KDM6B protects T-ALL cells from NOTCH1-induced oncogenic stress.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm resulting from the malignant transformation of T-cell progenitors. While activating NOTCH1 mutations are the dominant genetic drivers of T-ALL, epigenetic dysfunction plays a central role in the pathology of T-ALL and can provide alternative mechanisms to oncogenesis in lieu of or in combination with genetic mutations. The histone demethylase enzyme KDM6A (UTX) is also recurrently mutated in T-ALL patients and functions as a tumor suppressor. However, its gene paralog, KDM6B (JMJD3), is never mutated and can be significantly overexpressed, suggesting it may be necessary for sustaining the disease. Here, we used mouse and human T-ALL models to show that KDM6B is required for T-ALL development and maintenance. Using NOTCH1 gain-of-function retroviral models, mouse cells genetically deficient for Kdm6b were unable to propagate T-ALL. Inactivating KDM6B in human T-ALL patient cells by CRISPR/Cas9 showed KDM6B-targeted cells were significantly outcompeted over time. The dependence of T-ALL cells on KDM6B was proportional to the oncogenic strength of NOTCH1 mutation, with KDM6B required to prevent stress-induced apoptosis from strong NOTCH1 signaling. These studies identify a crucial role for KDM6B in sustaining NOTCH1-driven T-ALL and implicate KDM6B as a novel therapeutic target in these patients.

Txnip Enhances Fitness of Dnmt3a-Mutant Hematopoietic Stem Cells via p21.

Clonal hematopoiesis (CH) refers to the age-related expansion of specific clones in the blood system, and manifests from somatic mutations acquired in hematopoietic stem cells (HSCs). Most CH variants occur in the gene DNMT3A, but while DNMT3A-mutant CH becomes almost ubiquitous in aging humans, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is lacking. Here, we used interferon gamma (IFNγ) as a model to study the mechanisms by which Dnmt3a mutations increase HSC fitness under hematopoietic stress. We found Dnmt3a-mutant HSCs resist IFNγ-mediated depletion, and IFNγ-signaling is required for clonal expansion of Dnmt3a-mutant HSCs in vivo. Mechanistically, DNA hypomethylation-associated overexpression of Txnip in Dnmt3a-mutant HSCs leads to p53 stabilization and upregulation of p21. This preserves the functional potential of Dnmt3a-mutant HSCs through increased quiescence and resistance to IFNγ-induced apoptosis. These data identify a previously undescribed mechanism to explain increased fitness of DNMT3A-mutant clones under hematopoietic stress. SIGNIFICANCE: DNMT3A mutations are common variants in clonal hematopoiesis, and recurrent events in blood cancers. Yet the mechanisms by which these mutations provide hematopoietic stem cells a competitive advantage as a precursor to malignant transformation remain unclear. Here, we use inflammatory stress to uncover molecular mechanisms leading to this fitness advantage.See related commentary by De Dominici and DeGregori, p. 178. This article is highlighted in the In This Issue feature, p. 171.

A Humanized Animal Model Predicts Clonal Evolution and Therapeutic Vulnerabilities in Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are chronic blood diseases with significant morbidity and mortality. Although sequencing studies have elucidated the genetic mutations that drive these diseases, MPNs remain largely incurable with a significant proportion of patients progressing to rapidly fatal secondary acute myeloid leukemia (sAML). Therapeutic discovery has been hampered by the inability of genetically engineered mouse models to generate key human pathologies such as bone marrow fibrosis. To circumvent these limitations, here we present a humanized animal model of myelofibrosis (MF) patient-derived xenografts (PDX). These PDXs robustly engrafted patient cells that recapitulated the patient's genetic hierarchy and pathologies such as reticulin fibrosis and propagation of MPN-initiating stem cells. The model can select for engraftment of rare leukemic subclones to identify patients with MF at risk for sAML transformation and can be used as a platform for genetic target validation and therapeutic discovery. We present a novel but generalizable model to study human MPN biology. SIGNIFICANCE: Although the genetic events driving MPNs are well defined, therapeutic discovery has been hampered by the inability of murine models to replicate key patient pathologies. Here, we present a PDX system to model human myelofibrosis that reproduces human pathologies and is amenable to genetic and pharmacologic manipulation. This article is highlighted in the In This Issue feature, p. 2945.

Erratum: PTEN deletion drives acute myeloid leukemia resistance to MEK inhibitors.

[This corrects the article DOI: 10.18632/oncotarget.27206.].

Divergent Effects of Dnmt3a and Tet2 Mutations on Hematopoietic Progenitor Cell Fitness.

The DNA methylation regulators DNMT3A and TET2 are recurrently mutated in hematological disorders. Despite possessing antagonistic biochemical activities, loss-of-function murine models show overlapping phenotypes in terms of increased hematopoietic stem cell (HSC) fitness. Here, we directly compared the effects of these mutations on hematopoietic progenitor function and disease initiation. In contrast to Dnmt3a-null HSCs, which possess limitless self-renewal in vivo, Tet2-null HSCs unexpectedly exhaust at the same rate as control HSCs in serial transplantation assays despite an initial increase in self-renewal. Moreover, loss of Tet2 more acutely sensitizes hematopoietic cells to the addition of a common co-operating mutation (Flt3ITD) than loss of Dnmt3a, which is associated with a more rapid expansion of committed progenitor cells. The effect of Tet2 mutation manifests more profound myeloid lineage skewing in committed hematopoietic progenitor cells rather than long-term HSCs. Molecular characterization revealed divergent transcriptomes and chromatin accessibility underlying these functional differences.

PTEN deletion drives acute myeloid leukemia resistance to MEK inhibitors.

Kinases such as MEK are attractive targets for novel therapy in cancer, including acute myeloid leukaemia (AML). Acquired and inherent resistance to kinase inhibitors, however, is becoming an increasingly important challenge for the clinical success of such therapeutics, and often arises from mutations in the drug-binding domain of the target kinase. To identify possible causes of resistance to MEK inhibition, we generated a model of resistance by long-term treatment of AML cells with AZD6244 (selumetinib). Remarkably, resistance to MEK inhibition was due to acquired PTEN haploinsufficiency, rather than mutation of MEK. Resistance via this mechanism was confirmed using CRISPR/Cas9 technology targeting exon 5 of PTEN. While PTEN loss has been previously implicated in resistance to a number of other therapeutic agents, this is the first time that it has been shown directly and in AML.

Early gestational ethanol exposure in mice: Effects on brain structure, energy metabolism and adiposity in adult offspring.

We examined whether an early-life event - ethanol exposure in the initial stages of pregnancy - affected offspring brain structure, energy metabolism, and body composition in later life. Consumption of 10% (v/v) ethanol by inbred C57BL/6J female mice from 0.5 to 8.5 days post coitum was used to model alcohol exposure during the first 3-4 weeks of gestation in humans, when pregnancy is not typically recognized. At adolescence (postnatal day [P] 28) and adulthood (P64), the brains of male offspring were scanned ex vivo using ultra-high field (16.4 T) magnetic resonance imaging and diffusion tensor imaging. Energy metabolism and body composition were measured in adulthood by indirect calorimetry and dual-energy X-ray absorptiometry (DXA), respectively. Ethanol exposure had no substantial impact on white matter organization in the anterior commissure, corpus callosum, hippocampal commissure, internal capsule, optic tract, or thalamus. Whole brain volume and the volumes of the neocortex, cerebellum, and caudate putamen were also unaffected. Subtle, but non-significant, effects were observed on the hippocampus and the hypothalamus in adult ethanol-exposed male offspring. Ethanol exposure was additionally associated with a trend toward decreased oxygen consumption, carbon dioxide production, and reduced daily energy expenditure, as well as significantly increased adiposity, albeit with normal body weight and food intake, in adult male offspring. In summary, ethanol exposure restricted to early gestation had subtle long-term effects on the structure of specific brain regions in male offspring. The sensitivity of the hippocampus to ethanol-induced damage is reminiscent of that reported by other studies - despite differences in the level, timing, and duration of exposure - and likely contributes to the cognitive impairment that characteristically results from prenatal ethanol exposure. The hypothalamus plays an important role in regulating metabolism and energy homeostasis. Our finding of altered daily energy expenditure and adiposity in adult ethanol-exposed males is consistent with the idea that central nervous system abnormalities also underpin some of the metabolic phenotypes associated with ethanol exposure in pregnancy.

Inflammatory cytokines promote clonal hematopoiesis with specific mutations in ulcerative colitis patients.

Epidemiological sequencing studies have revealed that somatic mutations characteristic of myeloid neoplasms can be detected in the blood of asymptomatic individuals decades prior to presentation of any clinical symptoms. This premalignant condition is known as clonal hematopoiesis of indeterminate potential (CHIP). Despite the fact these mutant clones become readily detectable in the blood of elderly individuals (∼10% of people over the age of 65), the overall rate of disease progression remains relatively low. Thus, in addition to genetic mutations, there are likely environmental factors that contribute to clonal evolution in people with CHIP. One environmental stress that increases with age is inflammation. Although chronic inflammation is detrimental to the long-term function of normal hematopoietic stem cells, several recent studies in animal models have indicated hematopoietic stem cells with CHIP mutations may be resistant to these deleterious effects. However, direct evidence indicating a correlation between increased inflammation and accelerated CHIP in humans is currently lacking. In this study, we sequenced the peripheral blood cells of a cohort of patients with ulcerative colitis, an autoimmune disease characterized by increased levels of pro-inflammatory cytokines. This analysis revealed that the inflammatory environment of ulcerative colitis promoted CHIP with a distinct mutational spectrum, notably positive selection of clones with DNMT3A and PPM1D mutations. We also show a specific association between elevated levels of serum interferon gamma and DNMT3A mutations. These data add to our understanding of how cell extrinsic factors select for clones with specific mutations to promote clonal hematopoiesis.

Targeted Next-Generation Sequencing for Detecting MLL Gene Fusions in Leukemia.

Mixed lineage leukemia (MLL) gene rearrangements characterize approximately 70% of infant and 10% of adult and therapy-related leukemia. Conventional clinical diagnostics, including cytogenetics and fluorescence in situ hybridization (FISH) fail to detect MLL translocation partner genes (TPG) in many patients. Long-distance inverse (LDI)-PCR, the "gold standard" technique that is used to characterize MLL breakpoints, is laborious and requires a large input of genomic DNA (gDNA). To overcome the limitations of current techniques, a targeted next-generation sequencing (NGS) approach that requires low RNA input was tested. Anchored multiplex PCR-based enrichment (AMP-E) was used to rapidly identify a broad range of MLL fusions in patient specimens. Libraries generated using Archer FusionPlex Heme and Myeloid panels were sequenced using the Illumina platform. Diagnostic specimens (n = 39) from pediatric leukemia patients were tested with AMP-E and validated by LDI-PCR. In concordance with LDI-PCR, the AMP-E method successfully identified TPGs without prior knowledge. AMP-E identified 10 different MLL fusions in the 39 samples. Only two specimens were discordant; AMP-E successfully identified a MLL-MLLT1 fusion where LDI-PCR had failed to determine the breakpoint, whereas a MLL-MLLT3 fusion was not detected by AMP-E due to low expression of the fusion transcript. Sensitivity assays demonstrated that AMP-E can detect MLL-AFF1 in MV4-11 cell dilutions of 10-7 and transcripts down to 0.005 copies/ng.Implications: This study demonstrates a NGS methodology with improved sensitivity compared with current diagnostic methods for MLL-rearranged leukemia. Furthermore, this assay rapidly and reliably identifies MLL partner genes and patient-specific fusion sequences that could be used for monitoring minimal residual disease. Mol Cancer Res; 16(2); 279-85. ©2017 AACR.

Gene expression in the mouse brain following early pregnancy exposure to ethanol.

Exposure to alcohol during early embryonic or fetal development has been linked with a variety of adverse outcomes, the most common of which are structural and functional abnormalities of the central nervous system [1]. Behavioural and cognitive deficits reported in individuals exposed to alcohol in utero include intellectual impairment, learning and memory difficulties, diminished executive functioning, attention problems, poor motor function and hyperactivity [2]. The economic and social costs of these outcomes are substantial and profound [3], [4]. Improvement of neurobehavioural outcomes following prenatal alcohol exposure requires greater understanding of the mechanisms of alcohol-induced damage to the brain. Here we use a mouse model of relatively moderate ethanol exposure early in pregnancy and profile gene expression in the hippocampus and caudate putamen of adult male offspring. The effects of offspring sex and age on ethanol-sensitive hippocampal gene expression were also examined. All array data are available at the Gene Expression Omnibus (GEO) repository under accession number GSE87736.

Prenatal ethanol exposure alters adult hippocampal VGLUT2 expression with concomitant changes in promoter DNA methylation, H3K4 trimethylation and miR-467b-5p levels.

BACKGROUND: Maternal consumption of alcohol during pregnancy is associated with a range of physical, cognitive and behavioural outcomes in the offspring which are collectively called foetal alcohol spectrum disorders. We and others have proposed that epigenetic modifications, such as DNA methylation and post-translational histone modifications, mediate the effects of prenatal alcohol exposure on gene expression and, ultimately, phenotype. Here we use an inbred C57BL/6J mouse model of early gestational ethanol exposure equivalent, developmentally, to the first 3-4 weeks of pregnancy in humans to examine the long-term effects on gene expression and epigenetic state in the hippocampus. RESULTS: Gene expression analysis in the hippocampus revealed sex- and age-specific up-regulation of solute carrier family 17 member 6 (Slc17a6), which encodes vesicular glutamate transporter 2 (VGLUT2). Transcriptional up-regulation correlated with decreased DNA methylation and enrichment of histone H3 lysine 4 trimethylation, an active chromatin mark, at the Slc17a6 promoter. In contrast to Slc17a6 mRNA levels, hippocampal VGLUT2 protein levels were significantly decreased in adult ethanol-exposed offspring, suggesting an additional level of post-transcriptional control. MicroRNA expression profiling in the hippocampus identified four ethanol-sensitive microRNAs, of which miR-467b-5p was predicted to target Slc17a6. In vitro reporter assays showed that miR-467b-5p specifically interacted with the 3'UTR of Slc17a6, suggesting that it contributes to the reduction of hippocampal VGLUT2 in vivo. A significant correlation between microRNA expression in the hippocampus and serum of ethanol-exposed offspring was also observed. CONCLUSIONS: Prenatal ethanol exposure has complex transcriptional and post-transcriptional effects on Slc17a6 (VGLUT2) expression in the mouse hippocampus. These effects are observed following a relatively moderate exposure that is restricted to early pregnancy, modelling human consumption of alcohol before pregnancy is confirmed, and are only apparent in male offspring in adulthood. Our findings are consistent with the idea that altered epigenetic and/or microRNA-mediated regulation of glutamate neurotransmission in the hippocampus contributes to the cognitive and behavioural phenotypes observed in foetal alcohol spectrum disorders. Although further work is needed in both mice and humans, the results also suggest that circulating microRNAs could be used as biomarkers of early gestational ethanol exposure and hippocampal dysfunction.