Fabio Santoro

Histone deacetylase inhibitors as a new weapon in the arsenal of differentiation therapies of cancer

Absent or altered differentiation is one of the major features of cancer cells. Histone deacetylases (HDACs) play a central role in the epigenetic regulation of gene expression. Aberrant activity of HDACs has been documented in several types of cancers, leading to the development of HDAC inhibitors (HDACi) as anti-tumor drugs. In vitro and in vivo experimental evidences show that HDACi are able to resume the process of maturation in undifferentiated cancer cells, justifying their introduction as differentiating agents in several clinical trials. Modulation of cell fate by HDACi is observed at several levels, including the stem cell compartment: HDACi can act both on cancer stem cells, and with the rest of the tumor cell mass, leading to complex biological outputs. As a note of caution, when used as single agent, HDACi show only a moderate and limited biological response, which is augmented in combinatorial therapies with drugs designed against other epigenetic targets. The optimal employment of these molecules may be therefore in combination with other epigenetic drugs acting against the set of enzymes responsible for the set-up and maintenance of epigenetic information.

Pathogenic Effects of Human Herpesvirus 6 in Human Lymphoid Tissue Ex Vivo

ABSTRACT Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that has been suggested to act as a cofactor in the progression of human immunodeficiency virus disease. However, the lack of suitable experimental models has hampered the elucidation of the mechanisms of HHV-6-mediated immune suppression. Here, we used ex vivo lymphoid tissue to investigate the cellular tropism and pathogenic mechanisms of HHV-6. Viral strains belonging to both HHV-6 subgroups (A and B) were able to productively infect human tonsil tissue fragments in the absence of exogenous stimulation. The majority of viral antigen-expressing cells were CD4+ T lymphocytes expressing a nonnaive phenotype, while CD8+ T cells were efficiently infected only with HHV-6A. Accordingly, HHV-6A infection resulted in the depletion of both CD4+ and CD8+ T cells, whereas in HHV-6B-infected tissue CD4+ T cells were predominantly depleted. The expression of different cellular antigens was dramatically altered in HHV-6-infected tissues: whereas CD4 was upregulated, both CD46, which serves as a cellular receptor for HHV-6, and CD3 were downmodulated. However, CD3 downmodulation was restricted to infected cells, while the loss of CD46 expression was generalized. Moreover, HHV-6 infection markedly enhanced the production of the CC chemokine RANTES, whereas other cytokines and chemokines were only marginally affected. These results provide the first evidence, in a physiologically relevant study model, that HHV-6 can severely affect the physiology of secondary lymphoid organs through direct infection of T lymphocytes and modulation of key membrane receptors and chemokines.

A dual role for Hdac1: oncosuppressor in tumorigenesis, oncogene in tumor maintenance

Aberrant recruitment of histone deacetylases (HDACs) by the oncogenic fusion protein PML-RAR is involved in the pathogenesis of acute promyelocytic leukemia (APL). PML-RAR, however, is not sufficient to induce disease in mice but requires additional oncogenic lesions during the preleukemic phase. Here, we show that knock-down of Hdac1 and Hdac2 dramatically accelerates leukemogenesis in transgenic preleukemic mice. These events are not restricted to APL because lymphomagenesis driven by deletion of p53 or, to a lesser extent, by c-myc overexpression, was also accelerated by Hdac1 knock-down. In the preleukemic phase of APL, Hdac1 counteracts the activity of PML-RAR in (1) blocking differentiation; (2) impairing genomic stability; and (3) increasing self-renewal in hematopoietic progenitors, as all of these events are affected by the reduction in Hdac1 levels. This led to an expansion of a subpopulation of PML-RAR-expressing cells that is the major source of leukemic stem cells in the full leukemic stage. Remarkably, short-term treatment of preleukemic mice with an HDAC inhibitor accelerated leukemogenesis. In contrast, knock-down of Hdac1 in APL mice led to enhanced survival duration of the leukemic animals. Thus, Hdac1 has a dual role in tumorigenesis: oncosuppressive in the early stages, and oncogenic in established tumor cells.

NA-Seq: A Discovery Tool for the Analysis of Chromatin Structure and Dynamics during Differentiation

It is well established that epigenetic modulation of genome accessibility in chromatin occurs during biological processes. Here we describe a method based on restriction enzymes and next-generation sequencing for identifying accessible DNA elements using a small amount of starting material, and use it to examine myeloid differentiation of primary human CD34+ cells. The accessibility of several classes of cis-regulatory elements was a predictive marker of in vivo DNA binding by transcription factors, and was associated with distinct patterns of histone posttranslational modifications. We also mapped large chromosomal domains with differential accessibility in progenitors and maturing cells. Accessibility became restricted during differentiation, correlating with a decreased number of expressed genes and loss of regulatory potential. Our data suggest that a permissive chromatin structure in multipotent cells is progressively and selectively closed during differentiation, and illustrate the use of our method for the identification of functional cis-regulatory elements.

The DNA demethylating agent decitabine activates the TRAIL pathway and induces apoptosis in acute myeloid leukemia

Although epigenetic drugs have been approved for use in selected malignancies, there is significant need for a better understanding of their mechanism of action. Here, we study the action of a clinically approved DNA-methyltransferase inhibitor - decitabine (DAC) - in acute myeloid leukemia (AML) cells. At low doses, DAC treatment induced apoptosis of NB4 Acute Promyelocytic Leukemia (APL) cells, which was associated with the activation of the extrinsic apoptotic pathway. Expression studies of the members of the Death Receptor family demonstrated that DAC induces the expression of TNF-related apoptosis-inducing ligand (TRAIL). Upregulation of TRAIL, upon DAC treatment, was associated with specific epigenetic modifications induced by DAC in the proximity of the TRAIL promoter, as demonstrated by DNA demethylation, increased DNaseI sensitivity and histone acetylation of a non-CpG island, CpG-rich region located 2kb upstream to the transcription start site. Luciferase assay experiments showed that this region behave as a DNA methylation sensitive transcriptional regulatory element. The CpG regulatory element was also found methylated in samples derived from APL patients. These findings have been confirmed in the non-APL, AML Kasumi cell line, suggesting that this regulatory mechanism may be extended to other AMLs. Our study suggests that DNA methylation is a regulatory mechanism relevant for silencing of the TRAIL apoptotic pathway in leukemic cells, and further elucidates the mechanism by which epigenetic drugs mediate their anti-leukemic effects.

Functional-genetic dissection of HDAC dependencies in mouse lymphoid and myeloid malignancies.

Histone deacetylase (HDAC) inhibitors (HDACis) have demonstrated activity in hematological and solid malignancies. Vorinostat, romidepsin, belinostat, and panobinostat are Food and Drug Administration-approved for hematological malignancies and inhibit class II and/or class I HDACs, including HDAC1, 2, 3, and 6. We combined genetic and pharmacological approaches to investigate whether suppression of individual or multiple Hdacs phenocopied broad-acting HDACis in 3 genetically distinct leukemias and lymphomas. Individual Hdacs were depleted in murine acute myeloid leukemias (MLL-AF9;Nras(G12D); PML-RARα acute promyelocytic leukemia [APL] cells) and Eµ-Myc lymphoma in vitro and in vivo. Strikingly, Hdac3-depleted cells were selected against in competitive assays for all 3 tumor types. Decreased proliferation following Hdac3 knockdown was not prevented by BCL-2 overexpression, caspase inhibition, or knockout of Cdkn1a in Eµ-Myc lymphoma, and depletion of Hdac3 in vivo significantly reduced tumor burden. Interestingly, APL cells depleted of Hdac3 demonstrated a more differentiated phenotype. Consistent with these genetic studies, the HDAC3 inhibitor RGFP966 reduced proliferation of Eµ-Myc lymphoma and induced differentiation in APL. Genetic codepletion of Hdac1 with Hdac2 was pro-apoptotic in Eµ-Myc lymphoma in vitro and in vivo and was phenocopied by the HDAC1/2-specific agent RGFP233. This study demonstrates the importance of HDAC3 for the proliferation of leukemia and lymphoma cells, suggesting that HDAC3-selective inhibitors could prove useful for the treatment of hematological malignancies. Moreover, our results demonstrate that codepletion of Hdac1 with Hdac2 mediates a robust pro-apoptotic response. Our integrated genetic and pharmacological approach provides important insights into the individual or combinations of HDACs that could be prioritized for targeting in a range of hematological malignancies.

Epigenetic alterations in acute myeloid leukemias

Acute myeloid leukemia (AML) is a heterogeneous disease for which the standard treatment with cytotoxic chemotherapy has remained largely unchanged for over four decades, with unfavorable clinical results. Epigenetic alterations have been described in several AMLs, and in some cases their origin has been studied in detail mechanistically (such as in acute promyelocytic leukemia, caused by the promyelocytic leukemia–retinoic acid receptor‐α fusion protein). Recently, the advent of massive parallel sequencing has revealed that > 70% of AML cases have mutations in DNA methylation‐related genes or mutations in histone modifiers, showing that epigenetic alterations are key players in the development of most, if not all, AMLs, and pointing to the exploitation of new molecular targets for more efficacious therapies. This review provides a brief overview of the latest findings on the characterization of the epigenetic landscape of AML and discusses the rationale for the optimization of epigenetic therapy of AML.

HDAC3 activity is required for initiation of leukemogenesis in acute promyelocytic leukemia

HDAC3 activity is required for initiation of leukemogenesis in acute promyelocytic leukemia

Biological Features of HHV-6

Publisher Summary This chapter discusses the body of knowledge accrued over the last two decades on the biology of human herpesvirus-6 (HHV-6). HHV-6 has a restricted range of susceptible species, essentially limited to humans and selected nonhuman primates. Antibodies to HHV-6 or to a closely related simian herpesvirus have been demonstrated in monkeys and a simian HHV-6 homolog has been recently identified in mandrill and drill monkeys as well as chimpanzees. The biological features of HHV-6 have been extensively characterized using in vitro models, while in vivo studies are still limited, owing to the lack of suitable and easily accessible animal model systems. A major factor in determining the complexity of the biology of HHV-6 is the existence of two viral variants or subgroups, defined HHV-6A and 6B, which exhibit different biological features and possibly also reflected in the different disease associations described in vivo . The finding that both HHV-6 variants use the ubiquitous molecule CD46 as a membrane receptor is compatible with a broad cellular tropism. Productive infection, however, is limited to a small range of cells most likely by intracellular restriction factors acting beyond the viral entry step. The study of the pathogenesis of HHV-6 infection has been significantly hampered by the lack of physiologically relevant models. The systems most commonly used to grow HHV-6 in vitro , such as cord blood- and peripheral blood-derived mononuclear cells cultured in suspension, require the cells to be maximally stimulated with polyclonal activators in order to sustain HHV-6 replication, a condition that is unlikely to occur in vivo.