Jonathan D. Licht

MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts.

MicroRNAs comprise a broad class of small non-coding RNAs that control expression of complementary target messenger RNAs. Dysregulation of microRNAs by several mechanisms has been described in various disease states including cardiac disease. Whereas previous studies of cardiac disease have focused on microRNAs that are primarily expressed in cardiomyocytes, the role of microRNAs expressed in other cell types of the heart is unclear. Here we show that microRNA-21 (miR-21, also known as Mirn21) regulates the ERK–MAP kinase signalling pathway in cardiac fibroblasts, which has impacts on global cardiac structure and function. miR-21 levels are increased selectively in fibroblasts of the failing heart, augmenting ERK–MAP kinase activity through inhibition of sprouty homologue 1 (Spry1). This mechanism regulates fibroblast survival and growth factor secretion, apparently controlling the extent of interstitial fibrosis and cardiac hypertrophy. In vivo silencing of miR-21 by a specific antagomir in a mouse pressure-overload-induced disease model reduces cardiac ERK–MAP kinase activity, inhibits interstitial fibrosis and attenuates cardiac dysfunction. These findings reveal that microRNAs can contribute to myocardial disease by an effect in cardiac fibroblasts. Our results validate miR-21 as a disease target in heart failure and establish the therapeutic efficacy of microRNA therapeutic intervention in a cardiovascular disease setting.

Leukemic IDH1 and IDH2 Mutations Result in a Hypermethylation Phenotype, Disrupt TET2 Function, and Impair Hematopoietic Differentiation

Cancer-associated IDH mutations are characterized by neomorphic enzyme activity and resultant 2-hydroxyglutarate (2HG) production. Mutational and epigenetic profiling of a large acute myeloid leukemia (AML) patient cohort revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and a specific hypermethylation signature. Furthermore, expression of 2HG-producing IDH alleles in cells induced global DNA hypermethylation. In the AML cohort, IDH1/2 mutations were mutually exclusive with mutations in the α-ketoglutarate-dependent enzyme TET2, and TET2 loss-of-function mutations were associated with similar epigenetic defects as IDH1/2 mutants. Consistent with these genetic and epigenetic data, expression of IDH mutants impaired TET2 catalytic function in cells. Finally, either expression of mutant IDH1/2 or Tet2 depletion impaired hematopoietic differentiation and increased stem/progenitor cell marker expression, suggesting a shared proleukemogenic effect.

DNMT3A mutations in acute myeloid leukemia

BACKGROUND The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown. METHODS Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations. RESULTS A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P<0.001 for both comparisons). The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months vs. 41.1 months, P<0.001). DNMT3A mutations were associated with adverse outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 mutations, regardless of age, and were independently associated with a poor outcome in Cox proportional-hazards analysis. CONCLUSIONS DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome. (Funded by the National Institutes of Health and others.).

Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia

We report here that individuals with Noonan syndrome and juvenile myelomonocytic leukemia (JMML) have germline mutations in PTPN11 and that somatic mutations in PTPN11 account for 34% of non-syndromic JMML. Furthermore, we found mutations in PTPN11 in a small percentage of individuals with myelodysplastic syndrome (MDS) and de novo acute myeloid leukemia (AML). Functional analyses documented that the two most common mutations in PTPN11 associated with JMML caused a gain of function.

Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDK-inhibitor p21WAF1/CiP1.

Much of the predisposition to hereditary breast and ovarian cancer has been attributed to inherited defects in the BRCA1 tumour-suppressor gene. The nuclear protein BRCA1 has the properties of a transcription factor, and can interact with the recombination and repair protein RAD51 (ref. 8). Young women with germline alterations in BRCA1 develop breast cancer at rates 100-fold higher than the general population, and BRCA1-null mice die before day 8 of development,. However, the mechanisms of BRCA1-mediated growth regulation and tumour suppression remain unknown. Here we show that BRCA1 transactivates expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in a p53-independent manner, and that BRCA1 inhibits cell-cycle progression into the S-phase following its transfection into human cancer cells. BRCA1 does not inhibit S-phase progression in p21−/− cells, unlike p21+/+ cells, and tumour-associated, transactivation-deficient mutants of BRCA1 are defective in both transactivation of p21 and cell-cycle inhibition. These data suggest that one mechanism by which BRCA1 contributes to cell-cycle arrest and growth suppression is through the induction of p21.

Histone deacetylase inhibitors in cancer therapy: is transcription the primary target?

The study of HDIs in cancer, initially motivated by the study of aberrant transcriptional repression, is rapidly evolving. It is now apparent that the transcriptional effects of HDIs may be but one facet of their action. In specific forms of cancer such as the myeloid leukemias, HDIs may indeed be working as predicted to block histone deacetylases and potentiate acetylation at specific genes. However, the focus on histone acetylation neglects the many other facets of epigenetic regulation of gene expression. True targeting of transcription in leukemia and other tumors might require a combination of agents to modify chromatin, including DNA methyl transferase inhibitors, histone methylase inhibitors, HDIs, and specific stimulators of transcription factor activity.In the more common forms of cancer, HDIs are also promising therapeutic agents, but here their mechanism of action may be quite different. HDIs have pleiotropic effects on cells, with outputs of differentiation, growth arrest, and cell death all possible. It is not clear if transcription is the primary HDI target in these cells, or if other effects, perhaps triggered by aberrant mitosis, play a fundamental role. In patients, these effects may be dependent on the particular genetic lesions of the tumor, the dose and length of exposure of the tumor to drug, and the possibility that HDIs could affect secondary signaling pathways. A combination of basic, clinical, and translational studies will ultimately determine the clinical utility of these agents and their mechanism of action.

EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation

The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. Here, we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

MDS and secondary AML display unique patterns and abundance of aberrant DNA methylation

Increasing evidence shows aberrant hypermethylation of genes occurring in and potentially contributing to pathogenesis of myeloid malignancies. Several of these diseases, such as myelodysplastic syndromes (MDSs), are responsive to DNA methyltransferase inhibitors. To determine the extent of promoter hypermethylation in such tumors, we compared the distribution of DNA methylation of 14 000 promoters in MDS and secondary acute myeloid leukemia (AML) patients enrolled in a phase 1 trial of 5-azacytidine and the histone deacetylase inhibitor entinostat against de novo AML patients and normal CD34(+) bone marrow cells. The MDS and secondary AML patients displayed more extensive aberrant DNA methylation involving thousands of genes than did the normal CD34(+) bone marrow cells or de novo AML blasts. Aberrant methylation in MDS and secondary AML tended to affect particular chromosomal regions, occurred more frequently in Alu-poor genes, and included prominent involvement of genes involved in the WNT and MAPK signaling pathways. DNA methylation was also measured at days 15 and 29 after the first treatment cycle. DNA methylation was reversed at day 15 in a uniform manner throughout the genome, and this effect persisted through day 29, even without continuous administration of the study drugs. This trial was registered at www.clinicaltrials.gov as J0443.

Specific peptide interference reveals BCL6 transcriptional and oncogenic mechanisms in B-cell lymphoma cells.

The BTB/POZ transcriptional repressor and candidate oncogene BCL6 is frequently misregulated in B-cell lymphomas. The interface through which the BCL6 BTB domain mediates recruitment of the SMRT, NCoR and BCoR corepressors was recently identified. To determine the contribution of this interface to BCL6 transcriptional and biological properties, we generated a peptide that specifically binds BCL6 and blocks corepressor recruitment in vivo. This inhibitor disrupts BCL6-mediated repression and establishment of silenced chromatin, reactivates natural BCL6 target genes, and abrogates BCL6 biological function in B cells. In BCL6-positive lymphoma cells, peptide blockade caused apoptosis and cell cycle arrest. BTB domain peptide inhibitors may constitute a novel therapeutic agent for B-cell lymphomas.

The MMSET histone methyl transferase switches global histone methylation and alters gene expression in t(4;14) multiple myeloma cells

The multiple myeloma SET domain (MMSET) protein is overexpressed in multiple myeloma (MM) patients with the translocation t(4;14). Although studies have shown the involvement of MMSET/Wolf-Hirschhorn syndrome candidate 1 in development, its mode of action in the pathogenesis of MM is largely unknown. We found that MMSET is a major regulator of chromatin structure and transcription in t(4;14) MM cells. High levels of MMSET correlate with an increase in lysine 36 methylation of histone H3 and a decrease in lysine 27 methylation across the genome, leading to a more open structural state of the chromatin. Loss of MMSET expression alters adhesion properties, suppresses growth, and induces apoptosis in MM cells. Consequently, genes affected by high levels of MMSET are implicated in the p53 pathway, cell cycle regulation, and integrin signaling. Regulation of many of these genes required functional histone methyl-transferase activity of MMSET. These results implicate MMSET as a major epigenetic regulator in t(4;14)+ MM.