Biological studies Effect of Valproic acid on leukemia initiating cells (LICs). Aberrant histone acetylation was physiopathologically associated with the development of acute myeloid leukemias (AMLs). Reversal of histone deacetylation by histone deacetylase inhibitor (HDACis) activates a cell death program that allows tumor regression in mouse models of AMLs. We have used (in collaboration with H. de The, Paris) several models of PML-RARA-driven acute promyelocytic leukemias (APLs) to analyze the in vivo effects of valproic acid, a well-characterized HDACis. Valproic acid (VPA)-induced rapid tumor
288
IEO — Scientific Report 2011 — Ongoing research 2012
regression and sharply prolonged survival. However, discontinuation of treatment was associated to an immediate relapse. In vivo, as well as ex vivo, VPAinduced terminal granulocytic differentiation. Yet, despite full differentiation, leukemia-initiating cell (LIC) activity was actually enhanced by VPA treatment. In contrast to all-trans retinoic acid (ATRA) or arsenic, VPA did not degrade PMLRARA. However, in combination with ATRA, VPA synergized for PML-RARA degradation and LIC eradication in vivo. Our studies indicate that VPA triggers differentiation, but spares LIC activity, further uncouple differentiation from APL clearance and stress the importance of PML-RARA degradation in APL cure. Novel Technologies PAT-ChIP. Formalin-fixed, paraffin-embedded (FFPE) samples represent the gold standard for storage of pathology samples. We have set-up pathology tissue chromatin immunoprecipitation (PAT-ChIP), a technique for extraction and high-throughput analysis, by techniques such as ChIP-seq, of chromatin derived from FFPEsamples (in collaboration with M. Fanelli and PG Pelicci). Technically, the main challenge of PATChIPis the preparation of good-quality chromatin from FFPEsamples. Other steps have also been adapted from existing techniques to optimize their use for PAT-ChIPseq. PAT-ChIPprovides, for the first time, the chance to perform analyses of histone modifications and transcription factor binding on a genome-wide scale using patient-derived FFPEsamples. This technique therefore allows the immediate use of pathology archives (even those that are several years old) for epigenetic analyses and the identification of candidate epigenetic biomarkers or targets.
12. Molecular mechanisms of cell division (Andrea Musacchio) Our studies address the molecular mechanisms of cell division, the process whereby a single mother cell creates two identical daughter cells. Our approach is rooted in biochemical reconstitution, structural cell biology, and chemical biology. We study the reactions that ensure correct chromosome segregation, and the perturbations that prevent the correct execution of the cell division process. In the long run, these studies will shed light on the molecular basis of carcinogenesis. During cell division, the name mitosis denotes the process of physical separation of the genetic material. Cells enter mitosis with replicated copies of their chromosomes, known as sister chromatids. In mitosis, the sister chromatids form stable connections with molecular “cables” named microtubules. Microtubules
and microtubule motors are proteins that harness chemical energy to perform physical work. They are crucial components of the so-called mitotic spindle, a self-organizing structure that subtends to the entire process of cell division. The process of attachment of sister chromatids to the spindle is error-prone. Errors need to be identified and corrected if unbalances in the division of chromosomes are to be avoided. Two feedback control mechanisms, known as the spindle assembly checkpoint (SAC) and error correction (EC) ensure that each and every cell is given enough time to divide its chromosomes in two equal masses, preventing premature chromosome segregation. Furthermore, the EC mechanism sorts good attachments from bad ones, correcting the latter and selectively stabilizing the former. Together, EC and the SAC prevent improper cell division, protecting our cells from aneuploidy, unbalances in chromosome numbers that are all too frequently found in tumours. A fundamental question in the field of mitosis regards the relationship between feedback mechanisms and their regulation at kinetochores. Kinetochores are large protein scaffolds (>100 different proteins, each in multiple copies) that mediate the interaction of the sister chromatids with the mitotic spindle. Precisely how kinetochores regulate the SAC and EC is unclear, but the creation of intrakinetochore stretch, a structural deformation of the internal structure of the kinetochore when microtubules capture kinetochores, seems to be important. A 10-subunit protein assembly at the kinetochore, the KMN network, is crucially implicated in the control of the SAC and EC responses. The KMN network contains the main microtubule binding activity of the kinetochore, and it is therefore the logical site of control of the EC and the SAC. We have recently reconstituted most of the KMN network complex using recombinant proteins, and are now in the process of testing models for the interplay of these components on kinetochores under different conditions that simulate in vitro the attachment, or lack thereof, of microtubules. Small molecules are crucial tool for the characterization of the feedback mechanisms acting in mitosis. They allow acute inhibition of their targets, which in turn allows the observation of the effects of relevant perturbations in real time. In recent years, we characterized a small-molecule named reversine as a potent inhibitor of Mps1, a prominent SAC kinase. Cells treated with reversine exit mitosis prematurely, as they fail to maintain the SAC. Interestingly, we have recently shown that the inhibition of Aurora B and of Mps1 leads to a strongly synergistic effect on checkpoint inhibition that allows lowering the doses of inhibitor
used by a factor of 20 or more. The network properties underlying this behaviour are currently unclear and their identification represents a priority of our studies. Our long-term vision is that drug combinations will be exploited as a means to target cancer cells selectively and with low toxicity. To exploit the full potential of this approach, however, the molecular mechanism inhibited by different small molecules will need to be known in much greater detail.
Research Activities
in these processes. We also find that HDAC inhibition triggers Sae2 degradation by promoting autophagy that affects the DNA damage sensitivity of hda1 and rpd3 mutants. Rapamycin, which stimulates autophagy by inhibiting Tor, also causes Sae2 degradation. These findings led to a model where Rpd3, Hda1 and Gcn5 control chromosome stability by coordinating the ATR checkpoint and double-strand-break processing with autophagy, and point to new, ongoing studies in mammalian model systems. An additional, novel link has been established with the process of cell senescence. Two major mechanisms have been causally implicated in the establishment of cellular senescence: the activation of the DNA damage response (DDR) pathway and the formation of senescenceassociated heterochromatic foci (SAHF). In our studies (in collaboration with F. D’Adda di Fagagna, IFOM), we have showed that in human fibroblasts resistant to premature p16(INK4a) induction, SAHF are preferentially formed following oncogene activation but are not detected during replicative cellular senescence or on exposure to a variety of senescence-inducing stimuli. Oncogeneinduced SAHF formation depends on DNA replication and ATR (ataxia telangiectasia and Rad3-related). Inactivation of ATM (ataxia telangiectasia mutated) or p53 allows the proliferation of oncogene-expressing cells that retain increased heterochromatin induction. In human cancers, levels of heterochromatin markers are higher than in normal tissues, and are independent of the proliferative index or stage of the tumours. Pharmacological and genetic perturbation of heterochromatin in oncogeneexpressing cells increase DDR signalling and lead to apoptosis. In vivo, a histone deacetylase inhibitor causes heterochromatin relaxation, increased DDR, apoptosis and tumour regression. These results indicate that heterochromatin induced by oncogenic stress restrains DDR and suggest that the use of chromatin-modifying drugs in cancer therapies may benefit from the study of chromatin and DDR status of tumours.
13. Chronic inflammation and cancer: Transcriptional mechanisms as potential therapeutic targets (Gioacchino Natoli) Epidemiological and experimental data demonstrated a direct link between chronic inflammation and the development of several types of cancers. Moreover, cancers often contain an inflammatory infiltrate that is hijacked by tumor cells to promote angiogenesis, tissue invasion and cell proliferation. In vivo experiments in mouse models have demonstrated that modulation of tumor properties by inflammatory cells requires the transcription factors of the NF-kB family, which are essential to mount the transcriptional program underlying the inflammatory response. In turn, this has led to the suggestion that anti-NF-kB drugs may be used in tumor therapy. Drugs disabling the whole NF-kB signaling pathway are already being tested in clinical trials, but several concerns about their safety have been raised because of the many physiological responses in which NF-kB is required, most notably the defense against microbial infections. Conversely, therapies blocking the induction of subsets of inflammatory genes relevant to disease (and specifically cancer) pathogenesis regulated genes should provide more restricted and predictable effects, but the molecular bases for their design are not available. Our unit is interested in understanding the mutual relationships between the transcriptional response underlying the inflammatory response and the epigenome, namely the collection of chromatin modifications that maintain and propagate across mitosis the program of gene expression characteristic of a given cell type. On the one hand chromatin controls recruitment of transcription factors to inflammatory genes and generates tissue-specific and temporal profiles of inflammatory gene expression. On the other, inflammation affects epigenetic control in bystander tissue cells, thus leading to alteration of tissue differentiation in chronically inflamed tissues (e.g. intestinal metaplasia in chronic gastritis and Barrett’s esophagus in reflux esophagitis). Such alterations often precede and are associated with the
IEO — Scientific Report 2011 — Ongoing research 2012
289