Chromatin-Based Classification of Genetically Heterogeneous AMLs into Two Distinct Subtypes with Diverse Stemness Phenotypes.

Global investigation of histone marks in acute myeloid leukemia (AML) remains limited. Analyses of 38 AML samples through integrated transcriptional and chromatin mark analysis exposes 2 major subtypes. One subtype is dominated by patients with NPM1 mutations or MLL-fusion genes, shows activation of the regulatory pathways involving HOX-family genes as targets, and displays high self-renewal capacity and stemness. The second subtype is enriched for RUNX1 or spliceosome mutations, suggesting potential interplay between the 2 aberrations, and mainly depends on IRF family regulators. Cellular consequences in prognosis predict a relatively worse outcome for the first subtype. Our integrated profiling establishes a rich resource to probe AML subtypes on the basis of expression and chromatin data.

Combined HAT/EZH2 modulation leads to cancer-selective cell death.

Epigenetic alterations have been associated with both pathogenesis and progression of cancer. By screening of library compounds, we identified a novel hybrid epi-drug MC2884, a HAT/EZH2 inhibitor, able to induce bona fide cancer-selective cell death in both solid and hematological cancers in vitro, ex vivo and in vivo xenograft models. Anticancer action was due to an epigenome modulation by H3K27me3, H3K27ac, H3K9/14ac decrease, and to caspase-dependent apoptosis induction. MC2884 triggered mitochondrial pathway apoptosis by up-regulation of cleaved-BID, and strong down-regulation of BCL2. Even aggressive models of cancer, such as p53-/- or TET2-/- cells, responded to MC2884, suggesting MC2884 therapeutic potential also for the therapy of TP53 or TET2-deficient human cancers. MC2884 induced massive apoptosis in ex vivo human primary leukemia blasts with poor prognosis in vivo, by targeting BCL2 expression. MC2884-treatment reduced acetylation of the BCL2 promoter at higher level than combined p300 and EZH2 inhibition. This suggests a key role for BCL-2 reduction in potentiating responsiveness, also in combination therapy with BCL2 inhibitors. Finally, we identified both the mechanism of MC2884 action as well as a potential therapeutic scheme of its use. Altogether, this provides proof of concept for the use of epi-drugs coupled with epigenome analyses to 'personalize' precision medicine.

Multi-omics profiling reveals a distinctive epigenome signature for high-risk acute promyelocytic leukemia.

Epigenomic alterations have been associated with both pathogenesis and progression of cancer. Here, we analyzed the epigenome of two high-risk APL (hrAPL) patients and compared it to non-high-risk APL cases. Despite the lack of common genetic signatures, we found that human hrAPL blasts from patients with extremely poor prognosis display specific patterns of histone H3 acetylation, specifically hyperacetylation at a common set of enhancer regions. In addition, unique profiles of the repressive marks H3K27me3 and DNA methylation were exposed in high-risk APLs. Epigenetic comparison with low/intermediate-risk APLs and AMLs revealed hrAPL-specific patterns of histone acetylation and DNA methylation, suggesting these could be further developed into markers for clinical identification. The epigenetic drug MC2884, a newly generated general HAT/EZH2 inhibitor, induces apoptosis of high-risk APL blasts and reshapes their epigenomes by targeting both active and repressive marks. Together, our analysis uncovers distinctive epigenome signatures of hrAPL patients, and provides proof of concept for use of epigenome profiling coupled to epigenetic drugs to 'personalize' precision medicine.

HDAC2 deregulation in tumorigenesis is causally connected to repression of immune modulation and defense escape.

Histone deacetylase 2 (HDAC2) is overexpressed or mutated in several disorders such as hematological cancers, and plays a critical role in transcriptional regulation, cell cycle progression and developmental processes. Here, we performed comparative transcriptome analyses in acute myeloid leukemia to investigate the biological implications of HDAC2 silencing versus its enzymatic inhibition using epigenetic-based drug(s). By gene expression analysis of HDAC2-silenced vs wild-type cells, we found that HDAC2 has a specific role in leukemogenesis. Gene expression profiling of U937 cell line with or without treatment of the well-known HDAC inhibitor vorinostat (SAHA) identifies and characterizes several gene clusters where inhibition of HDAC2 'mimics' its silencing, as well as those where HDAC2 is selectively and exclusively regulated by HDAC2 protein expression levels. These findings may represent an important tool for better understanding the mechanisms underpinning immune regulation, particularly in the study of major histocompatibility complex class II genes.

Anti-mullerian hormone is expressed by endometriosis tissues and induces cell cycle arrest and apoptosis in endometriosis cells.

BACKGROUND: The anti-mullerian hormone (AMH) is a member of the transforming growth factor ß (TGF-ß) superfamily, which is responsible of the regression of the mullerian duct. AMH is expressed in the normal endometrium, where, acting in a paracrine fashion, negatively regulates cellular viability. Our objective was to evaluate the in vitro effects of the treatment with AMH of endometriosic cells. METHODS: AMH expression in human endometriosis glands was evaluated by immunohistochemistry. RT-PCR has been used to quantify the expression levels of AMH and AMH RII isoforms, as well as of cytochrome P450 in both endometriosis epithelial and stromal cells Effects of AMH and AMH-cleaved treatment in endometriosis cells were evaluated by flow-cytometry analysis. Finally, it has been evaluated the effect of plasmin-digested AMH on cytochrome P450 activity. RESULTS: AMH and AMH RII isoforms, as well as cytochrome P450, were expressed in both endometriosis epithelial and stromal cells. Treatment of endometriosis stromal and epithelial cell growth with AMH was able to induce a decrease in the percentage of cells in S phase and increase percentage of cells in G1 and G2 phase; coherently, decreased cell viability and increased percentage of cells death fraction was observed. The plasmin-digested AMH was able to suppress most of the cytochrome P450 activity, causing an increase of pre-G1 phase and of apoptosis induction treating with plasmin-digested AMH in both cell lines, most marked in the epithelial cells. CONCLUSIONS: The data produced suggest a possible use of AMH as therapeutic agents in endometriosis.

Context-selective death of acute myeloid leukemia cells triggered by the novel hybrid retinoid-HDAC inhibitor MC2392.

HDAC inhibitors (HDACi) are widely used in the clinic to sensitize tumorigenic cells for treatment with other anticancer compounds. The major drawback of HDACi is the broad inhibition of the plethora of HDAC-containing complexes. In acute promyelocytic leukemia (APL), repression by the PML-RARα oncofusion protein is mediated by an HDAC-containing complex that can be dissociated by pharmacologic doses of all trans retinoic acid (ATRA) inducing differentiation and cell death at the expense of side effects and recurrence. We hypothesized that the context-specific close physical proximity of a retinoid and HDACi-binding protein in the repressive PML-RARα-HDAC complex may permit selective targeting by a hybrid molecule of ATRA with a 2-aminoanilide tail of the HDAC inhibitor MS-275, yielding MC2392. We show that MC2392 elicits weak ATRA and essentially no HDACi activity in vitro or in vivo. Genome-wide epigenetic analyses revealed that in NB4 cells expressing PML-RARα, MC2392 induces changes in H3 acetylation at a small subset of PML-RARα-binding sites. RNA-seq reveals that MC2392 alters expression of a number of stress-responsive and apoptotic genes. Concordantly, MC2392 induced rapid and massive, caspase-8-dependent cell death accompanied by RIP1 induction and ROS production. Solid and leukemic tumors are not affected by MC2392, but expression of PML-RARα conveys efficient MC2392-induced cell death. Our data suggest a model in which MC2392 binds to the RARα moiety and selectively inhibits the HDACs resident in the repressive complex responsible for the transcriptional impairment in APLs. Our findings provide proof-of-principle of the concept of a context-dependent targeted therapy.

The class I-specific HDAC inhibitor MS-275 modulates the differentiation potential of mouse embryonic stem cells.

Exploitation of embryonic stem cells (ESC) for therapeutic use and biomedical applications is severely hampered by the risk of teratocarcinoma formation. Here, we performed a screen of selected epi-modulating compounds and demonstrate that a transient exposure of mouse ESC to MS-275 (Entinostat), a class I histone deacetylase inhibitor (HDAC), modulates differentiation and prevents teratocarcinoma formation. Morphological and molecular data indicate that MS-275-primed ESCs are committed towards neural differentiation, which is supported by transcriptome analyses. Interestingly, in vitro withdrawal of MS-275 reverses the primed cells to the pluripotent state. In vivo, MS275-primed ES cells injected into recipient mice give only rise to benign teratomas but not teratocarcinomas with prevalence of neural-derived structures. In agreement, MS-275-primed ESC are unable to colonize blastocysts. These findings provide evidence that a transient alteration of acetylation alters the ESC fate.

MePR: a novel human mesenchymal progenitor model with characteristics of pluripotency.

Human embryo stem cells or adult tissues are excellent models for discovery and characterization of differentiation processes. The aims of regenerative medicine are to define the molecular and physiological mechanisms that govern stem cells and differentiation. Human mesenchymal stem cells (hMSCs) are multipotent adult stem cells that are able to differentiate into a variety of cell types under controlled conditions both in vivo and in vitro, and they have the remarkable ability of self-renewal. hMSCs derived from amniotic fluid and characterized by the expression of Oct-4 and Nanog, typical markers of pluripotent cells, represent an excellent model for studies on stemness. Unfortunately, the limited amount of cells available from each donation and, above all, the limited number of replications do not allow for detailed studies. Here, we report on the immortalization and characterization of novel mesenchymal progenitor (MePR) cell lines from amniotic fluid-derived hMSCs, whose biological properties are similar to primary amniocytes. Our data indicate that MePR cells display the multipotency potential and differentiation rates of hMSCs, thus representing a useful model to study both mechanisms of differentiation and pharmacological approaches to induce selective differentiation. In particular, MePR-2B cells, which carry a bona fide normal karyotype, might be used in basic stem cell research, leading to the development of new approaches for stem cell therapy and tissue engineering.