascular and transparent cornea serves as a good in vivo model to study the pathogenesis of cell hyperproliferation, inflammation, and angiogenesis. Corneal disease 1 (Dstncorn1) mice are homozygous for a spontaneous null allele of the destrin (Dstn) gene, which is also known as actin depolymerizing factor (ADF). These mice exhibit abnormalities in the cornea including epithelial cell hyperproliferation, stromal inflammation, and neovascularization. We previously identified that the transcription factor, serum response factor (SRF) and a number of its target genes are upregulated in the cornea of these mice. In this study, we show that conditional ablation of Srf in the corneal epithelium of a diseased Dstncorn1 cornea results in the rescue of the epithelial cell hyperproliferation, inflammation, and neovascularization phenotypes, delineating an epithelial cell-specific role for SRF in the development of all of these abnormalities. Our study also demonstrates that Dstn is genetically upstream of Srf and defines a new functional role for SRF as the master regulator of a hyperproliferative, inflammatory phenotype accompanied by neovascularization.

Abstract Objectives To overcome the challenges associated with molecular and cytogenetic (MG) education in hematopathology (HP), a monthly joint HP/MG conference with specific curricular goals was established and evaluated by the participants. Methods All cases from the HP/MG conference over 56 months were reviewed. To assess the educational impact, a survey was distributed to current/former HP/molecular genetic pathology fellows and faculty. Results During the study period, a total of 252 cases covering MG testing considered important for HP fellowship training were presented. The 100 most recent cases since 2018 discussed findings of diagnostic (85%), prognostic (40%), or therapeutic (10%) importance. A broad range of technologies were discussed such as karyotyping, cytogenetic fluorescence in situ hybridization studies, microarrays, polymerase chain reaction–based tests, next-generation sequencing, and Sanger sequencing. Twenty-three (95.8%) of 24 survey respondents agreed that the conference achieved all of its goals, and all agreed it was worth implementing. Conclusions This educationally based HP/MG conference supplements existing rotations, didactic presentations, and consensus case conferences and enhances MG education in HP without excessive time commitment or need for extensive in-house MG testing. It also contributes to enhancing HP knowledge among the MG faculty and fellows.