BACKGROUND & AIMS Alterations in methylation of protein-coding genes are associated with Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC). Dys-regulation of noncoding RNAs occurs during carcinogen-esis but has never been studied in BE or EAC. We applied high-resolution methylome analysis to identify changes at genomic regions that encode noncoding RNAs in BE and EAC. METHODS We analyzed methylation of 1.8 million CpG sites using massively parallel sequencing-based HELP tagging in matched EAC, BE, and normal esophageal tissues. We also analyzed human EAC (OE33, SKGT4, and FLO-1) and normal (HEEpic) esophageal cells. RESULTS BE and EAC exhibited genome-wide hypomethylation, significantly affecting intragenic and repetitive genomic elements as well as noncoding regions. These methylation changes targeted small and long noncoding regions, discriminating normal from matched BE or EAC tissues. One long noncoding RNA, AFAP1-AS1, was extremely hypomethylated and overexpressed in BE and EAC tissues and EAC cells. Its silencing by small interfering RNA inhibited proliferation and colony-forming ability, induced apoptosis, and reduced EAC cell migration and invasion without altering the expression of its protein-coding counterpart, AFAP1. CONCLUSIONS BE and EAC exhibit reduced methylation that includes noncoding regions. Methylation of the long noncoding RNA AFAP1-AS1 is reduced in BE and EAC, and its expression inhibits cancer-related biologic functions of EAC cells.

Key Points IL8-CXCR2 is overexpressed in purified stem cells from AML and MDS, and CXCR2 expression is associated with worse prognosis. Inhibition of CXCR2 by genetic and pharmacologic means leads to decreased viability in AML/MDS stem cells and in vitro and in vivo models.

Mutations of the splicing factor–encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor–associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be used therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity and support the development of therapies targeting splicing abnormalities.

Key Points North American ATLL has a distinct genomic landscape with a high frequency of prognostic epigenetic mutations, including EP300 mutations. ATLL samples with mutated EP300 have compromised p53 function and are selectively sensitive to decitabine treatment.

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.

The bone marrow microenvironment influences malignant hematopoiesis but how it promotes leukemogenesis has not been elucidated. Additionally, the role of the bone marrow stroma in regulating clinical responses to DNA methyltransferase inhibitors (DNMTi) is also poorly understood. In this study, we conducted a DNA methylome analysis of bone marrow-derived stromal cells from myelodysplastic syndrome (MDS) patients and observed widespread aberrant cytosine hypermethylation occurring preferentially outside CpG islands. Stroma derived from 5-azacytidine-treated patients lacked aberrant methylation and DNMTi treatment of primary MDS stroma enhanced its ability to support erythroid differentiation. An integrative expression analysis revealed that the WNT pathway antagonist FRZB was aberrantly hypermethylated and underexpressed in MDS stroma. This result was confirmed in an independent set of sorted, primary MDS-derived mesenchymal cells. We documented a WNT/β-catenin activation signature in CD34+ cells from advanced cases of MDS where it associated with adverse prognosis. Constitutive activation of β-catenin in hematopoietic cells yielded lethal myeloid disease in a NUP98-HOXD13 mouse model of MDS, confirming its role in disease progression. Our results define novel epigenetic changes in the bone marrow microenvironment which lead to β-catenin activation and disease progression of MDS.

Interleukin (IL)-32 was originally identified in natural killer cells and IL-2-activated human T lymphocytes. As T cells are activated in allogeneic transplantation, we determined the role of IL-32 in human mixed lymphocyte cultures (MLCs) and GVHD. In allogeneic MLCs, IL-32 increased two-fold in responding T cells, accompanied by five-fold increases of TNF␣, IL-6, and IL-8. After allogeneic hematopoietic cell transplantation, IL-32 mRNA levels in blood leukocytes were statistically significantly higher in patients with acute GVHD (n ‫؍‬ 10) than in serial samples from patients who did not develop acute GVHD (n ‫؍‬ 5; P ‫؍‬ .02). No significant changes in IL-32 levels were present in patients with treated (n ‫؍‬ 14) or untreated (n ‫؍‬ 8) chronic GVHD, compared with healthy controls (n ‫؍‬ 8; P ‫؍‬ .5, and P ‫؍‬ .74, respectively). As IL-32 is activated by proteinase-3 (PR3), we determined the effect of the serine protease inhibitor ␣-1 antitrypsin (AAT) on IL-32 levels and showed suppression of IL-32 and T-lymphocyte proliferation in MLCs. In an MHC-minor antigen disparate murine transplant model, preconditioning and postconditioning treatment with AAT resulted in attenuation or prevention of GVHD and superior survival compared with albumin-treated controls (80% vs 44%; P ‫؍‬ .04). These findings suggest that AAT modulates immune and inflammatory functions and may represent a novel approach to prevent or treat GVHD. (Blood. 2011;118(18):5031-5039) IntroductionAn extensive literature describes the role of proinflammatory cytokines in the manifestations of conditioning-related toxicity in patients undergoing hematopoietic cell transplantation (HCT) and the immunologic interactions of donor and recipient cells that follow HCT. We were interested in determining specifically the involvement of IL-32 in the "cytokine storm" that has been described in the peri-HCT and post-HCT period. 1 For one, we and others have shown that tumor necrosis factor ␣ (TNF), which is consistently up-regulated in transplant recipients, is a potent inducer of IL-32. 2 Conversely, IL-32 has been shown to induce TNF, 2-5 suggesting the possibility of an amplification loop between these two cytokines. Secondly, IL-32 was originally identified in IL-2-activated T lymphocytes and natural killer (NK) cells, 6 supporting a potential role in T-cell activation and function after allogeneic HCT. Furthermore, IL-32 is present in supernatants of IL-12, IL-18, and IL-12 plus IL-18-stimulated human NK cells and in 7 the supernatant of concanavalin A-stimulated human PBMCs. 7 In addition, in patients with myelodysplastic syndrome who exhibit excessive apoptosis in hematopoietic cells, we reported, in agreement with others, that silencing of endogenous IL-32 significantly reduced apoptosis and the expression of other proinflammatory mediators. 3,8 Based on these data, we postulated a role for IL-32 in alloactivation and in GVHD. As IL-32 is activated via partial cleavage by proteinase-3 (PR3), 9 we hypothesized that the naturally occurring serine protease...

Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.