Dissertations / Theses on the topic 'Inhibitor of HDAC6'

Abnormal epigenetic control is a common early event in tumour progression, and aberrant acetylation in particular has been implicated in tumourigenesis. Histone deacetylases (HDACs) are enzymes that regulate acetylation of chromatin and a variety of other non-histone substrates. Significantly, HDAC inhibitors are potent anti-proliferative agents and exhibit clinical activity in lymphoproliferative and haematological maligancy. However, the mechanistic details by which HDAC inhibitors affect proliferation remain to be elucidated. I have explored the cellular processes affected by HDAC inhibitors, and begun to illuminate a new pathway, regulated by HDAC, which impinges on the cellular effect of HDAC inhibitors. My results suggest that the proteins HR23B and Myd88 are important sensitivity determinants for HDAC inhibitor treatment, and that their interplay with HDAC6 dictates cell fate choice between survival by autophagy or apoptosis.

Rett syndrome is a severe neurodevelopmental disorder. The condition affects approximately one in every 10.000 females and is only rarely seen in males. Causative mutations in the transcriptional regulator MeCP2 have been identified in more than 95% of classic Rett patients; mutations in CDKL5 are responsible for the early onset seizures Rett variant and mutations in FOXG1 gene lead to the congenital Rett variant. To shed light on molecular mechanisms underlying Rett syndrome onset and progression in disease-relevant cells, we took advantage of the breakthrough genetic reprogramming technology and we investigated changes in iPSC-derived neurons from patients with different MECP2 and FOXG1 mutations and in the brain of Foxg1+/- mice. In total brains from Foxg1+/ − mutants we noticed a statistically significant overexpression of a group of neuropeptides expressed in the basal ganglia, cortex, hippocampus and hypothalamus: Oxytocin (Oxt), Arginine vasopressin (Avp) and Neuronatin (Nnat).Moreover, in iPSC-derived neuronal precursors and neurons mutated in FOXG1 and in Foxg1+/− mouse embryonic brain (E11.5) compared to wild type controls we found an increase in the expression of GluD1 and inhibitory synaptic markers, such as GAD67 and GABA AR-α1 and a decreased expression of excitatory synaptic markers, such as VGLUT1, GluA1, GluN1 and PSD-95, suggesting an excitation/inhibition imbalance in the developing brain of the congenital RTT variant. Furthermore, we investigated transcriptome changes in neurons differentiated from MECP2 mutated iPSC-derived neurons and we noticed a prominent GABAergic circuit disruption and a perturbation of cytoskeleton dynamics. In particular, in MECP2-mutated neurons we identified a significant decrease of acetylated α-tubulin which can be reverted by treatment with a selective inhibitor of HDAC6, the main α-tubulin deacetylase. Taken togheter, these findings contribute to shed light on Rett pathogenic mechanisms and provide hints for the definition of new therapeutic strategies for Rett syndrome.

Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that acetylates several proteins that are involved in the immune response. HDAC6 inhibition has been shown in various models to decrease inflammation by altering various proteins involved in the dysregulation of B and T cell responses. In our current studies we sought to determine if HDAC6 inhibition would decrease disease in lupus-prone mice using two murine mouse models of SLE: MRL/lpr mice and NZB/W F1 mice. Both mouse models were fed a rodent diet formulated with the selective HDAC6 inhibitor ACY-738 (N-hydroxy-2-(1-phenylcycloproylamino) pyrimidine-5-carboxamide). NZBW mice received 18 weeks of treatment starting at 16-weeks-of-age and had an average of 57.3 +/- 14.6 ng/mL of ACY-738 in the plasma. MRL/lpr mice received 7 weeks of treatment starting at 11-weeks-of-age and had an average of 78.5 +/- 17.3 ng/mL of ACY-738 in the plasma. Controls received either dexamethasone 5x a week or were left untreated. As the mice aged, body weight, urine protein, and blood sera was collected weekly. Spleen cells were isolated following euthanasia for flow cytometry and kidneys were also collected for histological analyses. We found that in both mouse models that mice treated with ACY-738 had reduced splenic weight and IgG immunoglobulin isotypes. MRL/lpr mice that were treated with ACY-738 had a reduction in the number of IL-17+, ROR-gamma-t TH17 cells. NZBW/ F1 mice that received ACY-738 treatment also had a reduction in the TH17 cells and we observed a significant reduction in kidney pathology. Selective HDAC6 targeting may warrant future investigations as a potential therapeutic target for the treatment of SLE.
Master of Science

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by abnormalities in multiple components of the immune system resulting in progressive damage to multiple organs. Current treatments for SLE are often intensive and result in side effects and the potential for continued flares and progression of disease. Histone deacetylase (HDAC) enzymes control multiple cellular functions by removing acetyl groups from lysine residues in various proteins. HDAC inhibitors have been investigated as a potential treatment for SLE with promising results, however selective HDAC6 inhibition (HDAC6i) has become a leading candidate for pharmacologic inhibition to reduce the potential for side effects. We hypothesize that HDAC6i will decrease SLE disease by targeting substrates of HDAC6 in multiple components of immunity and organ systems. NZB/W mice were treated with ACY-738 or ACY-1083, followed by evaluation of multiple disease parameters and mechanisms involved in disease pathogenesis within the kidney, bone marrow, and spleen. Within the kidney, HDAC6i decreased glomerular pathology scores, proteinuria, and IgG and C3 deposition. Within glomerular cells, HDAC6i increased alpha-tubulin acetylation and decreased nuclear NF-κB. Within the spleen, there was a dose-dependent decrease in the frequency of Th17 cells and a mild decrease in the frequency of Treg cells. Concurrently, there were decreased levels of IL-12/IL-23 and minimal decreases in TGF-β in the serum. Within the bone marrow, B cell development through Hardy fractions exhibited accelerated progression through later stages as NZB/W mice aged. This accelerated progression may allow B cells to bypass important regulatory checkpoints in maintaining immune tolerance and contribute to autoimmunity. Treatment with an HDAC6i corrected the aberrant B cell development in the bone marrow and RNAseq analysis unveiled six genes (Cebpb, Ccr9, Spib, Nfil3, Lgals1, and Pou2af1) that may play a role in the aforementioned abnormalities. Overall, these findings show that HDAC6i decreased disease in NZB/W mice by targeting multiple components of the immune response, including glomerular cells, T cell subsets in the spleen, and bone marrow B cells. In conclusion, selective HDAC6i is an excellent candidate for pharmacologic therapy for SLE because it targets multiple immune abnormalities involved in SLE pathogenesis while remaining selective and safe.
Ph. D.

Les patients atteints de leucémie myéloïde chronique (LMC) breakpoint cluster region-Abelson (BCR-ABL)+ sont traités avec des inhibiteurs de tyrosine kinase (ITK), comme l’imatinib, cependant certains développent des résistances et des effets secondaires sévères. Des traitements combinés à base d’inhibiteurs d’histone désacétylase (HDAC)6 (HDAC6i), pouvant potentiellement réduire l’expression de BCR-ABL, apparaît être une approche intéressante pour prévenir l’apparition de résistances aux ITK. De plus, l’implication d’HDAC6 dans les voies de dégradation des protéines rend son inhibition couplée à celle du protéasome susceptible de sensibiliser les cellules aux ITK. Notre hypothèse est que la combinaison ITK-HDAC6i pourrait être efficace pour le traitement de la LMC. Dans un premier temps, les effets anti-cancéreux d’un HDAC6i identifié dans notre laboratoire, le composé 7b, à celui de référence, la tubacine, en combinaison avec l’imatinib ont été comparés. La combinaison imatinib-7b a généré des effets anti- cancéreux plus importants que la combinaison imatinib-tubacine et a provoqué une mort synergique apoptotique dépendante des caspases dans les cellules K-562 et réduit la proportion de cellules souches leucémiques alors qu’elle n’a eu qu’un effet modéré sur des cellules saines. Enfin, la combinaison a diminué plus fortement la capacité de formation de colonies et la masse tumorale des cellules de LMC respectivement en milieu semi-solide et dans des poissons zèbres xénogreffés, par rapport aux composés seuls. D’un point de vue mécanistique, la combinaison induit l’ubiquitination et la dégradation de BCR-ABL, et la dérégulation de protéines de ses voies de signalisation impliquées dans la prolifération et la survie cellulaire. La protéine HDAC6 possédant deux sites catalytiques, nos résultats tendent à montrer que le composé 7b cible le deuxième. Dans un second temps, une étude a été initiée sur un nouvel HDAC6i de type hydroxamate, le MAKV-15, qui diminue l’expression de BCR-ABL, et qui en pré-traitement avec le bortezomib, sensibilise les cellules à l’imatinib, entrainant une augmentation de la mort apoptotique dépendante des caspases dans les cellules sensibles et résistantes à l’imatinib. Enfin, nos résultats suggèrent que l’inhibition d’HDAC6 potentialise l’effet de l’imatinib, pourrait prévenir l’apparition de résistances et que de telles combinaisons pourraient représenter une approche thérapeutique prometteuse pour les patients atteints de LMC
Breakpoint cluster region-Abelson (BCR-ABL)+ chronic myeloid leukemia (CML) patients receive tyrosine kinase inhibitors (TKIs) such as imatinib as the first-line treatment; however, some patients develop resistances and severe adverse effects. Combination treatments, especially with histone deacetylase (HDAC)6 inhibitors (HDAC6i), appear as an attractive option to prevent TKI resistances considering the capacity of HDAC6i to downregulate BCR-ABL. Moreover, HDAC6 is implicated in protein degradation pathways, so that its inhibition combined with that of the proteasome could sensitize cells to TKIs. Thus, we hypothesized that HDAC6i combined to TKIs could be effective for CML treatment. In the first part, we compared the anti-CML effects of a HDAC6i identified in our laboratory, compound 7b, to the reference HDAC6i tubacin, in combination with imatinib. Results showed that the imatinib-7b combination generated stronger anti- CML effects than imatinib-tubacin. Especially, the imatinib-7b combination elicited a potent synergistic caspase- dependent apoptotic cell death and drastically reduced the proportion of cancer stem cells in K562 CML cells, whereas it only moderately impacted various healthy cell models. Ultimately, the imatinib-7b combination decreased more potently the colony forming capacities and tumor mass formation of CML cells in a semisolid methylcellulose medium and in xenografted zebrafishes, respectively, compared to each compound alone. Mechanistically, the combination induced BCR-ABL ubiquitination and downregulation leading to a dysregulation of multiple key proteins of its downstream pathways involved in CML proliferation and survival. Results tend to demonstrate that 7b could target the second site. In the second part, we initiated a study of a novel hydroxamate-based HDAC6i, MAKV-15, and preliminary results demonstrated it triggered BCR-ABL downregulation. Accordingly, in pre-treatment with bortezomib it sensitizes CML cells to imatinib leading to enhanced caspase-dependent apoptotic death in imatinib-sensitive and imatinib-resistant CML cells. Considering that HDAC6 is reported to possess two functional catalytic sites, we finally attempted to determine which catalytic site is targeted by these HDAC6i. Taken together, our results suggest that HDAC6i potentiate the effect of imatinib and could overcome TKI resistance in CML cells and therefore such combination may represent a promising therapeutic approach for CML patients

L’épigénétique représente les modifications de l’expression génique, sans altérer la séquence nucléique de l'ADN. L'un des mécanismes de régulation est le remodelage de la chromatine qui s’effectue via les histones acétyltransférases et les histones désacétylases (HDAC) permettant ou non la transcription de gènes. Une expression anormale des HDAC est corrélée à de nombreuses maladies (dépendance à l'alcool, inflammation ainsi que les maladies cardiovasculaires et neurodégénératives, cancers…). Il est primordial de cibler la sélectivité d’une isoforme parmi les 11 connues des HDAC zinc dépendantes pour éviter les effets secondaires. Le but de la recherche était de concevoir et de synthétiser de nouveaux composés, de vérifier leur activité inhibitrice vis-à-vis des HDAC de classe I ou II et leur cytotoxicité sur quatre lignées cellulaires: HaCaT, V79-4, SH-SY5Y et PC12. Ainsi, nous nous sommes concentrés sur les pharmacomodulations du ZBG, de l’espaceur et de la tête de molécules connues tels que le MS-275 (sélectif de la classe I des HDAC), les SAHA et TSA (espaceur en C5 ou C6) avec une forte activité inhibitrice vis-à-vis des HDAC, mais non sélectifs. Nous nous sommes concentrés sur les pharmacomodulations de l'HDACI connu modifiant le domaine de liaison au zinc ZBG (sulfonylhydrazide, catéchol), la nature de l’espaceur (alkyl, aryl) et le groupe de reconnaissance de surface (bis-aryl, adamantyl, indolopyridazinone). Une bibliothèque de 57 nouveaux composés a été créée en trois séries. Aucun d'entre eux n'a montré d'activité inhibitrice satisfaisante. Les composés sélectionnés n'ont pas montré d'activité cytotoxique sur les lignées de cellules neuronales. Sur la base de cette recherche, il est possible de créer de nouveaux composés dans la série indolopyridazinone afin de les tester
Epigenetics represents changes in gene expression without altering the nucleic sequence of DNA. One of the main mechanisms of regulation of gene expression is chromatin remodeling via histone acetyltransferases and histone deacetylases (HDAC), which may or may not allow gene transcription. An abnormal expression of HDACs is correlated with many diseases (alcohol dependence, inflammation as well as cardiovascular and neurodegenerative diseases, cancers...). It is essential to target the selectivity of one isoform among the 11 known zinc-dependent HDACs to avoid side effects. The aim of the research was to design and synthesize new compounds, verify their inhibitory activity against class I or II HDACs and their cytotoxicity on four cell lines: HaCaT, V79-4, SH-SY5Y and PC12. We focused on the pharmacomodulations of ZBG, the linker and the cap of known molecules such as MS-275 (selective for class I of HDACs), SAHA and TSA (spacer in C5 or C6) with a strong inhibitory activity towards HDACs, but not selective. We concentrated on the pharmacomodulations of known HDACI modifying the zinc binding domain (sulfonylhydrazide, catechol), the nature of the spacer (alkyl, aryl) and the surface recognition group (bis-aryl, adamantyl, indolopyridazinone). A library of 57 new compounds was designed in three series. None of them showed satisfactory inhibitory activity. The selected compounds did not show cytotoxic activity on neuronal cell lines. Based on this research, it is possible to create new compounds in the indolopyridazinone series in order to test them

Le déséquilibre HAT/HDAC aurait une influence sur le développement de certains cancers ainsi que dans l’addiction à l’alcool ou à la cocaïne. En inhibant les histones désacétylases, le taux d'acétylation de la chromatine augmente ce qui permet l’accès aux facteurs de transcription et l'expression des gènes. Aujourd'hui, il existe de nombreux inhibiteurs d'HDAC de structures diverses, mais ils ne sont pas spécifiques et présentent des effets secondaires importants. Les inhibiteurs d'HDAC comme le butyrate de sodium ou le MS-275 ont montré une modification de la dépendance à l'alcool chez le rat. MS-275 inhibe principalement la classe I de HDAC et conformément à ces observations nous nous intéressons aux inhibiteurs les plus sélectifs de la classe I tels que le Largazole thiol et le RedFK228. Notre but est de synthétiser de nouveaux cyclodepsipeptides analogues afin d'obtenir un inhibiteur sélectif de la classe I. Les HDAC de la classe I sont Zn-dépendants, ces analogues auront un groupement sulfonylhydrazide ayant une bonne affinité pour l’ion Zn2+ (ZBG). Il sera relié au cyclodepsipeptide par un bras espaceur dont la longueur sera adaptée (n = 2, 3). Une autre pharmacomodulation concerne l'incorporation d’hétérocycles différents (oxazole, thiazole et pyridine). Les inhibitions de ces composés ont pu être testées sur HDAC1, HDAC3 et HDAC6. Un composé a une spécificité pour HDAC3 et un autre a une spécificité pour HDAC1. Les tests sur des rats "binger" permettent de penser que HDAC1 est impliqué dans ce model de consommation et non HDAC3
The imbalance HAT/HDAC would influence the development of cancers and alcohol or cocaine addiction. HDAC inhibition allows increase of both acetylation rate and gene expression. Today, there are many structurally diverse potent, but non-specific HDAC inhibitors displaying important side-effects. HDAC inhibitors such as sodium butyrate or MS-275 have been shown to alter the alcohol dependence in the rat. MS-275 inhibits mainly class I of HDAC and in line with these observations we are interested in more selective class I inhibitors such as Largazole thiol and RedFK228. Our purpose is to synthesize new cyclodepsipeptides analogues in order to obtain selective class I inhibitor. HDAC class I is a Zn-dependent enzyme and our target molecules have sulfonylhydrazide function as efficient Zinc binding group (ZBG). Additional pharmacomodulations concern the incorporation of different heterocycles (oxazole, thiazole, pyridine) and varying linker lengths (n = 2, 3). Inhibitions of these compounds have been tested on HDAC1, HDAC3 and HDAC6. A compound has specificity for HDAC3 and another has specificity for HDAC1. Tests on rats "binger" suggest that HDAC1 is involved in this model of consumption and not HDAC3

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xix, 116 p.; also includes graphics. Includes bibliographical references (p. 106-116). Available online via OhioLINK's ETD Center

Histone deacetylases (HDACs) are key mediators of gene expression and, thus, major regulators of cell function. As such, HDACs play a role in orchestrating tumor biology, and the use of small inhibitors targeting theses proteins is attractive for the field of cancer therapy. Indeed, several HDAC inhibitors have received FDA-approval for the treatment of malignancies, while a myriad of these compounds continue to be evaluated in clinical trials. Besides their direct impact on tumor growth, HDAC inhibitors have been shown to increase immunogenicity of cancer cells, facilitating generation of a productive immune response against tumors. Immunotherapeutic approaches take advantage of the intrinsic ability of the immune system to manifest an anti-tumor response. Mechanisms of immune escape are often developed by cancer cells, neutralizing activity of the immune system. For example, upregulation of the PD1 ligands PDL1 and PDL2 by tumor cells negatively regulates the anti-tumor functions of PD1-expressing infiltrating T-cells. Importantly, strategies targeting this inhibitory axis have shown outstanding clinical benefit for the treatment of solid and hematological malignancies. The mechanisms by which HDAC inhibitors modulate tumor and immune cells biology were explored herein. Initially, treatment of melanoma cells with pan- and class I-selective HDAC inhibitors resulted in upregulation of PDL1 and PDL2 molecules. These effects were observed in mouse and human cell lines, as well as in tumor cells resected from metastatic melanoma patients. This upregulation was robust and sustained, lasting at least 96 hours in vitro, and validated in vivo using a B16F10 syngeneic mouse model. Enhanced expression of PDL1 mediated by HDAC inhibitors was found to result from enhanced histone acetylation at the PDL1 gene promoter region. Combination therapy of HDAC inhibition and PD1 blockade was explored in the tumor setting, leading to synergistic effects in terms of reducing melanoma progression and increasing survival of B16F10 melanoma-bearing mice. These data provide a clinical rationale for combination therapy of epigenetic modifiers (e.g. HDAC inhibitors) and PD1 blockade as means to augment cancer immunotherapy, improving patient outcomes. As a second pillar of this research, the impacts of HDAC-selective inhibition were explored on immune cell biology, since the broad nature of pan-HDAC inhibitors was shown to be detrimental to T-cells in vitro and in vivo. Based on screening assay results, novel implications of treating melanoma patient T-cells ex vivo with the HDAC6-selective inhibitor ACY1215 were investigated. Treatment with this compound was unique among pan- and isotype-selective HDAC inhibitors in modulating T-cell cytokine production and showing minimal impact of T-cell viability. ACY1215 tempered Th2 cytokine production (i.e. IL-4, IL-6 and IL-10), and maintained Th1 effector cytokines (e.g. IFNγ and IL-2). Furthermore, ACY1215 increased expression of surface markers, including CD69 activation marker and ICOS co-stimulatory molecule. In addition, ACY1215 treatment enhanced accumulation of central memory T-cells during ex vivo expansion of tumor infiltrating T-cells harvested from resected tumors of metastatic melanoma patients. Importantly, ACY1215-mediated inhibition improved tumor-killing capacity of T-cells. These results highlight an unexplored ability of selective HDAC inhibitor ACY1215 to augment T-cell expansion during protocols of adoptive cell therapy. While the discoveries presented here warrant further investigation of cellular and molecular mechanisms associated with ACY1215-treated T-cells, the clinic implications are clear and rapidly translatable.

As our understanding of cancer biology increases and novel therapies are developed, an increasing number of predictive biomarkers are becoming clinically available. Aberrant acetylation has been strongly linked to tumourigenesis and the modulation of acetylation through targeting histone deacetylase (HDAC) has led to the introduction of many HDAC inhibitors. To date, two have had regulatory approval for the treatment of cutaneous T cell lymphoma (CTCL). Modifications in chromatin control underpin the mechanism of action of HDAC inhibitors. A genome wide loss-of-function screen identified HR23B as a gene that governs sensitivity to HDAC inhibitors. HR23B shuttles ubiquitinated cargo proteins to the proteasome and elevated levels may contribute to cell death mediated by this pathway. It also governs cell sensitivity to drugs that act directly on the proteasome. HDAC inhibitors influence proteasome activity and there may be a synergistic interaction with proteasome inhibitors. HR23B and HDAC6 interact and HDAC6 may be a negative regulator of apoptosis and a positive regulator of autophagy and through its ability to down-regulate HR23B, may impact on the cellular outcome of HDAC inhibitor treatment. Expression of HR23B has been correlated with clinical response to HDAC inhibitors in a retrospective analysis of CTCL patients. The tissue expression of HR23B and the autophagy marker LC3 has been investigated and there may be a reciprocal relationship in their expression in some tumours which may provide prognostic information and patients with low HR23B expression but high levels of autophagy appear to have a particularly poor prognosis. Well designed, biomarker-driven prospective clinical trials are needed to clarify the predictive and prognostic roles of HR23B.

Platinum-based anticancer drugs, such as cisplatin, carboplatin, and oxaliplatin, have been approved for clinical use worldwide for decades. Despite their enormous success, their widespread application is hindered by either cross-resistance or toxic side effects, including nephrotoxicity and neurotoxicity. The need to overcome these drawbacks has stimulated the search for new platinum-based drugs. This dissertation will start with the accidental discovery of cisplatin, followed by an introduction of other platinum-based anticancer agents, including the action mechanism, general structures, and development history. Picoplatin is a newer generation of platinum-based anticancer agent. The bulky 2-methylpyridine as a non-leaving group on picoplatin could reduce the detoxification effect caused by thiol-containing species, such as glutathione and metallothionein, thus may grant picoplatin the ability to overcome cisplatin resistance. A convenient synthesis route for picoplatin derivatives has been developed. 11 new picoplatin derivatives have been designed by varying the bulkiness of the non-leaving amine group. All complexes have been characterized by different instrumentations, including MS, 1H NMR, 13C NMR, 195Pt NMR, HMQC, X-ray crystallography, and elemental analysis. Different bioassays, such as DNA binding, cell viability, and cellular accumulation, have been applied to evaluate their efficacy on cisplatin-sensitive ovarian cancer cell line A2780 and cisplatin-resistant ovarian cancer cell line A2780cis. The newly designed picoplatin derivatives show comparable efficacy with that of picoplatin and less resistance compared with cisplatin. The study of picoplatin derivatives laid the foundation toward the research of bifunctional platinum-based anticancer agents by incorporating histone deacetylase (HDAC) inhibition. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are a pair of important enzymes in epigenetic regulation. They work in harmony to acetylate and deacetylate histone lysine residues, resulting in a more relaxed or more condensed chromatin structure, respectively. HDAC has been found to be overexpressed in some cancer cells. Since 2006, 5 HDAC inhibitors (HDACi) have entered clinical use for cancer treatment. 19 new HDACi with additional coordination sites on the phenyl cap have been designed, synthesized, and evaluated. A few of the new HDACi show comparable or even better HDAC inhibition than that of Vorinostat (SAHA, the first FDA approved HDACi). A logical design would involve the installation of HDACi on the platinum center as a non-leaving group ligand. When the bifunctional drug reaches the cancer cell, the synergistic effect could be maintained as the relaxed chromatin structure makes DNA more susceptible to be attacked by the platinum centers, thus increase the anticancer activity and possibly selectivity toward cancer cells. 6 Pt-HADCi conjugates have been designed and synthesized. Dual functions of the new Pt-HDACi have been confirmed by DNA electrophoresis assay and HDAC inhibition assay. One of the Pt-HDACi (CF-101) shows comparable cytotoxicity with cisplatin and less resistance, which could be used as the lead compound for further structural modification and in vivo studies.

Research performed over the last decade has highlighted the role of HDAC inhibitors (HDACis) as modulators of transcriptional activity and as a potential new class of therapeutic agents against many types of malignacies including breast cancer. These drugs inhibit histone deacetylases, leading to derepression of transcription of various genes that are important for cell cycle arrest and cell death. Trichostatin A (TSA) is one of the best established HDAC inhibitors and has been shown to exhibit potent differentiating and anti-proliferative properties. My data demonstrated that treatment of the MCF-7 breast cancer cell line with TSA causes G2/M phase cell cycle arrest. I characterised the novel F-Box protein called FBXL20 and identified it as a direct target of TSA. This protein is part of a novel E3 ligase complex as it binds Skpl, CUL-I and ROC-I and forms a classical SCF complex that is responsible for ubiquitination and targeting proteins for degradation by the 26S proteasome. I further studied the differences between FBXL20 in human cells and its isoform in rat cells. My data showed that FBXL20 is localised in the cytoplasm, concentrated around the nucleus and plays a role in the TSA-induced effects in MCF-7 cells, through regulating the pro-apoptotic protein Bim. Silencing FBXL20 abolished the G2/M arrest caused by TSA treatment. Although FBXL20 is a similar protein to Skp2 they are regulated by different proteins and exert different functions. These findings provide novel data to demonstrate that known and novel HDACis induce G2/M arrest followed by cell death and that this arrest is dependent on the novel FBXL20 protein in breast cancer cells. Using these newly defined properties of HDACis, I screened a panel of potential HDACis and identified at least one to be more potent than SAHA, which is currently used in the clinical setting and showed that it is able to inhibit proliferation, cause cell cycle arrest and cell death of breast cancer cells.

In Acute Myeloid Leukemia (AML), the dysregulation of the Hh signaling is involved in the development and expansion of leukemic cancer cells and influences the response to therapeutic agents. Notably, the FDA approved only one Hh inhibitor (glasdegib) as a therapeutic strategy for AML treatment, and the majority of patients eventually relapse, underlying the urgency of discovering new therapeutic targets. One characteristic of the Hh pathway is its localization on the primary cilium membrane (PC), a microtubule-based organelle expressed by almost all non-proliferating mammalian cells. Indeed, centrosomes participate in a mutually exclusive manner in the formation of PC or mitotic spindle. Cancer cells, including AML cell lines, are characterized by a high rate of proliferation and fail to present the PC on their surface. Novel approaches to restore the PC on the surface of cancer cells are emerging, and most of them target the histone deacetylase HDAC6. Indeed, HDAC6 inhibition prevents the reabsorption of the PC and blocks cell proliferation. Since HDAC6 inhibitors (i.e., TubastatinA) are already used to treat other tumors, they might also be promising in AML treatment. In this work, analyzing the blood samples of 36 adult AML patients, we demonstrated that Hh target genes (GLI1, PTCH1), HDAC6, and the Multi- Drug-Resistant genes (MDRs) ABCC1 and ASXL1 were more expressed than in healthy donors (HD). In addition, through in silico analyses, we verified that in AML patients the expression of Hh/HDAC6 and MDRs genes were positively correlated. We also detected the same genetic regulation in in vitro models of AML, as cell lines with higher Hh expression (U937 and THP-1) showed higher levels of HDAC6 and MDRs than cell lines with low Hh expression (NB-4 and OCI-AML2). We generated a zebrafish model with Hh hyperactivation through the injection of the shh mRNA to functionally investigate the effect of Hh/HDAC6 dysregulation, and we confirmed the increased expression of hdac6 and MDRs. Moreover, in the zebrafish reporter line for the hematopoietic stem precursor cells (HSPCs) the Tg(CD41:GFP) line, we found that Hh hyperactivation induces the hyperproliferation and expansion of the HSPCs in the caudal hematopoietic tissue, a phenotype that resembles the expansion of leukemic blast of AML patients. Interestingly, we rescued this hematopoietic defect by treating the embryos with the HDAC6 inhibitor TubastatinA but not with the Hh inhibitor cyclopamine. By the generation of a zebrafish model carrying the overexpression of the human HDAC6 mRNA, we demonstrated that HDAC6 alone can induce the hyperproliferation of the HSPCs population and that this phenotype is specific as, through its inhibition, we rescued the hematopoietic defect. Since we observed that an increased proliferation rate elicited the expansion of HSPCs, we hypothesized an implication of the PC. Indeed, we demonstrated that zebrafish HSPCs present the PC, therefore suggesting that HDAC6 controls HSPCs proliferation, through PC's stabilization on their surface. For the first time, we described a role for HDAC6 in HSPCs expansion and identified it as a promising target for AML patients. Indeed, HDAC6 inhibition was also efficient in reducing the viability of leukemic cell lines and HSPCs expansion in well-established AML zebrafish models, carrying the overexpression of genes frequently mutated in AML patients: NPMc+ and FLT3-ITD. Moreover, in these zebrafish models, we demonstrated the efficacy of combination therapy with the standard chemotherapeutic agent cytarabine and HDAC6 inhibition. In conclusion, we identified a positive correlation between the Hh signaling, HDAC6 and the MDR genes in AML patients. In the zebrafish model, we reported that both Hh or HDAC6 overexpression drive the hyperproliferation of the HSPCs population, a phenotype that is rescued only through HDAC6 inhibition. HSPCs hyperproliferation and the rescue through the specific HDAC6 inhibition, might be explained by alteration in the PC, that we described to present in the HSPCs. As reporter for Hh inhibition, we demonstrated that also HDAC6 inhibition efficiently reduces the expression of MDR genes. Therefore, we hypothesize the use of HDAC6 inhibitor to counteract AML resistance mechanisms. Finally, we described that HDAC6 inhibition shows high potency in specific AML condition, and that can be a suitable target for the assessment of new combination therapies with standard chemotherapic agents.

Mechanisms underlying the interactions between the proteasome inhibitor carfilzomib and HDAC inhibitors were examined in both germinal center (GC) and activated B-cell (ABC) subtypes of human diffuse large B-cell lymphoma (DLBCL). Simultaneous exposure to minimally toxic concentrations of carfilzomib and HDAC inhibitor vorinostat resulted in the release of mitochondrial pro-apoptotic proteins SMAC and cytochrome c, pro-apoptotic caspase activation, and synergistic induction in apoptosis in both ABC and GC DLBCL subtypes. These events were associated with a marked increase in the stress kinase JNK, ROS generation, G2-M cell cycle arrest, as well as induction of DNA damage. Genetic knockdown of JNK resulted in a significant decrease in carfilzomib/vorinostat induced cell death. Co-administration of the antioxidant MnTBAP significantly reduced carfilzomib/vorinostat induced cell death, and resulted in a marked decrease in caspase-3 as well as a striking decrease in JNK phosphorylation. Tumor growth reduction was also observed in animal models that were treated with a combined regimen of carfilzomib and vorinostat. Finally, the combined treatment of carfilzomib/vorinostat was able to overcome any cross-resistance to carfIlzomib in bortezomib resistant cells. Collectively, these finding indicate that the combined regimen of carfilzomib and HDAC inhibitors promote lethality in ABC and GC human DLBCL cells by a variety of mechanisms both in vitro and in vivo. Further studies are necessary for clinical development of this drug regimen.

Histone deacetylase (HDAC) inhibition has recently emerged as a novel therapy for cancer treatment. However, currently approved histone deacetylase inhibitors (HDACi) are pan-inhibitors thus inhibiting all 11 zinc dependent HDAC isoforms including those not involved in tumorigenesis. These inhibitors are also associated with various side effects including a potentially fatal cardiotoxicity. To address these issues, isoform selective HDACi were designed and synthesized. The use of 3-hydroxy-pyridin-2-thione (3HPT) as zinc chelation group resulted in small molecules devoid of HDAC1 inhibition but active against HDAC6 and/or 8. Selected 3HPT containing HDACi displayed anticancer activity against various cancer cell lines including DU145, LNCaP and Jurkat. Surprisingly, the lead-compounds were very potent against Jurkat Jγ cells which are resistant to SAHA-induced apoptosis. HDACi were also targeted to cancer cells using folic or pteroic acids as targeting groups. Incorporation of the folic acid into the HDACi pharmacophoric model resulted in inhibitors selective for HDAC6, whereas pteroic-based HDACi inhibited both HDAC1 and 6. Only the pteroic-based inhibitors displayed anticancer activities against folate receptor overexpressing tumors such KB and HeLa. Furthermore, cell-based studies established the inhibition of HDAC1 as the basis for the anticancer activities of the pteroic-based HDACi.

Malaria is a leading cause of morbidity and mortality, causing more than 400,000 deaths per year. Malaria is caused by parasites of the Plasmodium genus with most deaths due to P. falciparum infection. The control of malaria is complicated by the lack of a widely effective vaccine, the spread of mosquito resistance to insecticides and Plasmodium parasite resistance to available drugs, including the gold standard artemisinin-combination therapies. Thus, there is an urgent requirement for the development of new antimalarials, in particular those with different modes of action to existing drugs to limit potential problems of cross-resistance. Plasmodium species have a complex lifecycle that includes transmission from the female Anopheles mosquito vector to a human host requiring significant morphological changes. These morphological changes are associated with stage-specific changes in transcription regulated by epigenetic mechanisms. The proteins involved in these processes are potential new therapeutic targets for malaria. This includes histone deacetylases (HDACs), which together with histone acetyltransferases (HATs), are involved in reversible posttranslational acetylation of histone and non-histone proteins, regulating transcription and other cellular processes. To date, over 650 HDAC inhibitors have been investigated for in vitro activity against malaria parasites. Some inhibitors, particularly those with a hydroxamic acid zinc-binding group that targets inhibitors to the HDAC active site, have demonstrated low nM in vitro potency against P. falciparum and selectivity for the parasite over human cells. However, antiplasmodial HDAC inhibitor drug development has been hindered by factors including the lack of recombinant P. falciparum HDACs (only one available and purity is low), the lack of HDAC crystal structures (none available) and low throughput activity assays that are largely indirect measures of HDAC inhibition. Without these tools, mode of action studies, the rational design of new and improved inhibitors and the prioritisation of compounds for preclinical testing remains difficult. To address some of these challenges and further progress the development of antimalarial HDAC inhibitors, the current study employed a multi-pronged approach, including: (i) investigating the in vitro and in vivo activity of new HDAC inhibitors; (ii) establishing a higher throughput ELISA method to analyse P. falciparum lysine acetylation alterations and; (iii) developing a quantitative structure-activity relationship (QSAR) model based on classification algorithms. HDAC inhibitors typically have a pharmacophore comprising a zinc-binding group that interacts with the zinc ion in the active site of the enzyme, a linker unit and a cap group promoting hydrophobic interaction with amino acid residues at the entry of the active site. Here, a set of 26 new HDAC inhibitors with a peptoid-based scaffold was tested in vitro against drug sensitive asexual intraerythrocytic-stage P. falciparum 3D7 parasites. The set are analogues of compounds that have previously shown in vitro dual-stage antiplasmodial activity against asexual intraerythrocytic and exoerythrocytic stages and includes 16 compounds with a hydroxamic acid zinc-binding group and 10 prodrugs of this compound class. The unprotected hydroxamate-based inhibitors demonstrated growth inhibition of P. falciparum 3D7 asexual intraerythrocytic-stage parasites in the nanomolar to micromolar range (50% growth inhibition values (PfIC50) 0.008-1.04 μM) and up to 1,250-fold selectivity (selectivity indices (SI; PfIC50/human cell IC50): 10-1,250) for the parasite compared to human cells. Structure-activity relationship (SAR) analysis of cap region residues (carbonyl region, carboxylic region and isocyanide region) indicated that benzyl groups in the isocyanide region and alkyl groups in the para position of the carboxylic region are associated with increased antiplasmodial activity. In addition, methyl groups in the carbonyl region of the cap group demonstrated reduced cytotoxicity against neonatal foreskin fibroblasts (NFF), however, also somewhat reduced activity against asexual blood-stage parasites. Work by collaborators demonstrated micromolar in vitro activity of several compounds of this set against exoerythrocytic P. berghei parasite forms indicating dual-stage activity. The compound with the greatest dual-stage activity displayed an IC50 of 8 nM against asexual blood-stage P. falciparum and an IC50 of 60 nM against exoerythrocytic P. berghei in vitro. Compounds with PfIC50 of 100 nM or lower were tested against the multi-drug resistant P. falciparum Dd2 line (resistant to chloroquine, pyrimethamine, mefloquine, and other antimalarial drugs), and demonstrated a resistance index (RI) <1 indicating a lack of cross-resistance by this parasite line. The same subset of compounds was investigated for their ability to hyperacetylate P. falciparum histone H4; differential effects were observed with some compounds causing up to ~2.5-fold hyperacetylation compared to untreated controls. 10 prodrug peptoid-based HDAC inhibitors were also investigated. The prodrug strategy seeks to make the hydroxamic acid-based inhibitors more stable and bioavailable for in vivo applications as they are prone to degradation processes such as hydrolysis or reduction. These compounds were synthesised with masked hydroxamate functionalities that may undergo activation in vitro. Preliminary data demonstrated in vitro PfIC50 of 0.014-1.75 μM and 6-642-fold selectivity for the parasite over human fibroblasts. Three of these compounds displayed PfIC50 <0.1 μM and SI >100 and may therefore be of interest in further studies. Based on the in vitro antiplasmodial activity, selectivity and chemical diversity in the cap region, five peptoid-based compounds (3a, 3c, 3f, 3m, 3n, Pf3D7 IC50 0.008-0.034 μM, SI 97-625) were further investigated for in vivo efficacy against Plasmodium parasites. In addition, four analogues of the tethered phenylbutyrate-based HDAC inhibitor AR42 (Pf3D7 IC50 0.02 μM, SI 39) were also investigated in vivo (JT21b, JT83, JT92a, JT94; Pf3D7 IC50 0.005-0.21 μM, SI 55-118, (data generated by Dr MJ Chua, personal communication)). AR42 is currently in phase 1 clinical trials against various types of cancer and demonstrates an improved pharmacokinetic profile compared to a number of clinically approved HDAC inhibitors (e.g. AR42 Cmax 14.7 μM compared to vorinostat Cmax 1.9 μM, AR42 t1/2 11.1 h compared to vorinostat t1/2 0.75 h; tested in mice). AR42 analogues were of interest as AR42 has previously been shown to cure Plasmodium infections in mice (Dr MJ Chua, Griffith Institute for Drug Discovery; unpublished). While the two analogue sets differ significantly in linker and cap group, both bear a hydroxamic acid zinc-binding group. Compounds were tested in groups of two female BALB/c mice infected with P. berghei ANKA infected erythrocytes. Dosing was via oral gavage at 25 mg/kg twice daily with four hours between dosing (beginning 2 h post infection) for four consecutive days. Peripheral blood parasitemia was monitored by microscopic evaluation of stained thin blood films from day four post infection. None of the peptoid-based HDAC inhibitors attenuated P. berghei growth in BALB/c mice by more than 33% (3f (31%) and 3n (33%) on day 6 post infection). Data from collaborators demonstrated 3n to have the best metabolic stability (t1/2 271 min, Clint 6 μL/min/mg in mice; Prof Finn Hansen, University of Bonn, Germany) which may have contributed to this compound’s improved activity compared to some other analogues. In comparison, AR42 and two if its analogues cured mice of infection (AR42, 1 of 2 mice; JT21b, 2 of 2 mice; JT83 2 of 2 mice), up until day 24 post infection, at which point the mice were euthanised. AR42 and analogues are the first demonstration of oral cures in mice with a HDAC inhibitor (manuscript in preparation) and these data will be pursued in future work to further develop this HDAC inhibitor chemotype for malaria. One of the current limitations in the field is the lack of recombinant P. falciparum HDACs and the need to rely on low throughput assays to demonstrate HDAC inhibitor action via reduced total deacetylase activity or in situ lysine acetylation alterations. While deacetylase assays do not allow the differentiation of compound effects, Western blot using different acetyl-lysine antibodies can reveal compound specific acetylation profiles. Here, two higher throughput methods, dot blot and ELISA, were investigated to assess the effects of HDAC inhibitors on lysine acetylation. Using the control hydroxamate HDAC inhibitor vorinostat (first HDAC inhibitor clinically approved for cancer), the ELISA method was demonstrated to be more reliable than dot blot in detecting acetylation changes in protein lysates from P. falciparum trophozoites exposed to compound for 3 h. ELISA was therefore used to investigate histone H3 and H4 lysine acetylation alterations following exposure of P. falciparum to six commercially available anti-cancer HDAC inhibitors (vorinostat, panobinostat, trichostatin A, romidepsin, entinostat and tubastatin A). All compounds have in vitro activity against asexual intraerythrocytic P. falciparum parasites (Pf3D7), with tubastatin A activity reported for the first time here (PfIC50 0.15 ± 0.03 μM). All compounds were also shown to inhibit >84% deacetylase activity using P. falciparum protein lysates in an in vitro assay at 1 μM, with the exception of entinostat (~50% inhibition at 1 μM); this compound was also the least active against the parasite (PfIC50 11.5 μM). Using ELISA, vorinostat, panobinostat, trichostatin A, romidepsin and entinostat were all found to cause a ~3-fold increase in the signal detected using an anti-tetra-acetyl-lysine antibody. In comparison, the only human HDAC6-specific inhibitor tested, tubastatin A, caused 1.8-fold histone H4 hyperacetylation compared to the control. Further investigations of the individual N-terminal H4 lysine residues using antibodies specific to acetylated lysine 5, 8, 12 or 16 revealed that all compounds, except tubastatin A, caused hyperacetylation using each antibody. No differential effect was observed for histone H3 acetylation, with all compounds causing an ~1.8-fold increased signal using an acetyl-H3 antibody. The new ELISA method developed here provides a higher throughput way to assess differential compound induced lysine acetylation alterations in P. falciparum and therefore represents a valuable new tool to aid the investigation of HDAC inhibitors for malaria. As discussed above, the lack of tools, such as recombinant P. falciparum HDAC proteins, crystal structures and homology models, has meant that the identification of antiplasmodial HDAC inhibitors has been limited to whole-cell screening approaches which can be time-consuming and costly. To begin to address this problem, quantitative structure-activity relationship (QSAR) models were developed based on logistic algorithms with the aim of providing a new tool to triage compounds for in vitro testing. A database of 457 antiplasmodial HDAC inhibitors was assembled with published data on PfIC50 and, for 292 of those compounds with data on plasmodial selectivity. Two independent prediction algorithms based on logistic regression were developed to classify (1) antiplasmodial activity or (2) selectivity of hydroxamate-based HDAC inhibitors. Seven different activity and five different selectivity models were built, each with individual decision cut-offs defining active/selective and non-active/unselective compounds (e.g. PfIC50: active compound <0.1 μM> non-active compound; SI: selective compound >100< unselective compound). Activity model A7 revealed the highest prediction performance by predicting 93% of the training compound set and 87% of the external test compound set correctly. Cross validation revealed a prediction accuracy of 91%. The most accurate selectivity model S4 demonstrated a slightly poorer prediction performance due to a much smaller initial data set as not all the HDAC inhibitors had reported selectivity information (64%). Despite this, the selectivity model demonstrated an internal prediction accuracy of 91%, a cross-validated (internal) prediction accuracy of 82% and an external prediction accuracy of moderate 72%. To validate the prediction performance of the activity model further, they were applied to a set of 22 experimentally untested compounds (validation set) and the prediction performance compared to their experimental antiplasmodial activity. Applying prediction model A7 to this compound set predicted three hit compounds (two of which were confirmed by experimental assay data) and 12 non-actives (confirmed for 11 based on experimental assay data). The experimental PfIC50 assessment revealed asexual blood-stage PfIC50s for the whole set in the nanomolar to micromolar range (PfIC50 0.006-8.45 μM; data from Dr MJ Chua), with the correctly predicted hits (S2_E10 and LD016) having PfIC50 <0.008 μM. Overall, virtual screen using QSAR model A7 identified 87% of the validation compounds correctly and revealed high prediction specificity, identifying 92% of the non-active compounds correctly. Due to a lack of available data sets with selectivity index information (and time constraints for this project), the selectivity models were not able to be tested with an external set. These activity and selectivity QSAR models are the first generated for antiplasmodial HDAC inhibitors. These models will aid the in silico assessment of antiplasmodial activity and selectivity of hydroxamate-based HDAC inhibitors and therefore represent useful new tools in the investigation of HDAC inhibitors for malaria. In summary, data presented in this thesis include the identification of novel antiplasmodial HDAC inhibitors with activity against asexual intraerythrocytic-stage P. falciparum parasites, in vivo data demonstrating oral cures in mice for two analogues of the anti-cancer HDAC inhibitor AR42, a new ELISA method to allow higher throughput assessment of HDAC inhibitor induced changes to histone lysine residues and the first antiplasmodial HDAC inhibitor QSAR models. HDAC inhibitors identified in this study with promising in vitro and in vivo antiplasmodial activity profiles are new starting points for further development of HDAC inhibitors for malaria. In addition, the in vitro and in silico approaches developed in this study are useful new tools to facilitate the discovery of HDAC inhibitors and the understanding of their biological effects on the parasite.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

Histone deacetylases (HDACs) are enzymes involved in the remodeling of chromatin. In recent years, inhibition of HDACs has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer. In fact, HDAC inhibitors (HDACi) promote apoptosis, induce cell cycle arrest and differentiation of tumor cells, by mechanisms which remain in part unknown. T-cell acute lymphoblastic leukemia (T-ALL) is a pediatric malignancy characterized by clonal expansion of lymphoid progenitors. Although the majority of pediatric T-ALL patients can be cured by current protocols, about one fourth of patients has chemotherapy-resistant disease or relapse after therapy and novel therapeutic approaches are required. In our study, we analyzed the effects of HDACi on seven transcription factors important in T-ALL pathogenesis (NOTCH1, NOTCH3, c-MYB, TAL1, TLX1, TLX3 and LMO2) using both established T-ALL cell lines and patient-derived T-ALL xenografts previously obtained in our laboratory. In particular, we focused on transcription factors that define specific T-ALL subgroups (TAL/LMO, TLX1, TLX3) and we included members of the Notch family (NOTCH1 and NOTCH3) and c-MYB in view of their transversal role in T-ALL. In vitro analysis highlighted transcriptional down-regulation of C-MYB and TAL1, a post-translation regulation of NOTCH1 and NOTCH3 and the regulation of the transcriptional activity of TLX1 and TLX3 following HDAC inhibition. These biochemical effects were linked to increased apoptosis and impaired proliferation both in T-ALL cell lines and patients-derived cells, partially dependent on NOTCH1 and NOTCH3. We next investigated the in vivo effects of an HDACi in T-ALL xenografts belonging to specific T-ALL subgroups. Interestingly, PD-TALL8 (TLX1) and PD-TALL16 (TLX3) had better response to treatment compared to PD-TALL12 and PD-TALL9 (TAL/LMO). In fact, the HDACi dramatically decreased leukemic cells infiltrating the spleen and the bone marrow in TLX-driven xenografts, whereas this drug had modest or minimal effects on TAL/LMO xenografts. Taken together, these results identify TLX1 and TLX3 T-ALL subgroups as potential candidates for therapeutic treatment with HDACi.
Le Istone Deacetilasi (HDACs) sono enzimi coinvolti nel rimodellamento della cromatina. Negli ultimi anni è emerso come l’inibizione delle HDACs potrebbe essere utilizzata come strategia per ripristinare l’alterata regolazione epigenetica che si riscontra nei tumori. Infatti, gli inibitori delle HDAC (HDACi) inducono apoptosi, arresto del ciclo cellulare e differenziamento delle cellule tumorali, ma i meccanismi molecolari alla base di questi fenomeni rimangono poco chiari. La leucemia linfoblastica acuta a cellule T (T-ALL) è un tumore pediatrico caratterizzato dall’espansione clonale di progenitori linfoidi. Nonostante la maggioranza dei pazienti pediatrici affetti da T-ALL siano curati in modo efficace utilizzando gli attuali protocolli terapeutici, circa un quarto dei pazienti manifesta resistenza alla terapia o presenta ricadute e dunque emerge la necessità di nuovi approcci terapeutici. In questo studio abbiamo analizzato gli effetti degli HDACi nei confronti di sette fattori di trascrizione implicati nella patogenesi della T-ALL (NOTCH1, NOTCH3, c-MYB, TAL1, TLX1, TLX3 and LMO2) utilizzando sia linee cellulari stabilizzate, sia modelli murini di T-ALL precedentemente sviluppati nel nostro laboratorio a partire da cellule di pazienti. In particolare, ci siamo concentrati su fattori trascrizionali che identificano specifici sottogruppi di T-ALL (TAL/LMO, TLX1 e TLX3) e abbiamo incluso nell’analisi due membri della famiglia dei recettori Notch (NOTCH1 and NOTCH3) e c-MYB in virtù del loro ruolo oncogenico in questa patologia. Le analisi in vitro hanno evidenziato diversi meccanismi di regolazione dei vari fattori da parte degli HDACi. TAL1 e c-MYB risultano regolati a livello trascrizionale, NOTCH1 e NOTCH3 presentano una regolazione post-traduzionale e, nel caso di TLX 1 e TLX3, è presente una regolazione diretta della loro capacità trascrizionale. Gli effetti a livello di proteina si legano all’induzione di apoptosi e all’inibizione della proliferazione sia nelle linee cellulari, sia nelle cellule derivate da paziente e risultano essere parzialmente dovute alla down-modulazione di NOTCH1 e NOTCH3. In seguito siamo andati ad indagare la risposta in vivo di un HDACi in xenografts di T-ALL appartenenti a specifici sottogruppi genetici. E’ interessante notare che il trattamento ha avuto il maggiore risultato nelle PD-TALL8 (TLX1) e nelle PD-TALL16 (TLX3) rispetto alle PD-TALL12 e le PD-TALL9 (entrambe TAL/LMO). Infatti, il trattamento con HDACi negli xenografts di tipo TLX determina una riduzione dell’infiltrazione da parte delle cellule leucemiche nella milza e nel midollo mentre gli effetti ottenuti negli xenografts TAL/LMO risultano modesti o addirittura nulli. In conclusione, i dati ottenuti identificano i pazienti di T-ALL appartenenti ai sottogruppi TLX1 e TLX3 come potenziali candidati per il trattamento a scopo terapeutico con HDACi.

Le récepteur des oestrogènes (RE) peut moduler l’expression de gènes impliqués dans les processus de prolifération et d’apoptose cellulaires. Cette régulation est possible par le recrutement de complexes corégulateurs. Dans ces complexes, l’activité répressive s’explique essentiellement par la présence d’histones désacétylases (HDAC). Cette famille de protéines est composée de 18 membres classés en 4 groupes. Cette répartition est due aux similarités structurales et de fonctions de ces enzymes. Il y a la classe I (HDAC 1, -2, -3, -8), la classe II (HDAC 4, -5, -6, -7, -9, -10) et la classe IV (HDAC 11) qui ont une activité Zn2+ dépendante alors que la classe III (Sirt1-7) recense les HDAC avec une activité NAD+ dépendante. Des résultats récents du laboratoire ont montré, qu’au niveau ARNm, il y avait un important différentiel d’expression de HDAC9 entre les lignées cellulaires de cancer du sein RE positive et négative ou résistante au tamoxifène. Durant ma thèse, j’ai démontré que la régulation de HDAC9, au niveau de son expression, comme au niveau de ses fonctions, affecte la signalisation oestrogénique en modulant l’expression et l’activité transcriptionnelle de REα. De plus, de nombreuses études ont montré l’activité antiangiogénique d’inhibiteurs de HDAC (HDI) à large spectre comme la TSA (Tricostatin A). La conception et l’identification de HDI, potentiellement sélectifs, comme agents anti-tumoraux et/ou anti-métastatique représente une nouvelle approche de thérapie seule ou combinée avec les produits déjà utilisés dans le traitement du cancer. Ainsi, afin d’identifier et caractériser de nouveaux HDI, j’ai établi un outil bioluminescent pour la détection d’inhibiteurs sélectifs de HDAC. Plusieurs lignées cellulaires Gal4-VP16-HDAC ont été générées dans ce but
The estrogen receptor (ER) can modulate the gene expression with consequences in the cell proliferation, apoptosis. This modulation is possible by the recruitment of coactivator or corepressor complexes. The repression activity is in particular explained by the histones deacetylases (HDACs). This protein family is composed by eighteen members who have been classified in four groups. These HDACs are subdivided on structural and functional similarities. The class I isoforms (HDACs 1, 2, 3 and 8), class II (HDACs 4, 5, 6, 7, 9, 10) and class IV (HDAC11) are Zn-dependent enzymes, whereas class III HDACs (Sirtuins 1-7) are NAD+-dependent. Recent data from the laboratory have shown, at the mRNA level, there is an enormous expression differential of HDAC9 between breast cancer cell line ER positive and negative or OHT resistant cell line. During my thesis, I demonstrated that the regulations of the HDAC9 on the level of its expression as of its role in the various breast cancer cell lines were implicated in the estrogen signaling. This regulation takes place at the transcriptional level and in the ERet#945; activity.In addition, using broad spectrum HDAC inhibitors (HDIs) such as TSA (Tricostatin A), many studies have shown that these inhibitors had antiangiogenic activity. Thus, the design or the identification of selective and potent HDAC inhibitors as agents anti-tumoral and/or anti-metastatic can emerge in a novel opportunity used alone or in combination with the already existing agents for the treatment of cancers. In order to identify and characterize new HDIs, my thesis works consisted to establish bioluminescent cell lines for screening HDAC inhibitors. Different cell GAL4-VP16-HDACs chimeras' models were generated to determine the selectivity of HDIs for the different HDACs

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease requiring a genetic predisposition coupled with an environmental trigger in order for initiation of disease. While the exact pathoaetiology has yet to be determined, both B and T cell dysregulation are thought to contribute to disease. Histone deacetylases (HDACs) are a class of enzymes that hydrolyze the lysine bound acetyl group in both histone and non-histone proteins thereby altering protein structure and function. While the use of pan-HDAC inhibitors has proven to be effective for the treatment of a number of acute diseases, they may not be viable as therapeutics for chronic disease due to cytotoxicity and adverse side effects following long term treatment. We sought to determine whether treatment with a class I and II HDAC inhibitor (HDACi) or a specific HDAC6i would be able to ameliorate disease in lupus-prone NZB/W mice. We found that both the class I and II HDACi (ITF2357) and the HDAC6i (ACY-738) were able to decrease SLE markers of disease including splenomegaly, proteinuria, and anti-dsDNA and IgG production in the sera. Treatment with ITF2357 resulted in an increase in the number of immunosuppressive regulatory T (Treg) cells and a decrease in the pro-inflammatory Th17 phenotype. Furthermore, ITF2357 was found to increase Foxp3 acetylation leading to increased Foxp3 stability allowing for differentiation into the Treg phenotype. ACY-738 treatment was able to correct aberrant bone marrow B cell differentiation while also increasing the number of splenic Treg cells in NZB/W mice. These results suggest that HDAC inhibition is able to ameliorate SLE in NZB/W mice by altering aberrant T and B cell differentiation. Additional studies were performed to further examine the expression and function of different HDAC isoforms in immune cells. Due to the ability of HDAC inhibition to decrease markers of SLE disease as well as alter B and T cell development and differentiation, we sought to determine if specific HDAC isoforms are altered in lupus vs non lupus mice in early and late disease states. We determined the level of class IIb HDAC (HDACs 6, 9, and 10) expression in bone marrow B cells, splenic B and T cells, and glomerular cells from early- and late-disease MRL/lpr lupus-prone mice compared to healthy, age-matched C57BL/6 control mice. Expression of HDAC6 and HDAC9 were significantly increased in all of the tissues tested from MRL/lpr mice. Furthermore, both cytoplasmic and nuclear HDAC activity was increased in diseased MRL/lpr mice, and HDAC activity and expression continued to increase as disease progressed. In vitro treatment with ACY-738, a selective HDAC6i, was able to decrease cytoplasmic HDAC activity and inhibit iNOS production. Furthermore, ACY-738 was able to alter apoptosis through increased Bax expression in B cells. Treatment with ACY-738 was also able to inhibit Hsp90 expression and decrease NF-κB nuclear translocation, which are both upregulated during active SLE. Our studies indicate that HDAC activity contributes to SLE pathogenesis and that the use of isoform-selective HDAC inhibitors may be a viable treatment for SLE.
Ph. D.

Les HDACs humains consistent en une famille de 18 membres différents groupés dans quatre classes. Classe I (HDAC 1-3. 8), classe II (HDAC4-7, 9,10 dont HDAC4, 5, 7 et 9 forment un sous-groupe dû à une organisation structurale commune, alors que HDAC6 est membre de classe IIb), classe III, également désignée sous le nom des sirtuines (SIRT1-7) et classe IV (HDAC11). Les classes I, II et IV partagent des caractéristiques communes, car tous leurs membres sont zinc-dépendants et montrent quelques similitudes de séquence, alors que dans la classe III HDACs il s’agit d’enzymes NAD+-dépendant sans homologie aux autres HDACs. Les pan-inhibiteurs tels que SAHA, qui est actuellement dans des essais cliniques en phase III et a été récemment approuvé pour le traitement du lymphome à cellule T cutané, bloquent les enzymes de classes I et II, alors que le MS275 est un inhibiteur sélectif de la sous-classe I et bloque les activités de HDAC 1, 2 et beaucoup moins efficacement HDAC 3. Des inhibiteurs sélectifs de la classe II ont été également produits, permettant ainsi la dissection des diverses activités de HDACs. Notamment, alors que l'induction de TRAIL semble être associée à l'inhibition des enzymes de la classe I dans les systèmes cancéreux, d'autres fonctions cellulaires dépendent de l'action des HDCAs classe II, comme la régulation de la différentiation, principalement la différentiation cardiaque. HDACis dans des systèmes de différentiation : C2C12 cellules, F9 cellules, cellules 3T3L1. Nous avons examiné MC1568, un nouvel inhibiteur des HDACs, spécifique pour la classe II, dans un modèle leucémique, les cellules U937, et divers systèmes de différentiation, tels que les cellules C2C12 pour la différentiation de muscle, les cellules F9 pour la différentiation endodermale et les cellules 3t3L1 pour la différentiation des adypocytes. De façon intéressante, nous avons constaté que dans les cellules C2C12 l'inhibiteur des HDAC classe 2 bloque l'action catalytique de la classe 2 de HDAC, mais stabilise l'interraction entre le facteur transcriptionnel MEF2D et HDAC4. MEF2D est le principal facteur transcriptionnel, responsable de la différentiation terminale de muscle, et par conséquent l’inhibition de son activité transcriptionnel bloque la différentiation du muscle. D'une manière semblable, dans les deux autres systèmes nous avons évalué l'inhibition de la différentiation et nous avons trouvé que l’activité transcriptionnelle de RAR et PPARγ, essentiels pour la différentiation des deux systèmes respectivement, a été bloqué in vitro dans le premier cas et in vivo dans le deuxième cas. Dans le cas de la différentiation induite par PPARγ, on a utilisé le système des cellules 3T3L1. Le traitement de ces cellules avec Troglitazone induit la différentiation d'adypocytes, alors que la présence du MC1568 le bloque complètement. En analysant le niveau de l'expression de PPARγ pour RT-PCR, nous avons constaté que son niveau a été fortement diminué après traitement avec MC1568. De façon intéressante, le traitement d'une souris exprimant stablement le gène de la luciferase sous le contrôle de l’élément de réponse de PPAR (PPRE-Luc), a bloqué l'activité transcriptionnelle de PPARγ, prouvant que le composé régule l'activité transcriptionnelle de récepteur nucléaire et sa transactivation. Inhibition des HDACs classe II dans des modèles de cancer - Cellules MCF7 Dans des systèmes de cancer, tels que les cellules MCF7 nous avons constaté que l'inhibiteur des HDACs classe 2, MC1568, induisait la sumoylation de HDAC4 ce qui augmente l’activité catalytique de HDAC4 comme cela a été précédemment démontré. La transfection d'un mutant spécifique empêchant la sumoylation de HDAC4 en cellules F9 a diminué la capacité de répression de RAR sur un de ces gènes cibles, le collagène IV. Nous avons démontré que le traitement avec MC1568 régule l'activité transcriptionelle des deux facteurs de transcription, RAR alpha et NF-KB, sur trois gènes cibles, IRF1, TNF et TRAIL. La sumoylation de HDAC4 par RANBP2 réprime transitoirement l'expression de ces gènes cibles. Cette répression est perdue lorsque HDAC4 est dégradé. Leucémie promyelocitaire aigüe (APL), cellules NB4 Les cellules NB4 sont un système cellulaire de leucémie promyelocitaire aigüe (APL). Cette maladie est caractérisée par la présence d'une protéine de fusion, PMLRAR alpha, avec une forte activité de répression due au recrutement augmenté des complexes corépresseurs. Les patients sont soignés avec la thérapie de différentiation par de l'acide rétinoïque (ATRA). Même si le traitement est efficace, il reste toujours les problèmes de résistance et toxicité, causés par les traitements plus longs, qui sont à l’origine du syndrome d'ATRA et des effets tératogènes. Dans les cellules NBA, nous avons constaté que la combinaison d’ATRA et MC1568 agit de manière synergique, induisant une mort indépendante de caspases. L'inhibiteur ZVAD (un pan-inhibiteur pour toutes les caspases) ne bloque pas la mort induite par le MC1568 seul ni par la combinaison des deux composés. La voie des caspases activée a été principalement celle de la caspase 8. En même temps, nous avons trouvé une importante libération du cytochrome C, démontrant la participation de la voie de mort intrinsèque dans la mort induite par ces deux composés. L'utilisation d'un inhibiteur spécifique, le NAC, réduit la mort induite par ATRA et MC1568, prouvant que la voie engagée est celle du NAC. Sachant qu'un inhibiteur de HDAC classe I comme le MS275 induit dans le même système une mort cellulaire caspase-dépendante, principalement via TRAIL, notre observation suggérerait que l'inhibition des HDAC classe 2 active une voie qui est principalement caspase-indépendante. Conclusions Dans mon travail de thèse j'ai évalué l'effet d'un inhibiteur sélectif des HDAC classe II dans plusieurs systèmes et j’ai défini ses effets sur la différenciation et l’apoptose. Cette analyse sera utile pour générer des nouveaux inhibiteurs sélectifs pour les HDACs pour le traitement contre le cancer avec un indice thérapeutique plus élevé que les pan-inhibiteurs des HDACs actuels
Human HDACs comprise a family of 18 different members which are grouped into four classes. Class I (HDAC 1-3,8), class II (HDAC4-7,9,10 of which HDAC4, 5, 7 and 9 form a class II a subgroup due to a common structural organization, while HDAC6 is member of class IIb), class III, also referred to as sirtuins (SIRT1-7) and class IV (HDAC11). Classes I, II and IV HDACs share common features, as all their members are zinc-dependent and exhibit some sequence similarities, while class III HDACs are NAD+-dependent enzymes without homology the other HDACs(1). Pan-inhibitors like SAHA, which is currently in phase III clinical trials and has recently been approved for treatment of cutaneous T cell lymphoma, inhibit both classes I and II enzymes, while MS275 is a subclass I selective inhibitor, which blocks the activities of HDAC 1, 2 and much less efficiently HDAC 3. Also class II selective inhibitors have been generated, marking the onset of a dissection of the various activities of HDACs. Notably, while induction of TRAIL appears to be associated with inhibition of class I enzymes in cancerous systems, other cellular functions involve the action of class II HDACs, like the regulation of the differentiation, mainly the cardiac differentiation. In differentiation systems: C2C12 cells, F9 cells, 3T3L1 cells. We tested a new compound HDAC inhibitor, specific for HDAC class 2 in one leukemic model, U937 cells, and various differentiation systems, such as the C2C12 cells for muscle differentiation, the F9 cells for endodermal differentiation and 3t3l1 cells for adypocyte differentiation. Interestingly we found that in C2C12 cells the HDAC class 2 inhibitor blocked the catalytic action of the HDAC class 2, but stabilized the interaction between the transcriptional factor MEF2D and HDAC4. MEF2D is the main transcriptional factor, responsible for the terminal muscle differentiation, and its transcriptional activity inhibition consequently blocked the muscle differentiation. In a similar way in both the other two systems we evaluated the differentiation inhibition and we found that transcriptional activity of RAR and PPARγ, respectively essential for the differentiation of the two systems, was blocked either in vitro in the first case either in vivo in the second case. In the case of the PPARγ induced differentiation, the experiments were assayed in the 3T3L1 cells. The treatment of these cells with Troglitazone induced adypocyte differentiation, while the presence of the MC1568 blocked it completely. Analyzing the Pparg’s expression level for RT-PCR, we found that its level was highly decreased after treatment with the MC1568. Interestingly the treatment of a mouse stably expressing a PPRE-luc, PPAR responsive elements luciferase reporter, blocked the transcriptional activity of PPARγ, showing in this case that the compound was regulating both the nuclear receptor’s transcriptional activity and the transactivation. In tumour model: MCF7 cells In cancer systems, such as MCF7 cells we found that the HDAC class 2 inhibitor MC1568 was inducing HDAC4 sumoylation, that, as previously showed (2) increases the HDAC4 catalytic action. The transfection of a specific HDAC4 sumoylation mutant in F9 cells decreased the repressory power on a note RAR target gene, Collagen IV. We were able to demonstrate that the treatment with MC1568 was regulating the activity of two transcriptional factor, RAR alpha and NF-KB, on three target genes, IRF1, TNF and TRAIL. The HDAC4 sumoylation mediated by RANBP2 repressed transiently the expression of these target genes, that were up-regulated after the complete degradation of HDAC4. Acute promyelocitic leukemia (APL), NB4 cells The NB4 cells is a cellular system of acute promyelocitic leukemia (APL). This disease is characterized by the presence of a fusion protein, PMLRAR alpha, with a strong repressory activity, mediated by the enhanced recruitment of corepressory complexes. The patients are treated with the differentiation therapy with retinoic acid (ATRA). Even if it is quite efficient to treat it, it still remains the problem of resistances, recidives and toxicity, done by the longer treatments, that give the, as said, ATRA syndrome and teratogenicity. In these cells we found that the combination among ATRA and MC1568 seems synergic, inducing a caspase independent cell death, even if in presence of caspases activation. The ZVAD inhibitor (pan-inhibitor for all caspases) failed to block the death induced by the MC1568 and by the combination among the two compounds. Analyzing the activation of the caspases it seemed that the pathway activated was mainly mediated by caspase 8. At the same time we found a strong release of cytochrome C, demonstrating the involvement of the mitochondria in the death induced by these two compounds. The use of a specific inhibitor, the NAC inhibitor, was able to reduce the death mediated by ATRA and MC1568, showing that the pathway was mainly regulated by the NAC dependent way. At this time, we are arrived to select the pathway regulated by this compound. Knowing that an HDAC inhibitor like MS275, specific inhibitor for class 1 HDACs, induce in the same system a caspase-dependent cell death, mainly regulated by TRAIL3, our observation would suggest that the class 2 HDAC inhibition is specifically regulating a pathway that is mainly caspase-independent. This observation could be usefull for treatment therapy, not only ammeliorating the treatment with retinoids, but overcoming possible resistances mediated by mutations and alteration in receptor expression in tumoral cells. Conclusions In my work I have evaluated the effect of a class 2 HDAC inhibitor in several systems, differentiative and cancerous, highlighting its effects in transcriptional regulation and cell death
Nell’uomo gli enzimi de acetilanti gli istoni appartengono ad una famiglia di 18 membri che sono raggruppati in 4 classi. La classe I (HDAC1-3,8), classe II (HDAC4-7,9,10 di cui HDAC4, 5,7,9 formano un subgruppo dovuta ad una comune organizzazione strutturale, mentre HDAC6 è un membro della classe 2), classe III, denominata come sirtuine (SIRT1-7) e classe IV (HDAC11). Le HDAC appartenenti alle classi I, II, IV sono comunemente dipendenti dallo zinco, mentre le HDAC appartenenti alla classe III sono enzimi dipendenti dal NAD +(1). SAHA, un pan inibitore delle HDAC, inibisce entrambi le classi I e II. Attualmente è in phase III clinical trials ed è stato recentemente approvato per il trattamento del linfoma cutaneo delle cellule T. MS275, invece, è un inibitore selettivo per la subclasse I, bloccando preferenzialmente HDAC1,2. Recentemente sono stati sintetizzati anche inibitori selettivi per la classe II, rendendo possibile lo studio delle varie funzioni delle differenti classi delle HDAC. Come è noto, mentre l’induzione di TRAIL sembra essere associata con l’inibizione di enzimi classe I in sistemi cancerosi, le HDAC di classe II sembrano essere coinvolte in altra funzioni cellulari, come la regolazione del differenziamento, principalmente quello cardiaco. Inibitori delle HDAC in sistemi differenziativi: le cellule C2C12, le cellule F9 e le cellule 3T3L1. Abbiamo testato un nuovo composto inibitore HDAC, specifico per la classe 2 in un modello leucemico, le cellule U937, e vari sistemi differenziativi, come le cellule C2C12 per il differenziamento cardiaco, le cellule F9 per il differenziamento endodermico e le cellule 3T3L1 per il differenziamento adipocitario. Abbiamo scoperto che nelle cellule C2C12 l’inibitore HDAC classe 2 stabilizzò l’interazione tra il fattore trascrizionale MEF2D ed HDAC4, pur bloccando l’azione catalitica delle HDAC classe II. MEF2D è il principale fattore trascrizionale resposabile del differenziamento cardiaco terminale e l’inibizione della sua attività trascrizionale conseguentemente bloccò il differenziamento muscolare. Negli altri due sistemi, similmente, noi abbiamo riscontrato l’inibizione del differenziamento ed abbiamo trovato che l’attività trascrizionale di RAR e PPARγ, essenziali per il differenziamento dei relativi sistemi differenziativi, fu bloccata sia in vitro nel primo caso che anche in vivo nel secondo. Nel caso del sistema differenziativo indotto da PPARγ, gli esperimenti furono fatti nelle 3T3L1. Il trattamento di queste cellule con Troglitazione indusse il differenziamento adipocitario, mentre la presenza dell’MC1568 lo bloccò completamente. Analizzando il livello di espressione di PPARγ per RT-PCR, abbiamo ritrovato che il suo livello era altamente decrementato dopo trattamento con MC1568. Abbiamo notato inoltre che il trattamento di un topo transgenico esprimente stabilmente il PPRE-luc, reporter luciferasi degli elementi responsivi a PPAR, bloccò completamente l’attività trascrizionale di PPARγ, mostrando in questo caso che il composto regolava sia l’attività trascrizionale del nuclear receptor sia il suo livello di espressione. In modelli cancerosi: le cellule MCF7 In sistemi cancerosi, così come le cellule MCF7 abbiamo riscontrato che lo specifico inibitore delle HDAC di classe II induceva la sumoilazione di HDAC4, che, come previamente mostrato (2) incrementa l’azione catalitica di HDAC4. La trasfezione di un mutante di HDAC4 specifico per il sito di sumoilazione nelle cellule F9 decrementò il potere repressorio su un noto target di RAR, CollagenIV. Abbiamo dimostrato che il trattamento con MC1568 regolò l’attività di due fattori trascrizionali, quali RAR alpha ed NF-KB, su tre geni target, IFR1, TNF e TRAIL. La sumoilazione di HDAC4 mediata da RANBP2 represse in modo transiente l’espressione di questi geni target, che furono up-regolati dopo la completa degradazione di HDAC4. Leucemia promielocitica acuta (APL), NB4 cells Le cellule NB4 è un sistema cellulare di leucemia promielocitica acuta (APL). Questa malattia è caratterizzata dalla presenza di una proteina di fusione, PMLRAR alpha, con un forte attività repressoria. I pazienti sono attualmente curati con la terapia differenziativa con acido retinoico (ATRA). Anche se è un trattamento molto efficiente, vi sono ancora casi di recidività, resistenze e tossicità al trattamento, dovuto principalmente ai trattamenti prolungati, dando luogo alla cosiddetta ATRA sindrome e teratogenicità. In queste cellule abbiamo scoperto il sinergismo tra ATRA e MC1568. La combinazione delle due droghe ha indotto una morte caspasi-indipendente, anche se in presenza di attivazione delle caspasi. L’inibitore ZVAD, infatti, fallì a bloccare la morte indotta dall’MC1568 e dalla combinazione tra i due composti. Analizzando l’attivazione delle caspasi, abbiamo riscontrato che il pathway maggiormente attivato è quello della caspasi 8. Allo stesso tempo abbiamo ritrovato un forte rilascio del citocromo C, dimostrando il coinvolgimento del mitocondrio nella morte indotta da questi due composti. L’uso di un inibitore specifico dei reattivi dell’ossigeno, N-acetyl-cisteina, ridusse significativamente la morte indotta, definendo il ruolo chiave dei ROS. Sapendo che un inibitore delle HDAC quale MS275, un inibitore specifico delle HDAC di classe 1, inducesse nello stesso sistema una morte caspasi dipendente, principalmente regolata da TRAIL3, la nostra osservazione suggerirebbe che l’inibizione specifica delle HDAC di classe II regola specificamente un pathway caspasi-indipendente, utile per migliorare il trattamento con retinoidi, ma soprattutto per curare quei pazienti con resistenze dovute a mutazioni o alterazione nella espressione del recettore in cellule tumorali. In Conclusione Nel mio lavoro ho studiato l’effetto di un inibitore specifico delle HDAC di classe II in svariati sistemi, differenziativi e cancerosi, concentrandomi sugli effetti nella regolazione trascrizionale e la morte cellulare

Histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated a wealth of biological effects, including anti-proliferative, anti-inflammatory, anti-parasitic, and cognition-enhancing activities. The recent FDA approvals of the inhibitors SAHA and FK-228 have validated HDACi clinical use in cutaneous T cell lymphoma, while numerous clinical trials are currently ongoing using HDACi against a variety of disease states. While the future of the HDAC field looks increasingly promising, there are lingering issues hindering broader use. Recent data point to dysregulation of specific HDAC isoforms in many disease states. However, most current HDACi are pan-inhibitors, lacking the specificity to target individual isoforms. Adding to this, there are currently 18 identified HDAC isoforms, and most lack a defined crystal structure, further complicating the task of designing isoform-specific inhibitors. Most importantly, HDACi have demonstrated a lack of efficacy against solid tumors in the clinic, a major obstacle to broader use in cancer therapy. Several of these issues could more fully be addressed through specific targeting of HDACi, and could bring HDACi into wider and more efficacious pharmaceutical use. Targeting the specific tissue or organelle where HDAC dysregulation occurs could confer greater efficacy in vivo. To this end, we have created four classes of compounds: (1) aryltriazolyl HDACi that potently inhibit HDAC activity and prostate cancer cell growth, (2) dual-targeted inhibitors of Topoisomerase II and HDAC and (3) dual-targeted inhibitors of Topoisomerase I and HDAC, both of which have potent inhibition against both target enzymes as well as cancer cell lines, and finally (4) macrocyclic HDACi that potently inhibit the growth of lung cancer cell lines and preferentially target lung tissue in vivo.

Friedreich ataxia (FRDA) is an inherited autosomal recessive neurodegenerative disorder caused by a GAA trinucleotide repeat expansion mutation within the first intron of the FXN gene. Normal individuals have 5 to 30 GAA repeats, whereas affected individuals have from approximately 70 to more than 1,000 GAA triplets. In addition to progressive neurological disability, FRDA is associated with cardiomyopathy and an increased risk of diabetes mellitus. Currently there is no effective therapy for FRDA and this is perhaps due to the lack of an effective system to test potential drugs. Therefore, the main aim of this thesis is to develop a novel cell culture system, to aid in rapid drug screening for FRDA. Firstly, I have demonstrated the establishment of novel cell culture systems, including primary fibroblasts, neural stem cells (NSC) and splenocytes, from FRDA YAC transgenic mouse models (YG8 and YG22). Then, I have shown the differentiation of NSCs into neurons, oligodendrocytes and astrocytes. The presence of these cells was confirmed by using cell specific immunofluorescence assays. I have also shown that both YG8 and YG22 rescue mice have less tolerance to hydrogen peroxide induced oxidative stress than WT mice, as similarly seen in FRDA patient fibroblasts. Recent findings indicate that FRDA is associated with heterochromatin-mediated silencing of the FXN gene accompanied by histone changes, flanking the GAA repeats. This suggested potential therapeutic use of compounds which can reduce the methylation and increase the acetylation of histone proteins. Therefore, using human and mouse primary fibroblast cell lines I have investigated the efficacy and tolerability of various DNA demethylating agents, GAA interacting compounds and class III histone deacetylase (HDAC) inhibitors. Although DNA demethylating agents showed increased FXN expression, no correlation between the level of DNA methylation and FXN expression was identified. Nevertheless, the use of GAA interacting compounds, particularly DB221, and the HDAC inhibitor, nicotinamide, have shown encouraging results, provoking us to use such compounds in future long-term in vivo studies. In addition, I have also investigated the long-term efficacy of two benzamide-type HDAC inhibitors, RGFA 136 and RGFP 109, on the FRDA YAC transgenic mice. No overt toxicity was identified with either drug, indicating a safe administration of these compounds. Both compounds produced improved functional analysis together with significantly reduced DRG neurodegeneration. However, neither of these compounds was shown to significantly increase the FXN mRNA expression. Nevertheless, elevated levels of frataxin protein in the brain tissues were obtained with RGFP 109, suggesting that RGFP 109 is capable of crossing the blood-brain barrier. I have also found increased levels of global acetylated H3 and H4 histone proteins in brain tissues, along with significant increase in aconitase enzyme activity, particularly with RGFP 109 treatments. Overall, these results support future clinical trial development with such compounds.

NAD+-Dependent histone deacetylases, also called sirtuins, are a family of enzymes that catalyse the cleavage of acetyl groups from lysine amino acid residues in histones and a variety of nonhistone proteins, playing a critical role in numerous biological processes. They have been found to be deregulated in several age-related diseases such as cancer and neurological disorders. Consequently, their role as therapeutic targets is widely explored. To date, seven different sirtuins (SIRT1-7) have been found in mammals, with SIRT1 and SIRT2 being the most studied. Nicotinamide (1), EX-527 (2) and 2-anilinobenzamide (3) have been reported as SIRT inhibitors (Figure 1). In an effort to discover alternative and selective nicotinamide pocket (C-pocket) sirtuin inhibitors, we decided to prepare and screen a small-molecule compound library based around the 10,11-dihydro-5H-dibenzo[b,f]azepine scaffold (4), structurally related to the above known SIRT inhibitors. Successfully, this approach led us to the identification of novel, highly selective low-micromolar SIRT2 inhibitors. In vitro screening assays revealed our best hit (5) to have an IC50 of 18 !M against SIRT2 and high selectivity over the SIRT1 isoform (Figure 2). Cellular screening assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed compound 5 to have antiproliferative activity at a concentration of 30 !M. Finally, docking studies with compound 5 were performed to predict its binding mode at the SIRT2 catalytic site and rationalise the observed isozyme Successfully, this approach led us to the identification of novel, highly selective low-micromolar SIRT2 inhibitors. In vitro screening assays revealed our best hit (5) to have an IC50 of 18 !M against SIRT2 and high selectivity over the SIRT1 isoform (Figure 2). Cellular screening assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed compound 5 to have antiproliferative activity at a concentration of 30 !M. Finally, docking studies with compound 5 were performed to predict its binding mode at the SIRT2 catalytic site and rationalise the observed isozyme selectivity. Further biological evaluation of hit molecule 5 is currently underway. 2) Towards the Discovery of Novel HDAC4 Inhibitors The class IIa HDAC4 enzyme is under extensive investigation, since its cellular functions have potential implications in human disease. Recently, it has been found that HDAC4 levels are significantly increased in platinum-resistant ovarian cancer cells and silencing of HDAC4 in those cell lines restores platinum sensitivity. Inhibition of this HDAC isozyme with pharmacological intervention could therefore potentially reverse clinically acquired platinum resistance in cancer cells. Hence, selective HDAC4 inhibitors have the potential to represent a novel class of anticancer agents, useful in combination therapy with other HDAC inhibitors or conventional chemotherapeutic agents. To date, only a limited number of HDAC4 inhibitors have been discovered and they all show dose-limiting toxicity, poor pharmacokinetics and metabolic instability, mainly due to the presence of reactive zinc-binding groups, such as the trifluoromethylketone and the hydroxamic acid moieties (Figure 3). In order to identify novel, non-toxic and selective HDAC4 inhibitors, we considered a novel molecular framework, bearing an alternative, drug-like zinc-binding group. A small compound library was synthesised and evaluated for its potential to inhibit the HDAC4 isoform, and restore platinum sensitivity in two different types of platinum-resistant ovarian cancer cell lines: PEO4 and SKOV3. Disappointingly, functional screens showed no HDAC4 inhibitory activity for the newly designed compounds, however a few molecules were found to be active in cellular assays. Remarkably, compounds 10 and 11 (Figure 4) were able to resensitise PEO4 and SKOV3 platinum-resistant ovarian cancer cell lines at low-micromolar concentrations, without showing any general cytotoxicity, even at concentrations up to 100 μM. Additional biological investigation is planned, with the aim to gain insights into the molecular mechanisms underlying this novel class of compounds.

HDAC inhibitors are known to have anti-inflammatory properties. HDAC inhibitors are used in combination with Oct4 to generate induced pluripotent stem cells. I hypothesized that polyesters based on simple aliphatic HDAC inhibitors like valproic acid (VPA) and phenylbutyric acid (PBA) can serve as alternatives to existing polyester biomaterials with improved anti-inflammatory properties and as scaffolds for generation of iPSCs when used in combination with layer-by-layer thin films delivering reprogramming transcription factors. Vinyl ester of phenylbutyric acid (VEPA) and vinyl ester of valproic acid (VEVA) were synthesized from their carboxylic acid precursors using an iridium complex catalyst at yields as high as 97% and 73%, respectively. Amorphous poly(VEPA) and poly(VEVA) polymers were prepared by free radical solution polymerization and characterized for molecular weight and glass transition temperature. Poly(VEPA) and poly(VEVA) microparticles of 20-40 um diameter were prepared by an emulsion-solvent evaporation method and examined under scanning electron microscopy (SEM). Their hydrolytic degradation was studied by dry weight loss and HDAC inhibitor release via high performance liquid chromatography (HPLC) in the presence of varied pH and lipase-containing buffers. No significant degradation occurred within 5 days, and an MTT assay conducted on HeLa cells in the presence of these microparticles confirmed an absence of cytotoxicity. Poly(VEPA) and poly(VEVA) microparticles were not found to be a suitable biomaterial for hypothesized applications in light of their poor degradation characteristics in vitro.

Hohe Telomeraseaktivität bedingt durch genomische TERT-Rearrangements definiert eine Gruppe an Hochrisiko-Neuroblastompatienten mit ungünstiger Prognose. Das Abzielen auf Telomerase ist ein hochpriorisierter Ansatzpunkt in der Therapie, für die es bislang keine klinisch erfolgreichen Inhibitoren gibt. Der Einsatz von epigenetisch wirksamen Histondeacetylase Inhibitoren (HDACi) stellt dabei eine interessante Therapieoption dar. In TERT-rearrangierten Neuroblastomzellen erzielte die Behandlung mit verschiedenen pan-, Klasse I oder spezifischen HDAC1/2 Inhibitoren eine Supprimierung der TERT mRNA Expression und der Telomeraseaktivität. RNA-Interferenz Studien bestätigten, dass HDAC1 und HDAC2 die TERT Expression positiv regulieren. Die transiente Überexpression von TERT zeigte einen partiellen Rescue des HDACi-bedingten anti-proliferativen Effekts. Der präventive und therapeutische Einsatz von HDACi Panobinostat verlangsamte das Xenografttumorwachstum, die TERT-Expression und Telomeraseaktivität in subkutanen NMRI-Foxn1nu/nu Mausmodellen des TERT-rearrangierten Neuroblastoms bei klinisch relevanten Dosen. Dies zeigt das translationale Potential und die klinische Durchführbarkeit der Panobinostat-Behandlung. ChIP Sequenzierung und Methylierungsanalyse zeigten keine bedeutenden Unterschiede der Histonmodifikationen und der Methylierung von CpG Dinukleotiden am TERT Lokus nach Panobinostatbehandlung. Die Inhibierung der de novo RNA Synthese zeigte, dass die Stabilität des TERT mRNA Transkripts nach Panobinostatbehandlung verringert war. Dies deutet darauf hin, dass die reduzierte Transkriptstabilität der zugrundeliegende molekulare Mechanismus ist. Zusammenfassend konnte gezeigt werden, dass die hohe Telomeraseaktivität in TERT-rearrangierten Neuroblastommodellen durch den Einsatz zugelassener HDACi supprimiert werden kann.
Telomerase activation by genomic TERT-rearrangements defines a subgroup of high-risk neuroblastomas with adverse outcome. Accordingly, telomerase activity presents a high-priority drug target with no currently available clinical inhibitors. It was assessed whether telomerase activity could be inhibited through histone deacetylase (HDAC) inhibition in models of TERT-rearranged neuroblastoma. Treatment with a panel of seven pan-, class I- or specific HDAC1/2 inhibitors suppressed TERT mRNA expression and telomerase activity in TERT-rearranged neuroblastoma cells at clinically achievable concentrations. RNA interference-based studies confirmed that HDAC1 and HDAC2 positively regulate TERT transcript levels. Enforced TERT expression partly rescued the anti-proliferative effect of HDAC inhibition indicating a causal role of TERT suppression in the HDAC inhibitormediated tumor-suppressive phenotype. Panobinostat treatment, in preventive and therapeutic settings, considerably attenuated tumor growth in subcutaneous TERT-rearranged neuroblastoma xenograft models in NMRI-Foxn1nu/nu mice and suppressed TERT transcript levels and telomerase activity at clinically relevant doses, thus demonstrating translational potential and clinical feasibility. ChIP sequencing detected no major differences in the chromatin context of the TERT locus between HDAC inhibitor-treated and control cells. Likewise, HDAC inhibition did not substantially alter the methylation profile in the TERT region. Blocking de novo RNA synthesis, however, reduced TERT mRNA transcript levels in HDAC inhibitor-treated cells, suggesting reduced TERT transcript stability as the underlying molecular mechanism. In summary, high-level telomerase activity caused by genomic rearrangements in neuroblastoma models is suppressed by treatment with clinically approved HDAC inhibitors, suggesting indirect druggability and a potential molecular rationale for therapeutic intervention.

Muscle invasive bladder cancer (MIBC) has a poor prognosis. Currently, therapy consists of radical radiotherapy or cystectomy with or without chemotherapy. The average age of patients with MIBC is high and older patients are less able to tolerate surgery or chemoradiation due to their impaired physical fitness and generally poor renal function. There is an urgent need to find new treatment regimes that are both tolerable and effective. The aims of this project were to investigate the radiosensitising effects of the histone deacetylase (HDAC) inhibitor panobinostat in bladder cancer cell lines, with the ultimate goal of proposing a novel radiosensitising therapy for MIBC, and to study the effects of panobinostat on the major DNA double strand break (DSB) repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ), to determine the predominant pathway targeted and to look further upstream at effects on the MRE11/RAD50/NBS1 (MRN) complex. The HDAC inhibitor panobinostat was found to be toxic in the low nanomolar range and significant radiosensitising effects were demonstrated at doses lower than IC50 in all the bladder cell lines studied. The radiosensitising effect of panobinostat was not influenced by TP53 status, which is generally regarded as an important determinant of bladder cancer response to radiotherapy. In the “synthetic lethality” bladder cancer cell model, panobinostat predominantly targets the HR pathway, reportedly the only proficient DNA repair pathway in MIBC. HR proteins RAD51 and CtIP were downregulated upon panobinostat treatment in a dose-dependent manner. Upstream, MRE11 and NBS1 proteins were also targeted by panobinostat, with levels slightly decreased in RT112 and T24 cells and in CAL29 cells post-ionising radiation. In summary, the HDAC inhibitor panobinostat was shown to be an efficient radiosensitiser in bladder cancer cells at low toxic doses and to predominantly target the HR pathway. These findings are promising and may contribute towards establishing a novel combination therapy of panobinostat with IR for MIBC patients.

Statins are widely used to lower cholesterol and thus reduce cardiovascular risk, although they increase diabetes risk. Butyrate, which is produced by gut bacteria and alters gene expression through epigenetic means, lowers cholesterol and protects against diabetes in animals. This thesis compares the effects of statins and butyrate on epigenetics, cholesterol metabolism and glucose metabolism in cultured cells. Both statins and butyrate lowered cellular cholesterol and impaired insulin secretion, although they acted through different mechanisms.

NTECEDENTS: els inhibidors d’histona deacetilasa (HDACi) han emergit els últims anys com potencials tractaments dirigits, amb la finalitat de revertir els canvis epigenètics associats a diferents tipus de càncer, incloent les neoplàsies hematològiques. La falta d’especificitat dels HDACi, però, dificulta la predicció dels seus efectes biològics i provoca una toxicitat no menyspreable, el que ha limitat el seu ús en la pràctica clínica. L’expressió de les HDACs no ha estat estudiada en les leucèmies agudes pediàtriques més que de manera parcial i en sèries amb un limitat número de pacients; per tant, són necessaris estudis que ajudin a aclarir el paper de cadascuna de les HDACs com a possibles biomarcadors i com a dianes terapèutiques, de cara a dirigir el tractament específic i personalitzat amb HDACi. HIPÒTESI: en aquest context, l’estudi global del perfil transcripcional de les HDACs en una sèrie àmplia i representativa de pacients pediàtrics amb diferents subtipus de leucèmia aguda permetria definir millor el seu impacte pronòstic i definir el seu rol com a dianes terapèutiques, ajudant a dirigir el tractament específic amb HDACi de forma més racional i individualitzada. OBJECTIUS: estudiar de forma global el perfil d’expressió de les HDAC1-11, SIRT1, SIRT7 i d’altres gens co-reguladors, MEF2C i MEF2D, en una sèrie de pacients pediàtrics amb leucèmia aguda diagnosticats i tractats en un sol centre. METODOLOGIA: anàlisi de l’expressió de l’mRNA de les HDAC1-11, SIRT1, SIRT7, MEF2C i MEF2D mitjançant PCR quantitativa en 211 pacients pediàtrics (0-18 anys), diagnosticats de leucèmia de novo des de l’any 2003 al 2017 en el nostre centre. Els pacients es van tractar uniformement d’acord als protocols consecutius de la Sociedad Española de Hematología y Oncología Pediátrica (SEHOP). Es va emprar un pool de pacients no-neoplàsics com a calibrador i també es va analitzar l’expressió dels diferents gens en cèl·lules CD34+ normals de moll d’os i a cèl·lules B CD19+ madures i limfòcits T madurs de sang perifèrica. RESULTATS: l’expressió de les HDACs va diferir en els diferents subtipus de cèl·lules hematopoètiques normals d’acord amb el grau de maduració i el llinatge. En els pacients amb leucèmia es va observar, en general, una sobreexpressió de les HDACs, amb perfils específics que correlacionaven amb característiques clíniques i biològiques i, fins i tot en algunes ocasions, amb la supervivència dels pacients. Així, alguns perfils d’expressió d’HDAC i el perfil de MEF2C semblaven reflectir, probablement, el llinatge i la maduresa dels blasts, mentre que d’altres identificaven la via oncogènica activa en les cèl·lules leucèmiques. Concretament, es va identificar un perfil d’expressió distintiu pels pacients amb reordenament del gen KMT2A (MLL), amb nivells elevats d’HDAC9 i MEF2D, independentment de l’edat, el partner del gen de fusió i el llinatge. A més, es va observar en els pacients amb LLA-B un pronòstic advers de la sobreexpressió de l’HDAC9, independentment de l’estat del gen KMT2A. CONCLUSIONS: en aquest treball s’han observat diferents perfils d’expressió segons els diferents subtipus de leucèmia aguda pediàtrica. Concretament, s’ha identificat un perfil distintiu dels pacients amb reordenament del gen KMT2A, amb la sobreexpressió de l’ HDAC9 i MEF2D, independentment del llinatge, de l’edat i del partner del gen de fusió. Aquests resultats aporten una imatge global de l’expressió de les HDACs en els pacients pediàtrics amb leucèmia aguda i recolzen el tractament dirigit amb HDACi més específics en seleccionats grups de pacients d’alt risc, com els pacients amb reordenament del gen KMT2A.
Histone deacetylase inhibitors (HDACi) emerged as promising drugs in leukaemia, but their toxicity due to lack of specificity limited their use. Therefore, there is a need to elucidate the role of HDACs in specific settings. The study of HDAC expression in childhood leukaemia could help to choose more specific HDACi for selected candidates in a personalized approach. We analysed HDAC1-11, SIRT1, SIRT7, MEF2C and MEF2D mRNA expression in 211 paediatric patients diagnosed with acute leukaemia. We found a global overexpression of HDACs, while specific HDACs correlated with clinical and biological features, and some even predicted outcome. Thus, some HDAC and MEF2C profiles probably reflected the lineage and the maturation of the blasts and some profiles pointed out specific oncogenic pathways active in the leukaemic cells. Specifically, we identified a distinctive signature for patients with MLL rearrangement, with high HDAC9 and MEF2D expression, regardless of age, MLL-partner and lineage. Moreover, we observed an adverse prognostic value of overexpression of HDAC9, regardless of MLL rearrangement. Our results provide useful knowledge on the complex picture of HDACs expression in childhood leukaemia and support the directed use of specific HDACi to selected paediatric patients with acute leukaemia.

Although curable in the majority of cases, Diffuse Large B-cell Lymphoma (DLBCL), the most prevalent Non-Hodgkin Lymphoma (NHL) throughout the world, is still fatal for 30-40% patients. This patient population could benefit from the addition of new drugs to the current DLBCL chemotherapy regimen. Histone deacetylase inhibitors (HDIs) are a promising group of drugs for the treatment of hematological malignancies. In the current study we tested the HDI PXD101 in a panel of the two most common DLBCL subtypes, GCB (germinal center) and ABC (activated B-cell like), ABC being the least curable subtype. Cell viability assays showed that PXD101 induces antiproliferative effects at submicromolar concentrations in DLBCL cell lines regardless of DLBCL subtype. Flow cytometry demonstrated that upon PXD101 treatment two GCB cell lines (DB and OCILY19) undergo G2M cell cycle arrest followed by apoptosis, while two GCB (SUDHL4 and SUDHL8) and one ABC (U2932) cell line undergo G1 arrest with little apoptosis. Further experiments demonstrated that upon PXD101 removal G1-arresting cells recover their normal proliferative state, while in G2M-arresting cells only 8h exposure to PXD101 is sufficient to induce considerable apoptosis. We classified as PXD101-resistant cell lines that re-enter the cell cycle after drug removal, and PXD101-sensitive cell lines that commit to apoptosis after short periods of drug exposure. Kinase assays established that upon PXD101 treatment G1 phase cyclin dependent kinase 2 (CDK2)-cyclin E complex activity significantly decreases in resistant but not in sensitive cells lines. Furthermore, pull-down assays revealed that CDK inhibitors (CDKIs) p21 and/or p27 in resistant, but not sensitive cell lines persistently bind the CDK2-cyclin E complex throughout PXD101 treatment, thereby explaining why resistant lines stop at the G1 phase. CDKIs induction by PXD101 was p53-independent. This is the first time that an in vitro model of sensitivity and resistance to HDIs in DLBCL is established. We have also performed preliminary genomic and proteomic analysis in DLBCL cell lines treated with PXD101. We anticipate that further analysis of the genomic response and the functional impact of protein acetylation induced by HDIs will offer additional insight into mechanisms of sensitivity and resistance to HDIs in DLBCL.

Epigenetic therapies of cancer have been intensely studied, because of the high frequency of epigenetic alterations found in tumor cells. Epigenetic modulators, such as histone deacetylases (HDAC) and DNA methyltransferase (DNMT), can be targeted by specific drugs with the intent to revert the epigenetic alterations induced by tumor progression. Although a few DNMT inhibitors have been approved for the therapy of some specific hematopoietic diseases, most of the results of clinical trials on other types of cancer have given limited results. The need for a better understanding of the molecular mechanism(s) underlying sensitivity to epigenetic therapies is therefore very strong. For obvious reasons, the use of preclinical models is mandatory to achieve this deeper understanding, to be translated to better clinical trials. We therefore used one form of acute myeloid leukemias (APL) as a preclinical model for epigenetic therapies. The choice of APL is due to the fact that the APL-associated fusion protein PML/RARα is known to induce epigenetic alterations in APL blasts by recruiting HDACs and DNMTs, making APL blasts obvious candidates for this kind of drugs. Monotherapies of either HDACi (VPA) or DNMTi (DAC) induced increased survival in APL mice and their combination were able to further prolong survival of APL mice, demonstrating the importance of hitting multiple targets of the epigenetic “Silencing Loop”. Both the studied epigenetic drugs also demonstrated an enhancement of the therapeutic effect of differentiating therapy with All Trans Retinoic Acid (ATRA). We also tried to identify the molecular mechanisms involved in anti-leukemic activity. Both VPA and DAC demonstrated an induction of the apoptotic response in leukemic cells through the upregulation of members of the Death Receptor pathways. When treating with VPA or DAC, we also observed some modifications in chromatin structure and DNA methylation pattern in a specific CpG rich region within the TRAIL promoter. These results suggest a partial direct epigenetic mechanism underlying TRAIL induction by DAC and VPA. We were able to confirm the results obtained in the APL model in an unrelated form of AML derived from the expression of AML1/ETO fusion protein, suggesting that a substantial fraction of AML patients could benefit from epigenetic treatments, and further reinforcing our view that the APL model represents a paradigm for the study of epigenetic drugs.

Les résultats insuffisants de la radiochimiothérapie conventionnelle dans les cancers bronchiques non à petites cellules (CBNPC) ont motivé l’évaluation d’une nouvelle stratégie de modulation pharmacologique de la radiosensibilité tumorale basée sur les modifications épigénétiques. Pour cela nous avons évalué la combinaison de la radiothérapie et d’un nouvel pan-inhibiteur des histones déacétylases (HDACi), l’abexinostat en préclinique in vitro et in vivo sur deux modèles de CBNPC puis en phase clinique précoce chez l’homme dans le cadre d’un essai de phase I. Nous avons d’abord montré que l’abexinostat augmente la radiosensibilité des cellules de CBNPC de manière dépendante de la séquence thérapeutique en normoxie et en hypoxie en augmentant l’apoptose caspase dépendante ainsi que les cassures doubles brins radio-induites et en réduisant la signalisation et la réparation de ces dommages de l’ADN. L’abexinostat potentialise également le retard de croissance tumorale induit par la radiothérapie in vivo dans des xénogreffes sous-cutanées de CBNPC avec un profil de toxicité acceptable. Nous avons également montré pour la première fois que la triple combinaison de radiothérapie, abexinostat et cisplatine potentialise le retard de croissance tumorale in vivo.Les premiers résultats in vitro et in vivo confortant le rationnel pour la combinaison abexinostat-radiothérapie nous avons réalisé étude de phase I exploratoire d’escalade de dose combinant l’abexinostat à la radiothérapie palliative hypofractionnée standard délivrant 30y en 10 fractions pour des tumeurs solides métastatiques. Parmi les 58 patients traités, d’âge médian 61 ans (20-82), on note 71% de stade M1 et 88% de patients ayant déjà reçu des traitements préalables par chimiothérapie et/ou radiothérapie et/ou chirurgie. La dose recommandée pour un essai de phase 2 que nous avons établie est de 90mg/m². Sur les 51 patients évaluables, on observe un taux de réponse globale de 7,8% (1 réponse complète (RC) et 3 réponses partielles (RP)) et un taux de réponse locorégionale de 11,8% (1 RC et 5 RP) avec un suivi médian de 16 semaines. Les patients présentant des lésions (cibles ou non) cérébrales ont présenté des taux de réponse encourageant avec notamment un patient en RC. Nous avons retrouvé peu d’effets secondaires de grade ≥3, les plus fréquents étant la thrombopénie (17,2%), la lymphopénie (12,1%) et l’hypokaliémie (6,9%). Au total, 6 patients (10%) ont interrompu leur traitement du fait des effets secondaires. Nous n’avons pas observé de prolongation de l’intervalle QTc de grade ≥3 et il n’y a pas eu d’interruption de traitement en rapport avec cet effet secondaire. Dans l’ensemble nos données in vitro et in vivo montrent une potentialisation de l’effet antitumoral par la combinaison d’abexinostat et radiothérapie. Chez les patients présentant des tumeurs solides avancées l’abexinostat oral en combinaison à la radiothérapie est bien toléré. Cette combinaison pourrait avoir un potentiel particulièrement intéressant dans le traitement des métastases cérébrales.De plus nos travaux précliniques suggèrent pour la première fois un effet prometteur d’une triple combinaison avec HDACi, cisplatine et radiothérapie qui justifie de plus amples investigations et pourrait guider de nouvelles stratégies thérapeutiques dans les CBNPC.Nos travaux s’inscrivent dans une stratégie de recherche translationnelle et montrent l’importance de la recherche préclinique pour les études d’association aux rayonnements ionisants. Seuls un développement préclinique rationnel et un développement clinique méthodique permettront l’émergence de combinaisons modulant la radiosensibilité tumorale de manière suffisamment efficace pour modifier nos standards de traitement et améliorer le pronostic de nos patients
Insufficient results of conventional chemoradiotherapy in non-small cell lung carcinomas (NSCLC) have encouraged assessment of new pharmacological strategies for modulation of radiosensitization based on epigenetic changes. We have investigated the combination of radiotherapy and a novel pan-histone deacetylase inhibitor (HDACi), abexinostat in vitro and in vivo in two NSCLC models and in an early phase clinical trial. Our findings demonstrate that abexinostat enhances radiosensitivity of NSCLC cells in a time dependent way in vitro in normoxia and hypoxia increasing radio-induced caspase dependent apoptosis and persistent DNA double strand breaks associated with decreased DNA damage signaling and repair. Interestingly, abexinostat potentiates tumor growth delay in vivo in combined modality treatments associating not only abexinostat and irradiation but also in the triplet combination of abexinostat, irradiation and cisplatin.We conducted an exploratory phase 1, dose-escalation study of abexinostat in combination with standard hypofractionated radiotherapy (30Gy in 10 fractions) in patients with advanced solid tumors treated in a palliative setting. Among 58 treated patients, the median age was 61.5 years (range, 20-82); 47% of the patients had M1 stage disease, and 95% had received previous chemotherapy alone or chemotherapy in combination with surgery and/or radiotherapy. The recommended phase 2 dose was determined to be 90 mg/m2. Of the 51 patients evaluable for response, best overall response was 8% (1 complete response [CR], 3 partial responses [PRs]), and best loco-regional response was 12% (1 CR and 5 PRs) at a median follow-up of 16 weeks. Of note, patients with target or non-target brain lesions showed encouraging responses, with 1 patient achieving a best loco-regional response of CR. Treatment-emergent grade ≥3 adverse events (AEs) were few, with most common being thrombocytopenia (17%), lymphopenia (12%), and hypokalemia (7%). Six patients (10%) discontinued treatment due to AEs. No grade ≥3 prolongation of the QTc interval was observed, with no treatment discontinuations due to this AE.Altogether, our data demonstrate in vitro and in vivo a potentiation of anti-tumor effect by abexinostat in combination with irradiation in NSCLC. Oral abexinostat combined with radiotherapy was well tolerated in patients with advanced solid tumors. The combination may have potential for treatment of patients with brain lesions.Moreover, our work suggest for the first time to our knowledge promising triple combination opportunities with HDACi, irradiation and cisplatin which deserves further investigations and could be of major interest in the treatment of NSCLC.Our studies which are part of a translational research strategy highlight the importance of preclinical investigations in the area of radiotherapy and drug combination research. Only rationale preclinical development and methodologically well conducted clinical development will allow new standards of treatment to emerge and improve patient’s prognostic

Therapeutic responses to Histone deacetylase (HDAC) inhibitors (HDACi) in many cancers are well described but development of resistance to HDACi is a major stumbling block. Whether HDACis induce epigenetic reprogramming and how this contributes to relapse is not reported. A CTCL cell line HuT78, and a CLL cell line MEC1, were used to develop HDACi resistant clones (RHuT78 and RMEC1 respectively) that persistently grow in the presence of the clinically used HDAC inhibitor Romidepsin. RHuT78 cells show perturbed trimethylation of histone H3 lysine K4 on Romidepsin treatment which linked to higher protein expression levels of the implicated demethylase KDM5A. Following on from these experiments, a qRT-PCR epigenetic gene expression array was used to quantify levels of 84 epigenetic gene transcripts in RHuT78 cells and significantly altered genes were taken forward for further investigation. Studies of gene expression patterns in parental, resistant and ‘drug holiday’ cell lines of both HuT78 and MEC1 led to particular interest in HDAC8, DNMT3A and DNMT3B. Functional studies showed that HDAC8 overexpression increased proliferation and resistance of HuT78 cells to Romidepsin. Parallel observations suggested an increase in proliferation of resistant cell lines cultured in the presence of the HDACi. This increased proliferation was seen even with lower concentrations of Romidepsin and argues against prolonged monotherapy using HDACis. Significantly, inhibitors of DNA methyltransferases synergised with Romidepsin in a dose and schedule dependent manner, reversing the changes in epigenetic gene expression associated with resistance and causing increased apoptosis in RHuT78 cells. Taken together this thesis identifies and characterises an unacknowledged contribution of epigenetic reprogramming to drug resistance and provides insights into the effects of Romidepsin on the epigenome that could potentially contribute to HDACi resistance.

Le développement embryonnaire est un processus hautement contrôlé où différentes voies de signalisation se coordonnent pour la construction d'un organisme. L'une des principales voies de signalisation impliquées dans ce processus est la voie canonique Wnt. La longue quête pour comprendre la cascade de signalisation Wnt/β-catenine a révélé que la réponse transcriptionelle induite par le signal Wnt/β-catenine est dépendante du contexte, ou compétence, cellulaire. Peu de choses sont connues sur les évènements moléculaires qui influencent cette compétence cellulaire. Dans les embryons de X. laevis Wnt/β-catenine est le signal inducteur pour l'Organisateur de Spemann. On ne sait pas ce qui limite l'activité Wnt dans ce territoire et par voie de conséquence la taille de l'Organisateur. Les résultats présentés dans ce manuscrit de thèse montrent que le facteur de transcription Barhl2 affecte le développement de l'organisateur de Spemann. Nous démontrons que Barhl2 inhibe l'activité Wnt via son interaction avec le corépresseur Groucho et le facteur de transcription Tcf, et mobilise l'activité de Hdac1 qui agit sur la structure chromatinienne. En utilisant des expériences in vitro et in vivo sur des cellules en culture et des embryons de Xénope nous démontrons que la régulation de Barhl2 sur les activités Groucho-Tcf est maintenue pendant l'embryogenèse et joue un rôle dans le confinement des progéniteurs neuraux dans le cerveau. Ensemble, nos résultats fournissent un mécanisme nouveau et important agissant sur le contrôle de l'activité transcriptionelle Wnt et la compétence des cellules à répondre à ce signal
Embryonic development is a highly controlled process where different signaling pathways participate into the elaboration of an organism. One of the main signaling pathways is the Wnt canonical pathway. The long-lasting search to understand Wnt/β-catenin transduction cascade revealed that the net transcriptional read out of Wnt/β-catenin signaling is highly dependent on the cellular context. In X. laevis embryos Wnt/β-catenin signaling is the informative signal for the Spemann Organizer induction. However little is known on what limits Wnt activity in this territory and consequently the size of the Spemann Organizer. The results presented in this manuscript provide evidence that the evolutionarily conserved transcription factor Barhl2 limits the development of the Spemann organizer. In this territory Barhl2 inhibits Wnt activity via its interaction with the co-repressor Groucho and the transcription factor Tcf. It participates to the recruitment of the chromatin remodeling enzyme, Hdac1 that represses the expression of Spemann organizer genes. Using a Xenopus tropicalis Tcf reporter line we demonstrate that Barhl2 inhibitory effect on Groucho-Tcf activities is maintained during embryogenesis and plays a role in the confinement of neural progenitors in the brain. Together, our results provide a new and important mechanism for the control of Wnt transcriptional activity

Il Mieloma Multiplo (MM) e’ un tumore ematologico che colpisce le plasma cellule (PCs) nel midollo osseo. Lo scambio di informazioni tra le MM-PCs e il microambiente nel midollo osseo, tra le quali quelle dovute alle interazioni cellula-cellula, il rilascio di fattori pro-sopravvivenza e vescicole extracellulari (EV), promuove la sopravvivenza tumorale e la resistenza ai farmaci. All’inizio la mia ricerca si e’ focalizzata sulla caratterizzazione del contenuto proteico delle EVs rilasciate dalle cellule di MM. Tra tutte le protein identificate, la glicoproteina CD44 e’ una delle piu’ abbondanti ed e’ stata gia’ associata, sia in vitro che in vivo, con la resistenza del mieloma multiplo al dexamethasone e alla lenalidomide. L’analisi di 200 campioni di siero estratti da pazienti affetti da mieloma multiplo mostra che il CD44 circolante e trasportato dalle MM-EVs correla con ISS stage e i livelli di β2microglobulina e costituisce un potenziale fattore prognostico, fornendo in questo modo il razionale per successive investigazioni di nuovi biomarker associati con lo stato della malattia. Nonostante le molte opzioni terapeutiche possibili, il MM e’ inevitabilmente associato con la resistenza e la scarsa efficacia farmacologica. Gli inibitori delle istoni-deacetilasi (HDACi) costituiscono una nuova classe di chemioterapici in valutazione in trials clinici per il trattamento di pazienti affetti da mieloma multiplo. Anche se gli studi preclinici sugli HDACi hanno dimostrato la loro attivita’ anti-mieloma, nella clinica i trattamenti con gli HDACi sono purtroppo limitati a causa della loro bassa tollerabilita’. Noi crediamo che gli HDACi possano costituire un valido supporto se impiegati in combinazione con lo standard terapeutico per il mieloma multiplo. In questa tesi mostro che un nuovo panHDACi, l’AR42, abbassa l’espressione del CD44. Questa down-regolazione e’ in parte mediata dal miR-9-5p, il quale sopprime l’espressione dell’insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), uno stabizzatore dell’ RNA messaggero del CD44. Abbiamo dimostrato che l’AR42 aumenta l’attivita’ della lenalidomide sia in cellule primarie di MM isolate da pazienti refrattari al trattamento con la lenalidomide sia in modello murino di mieloma multiplo. In conclusione, i nostri dati suggeriscono una nuova potenziale combinazione terapeutica che modula l’espressione del CD44 e che potrebbe aiutare ad attenuare la resistenza alla lenalidomide nei pazienti affetti da mieloma.
Multiple myeloma (MM) is a hematological malignancy of plasma cells (PCs) in the bone marrow. The interplay between MM-PCs and bone marrow microenvironment, including cell-cell contacts and release of pro-survival factors and extracellular vescicles (EV), promotes cancer cell survival and drug resistance. At first my research was focused on the characterization of the proteomic content of EVs secreted by MM cell lines. Among them, the glycoprotein CD44 is one of the most abundant proteins and has been already associated, both in vivo and in vitro, with lenalidomide and dexamethasone resistance in multiple myeloma. The analysis of serum samples from a cohort of 200 MM patients shows that circulating CD44 carried by MM-EVs correlates with ISS stage and β2microglobulin and constitutes a potential prognostic factor, thus providing the rationale to further explore novel molecular players associated with MM disease. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACi’s) are promising novel chemotherapeutics under evaluation in clinical trials for the treatment of MM patients. Although in preclinical studies HDACi’s have proven anti-myeloma activity, in the clinics single-agent HDACi treatments have been limited due to low tolerability. We believe that HDACi could constitute a valid support if used in combination with the MM state of care. In this thesis I show that a novel pan-HDACi AR42 downregulates CD44. Moreover, the CD44 downregulation is in part mediated by miR-9-5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we demonstrate that AR42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in MM mouse model. In conclusion, our observations suggest a potential novel combinatorial therapeutic approach modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients.

Systemic lupus erythematosus (SLE) is a chronic multifactorial inflammatory autoimmune disease with heterogeneous clinical manifestations. Among different manifestations, lupus nephritis (LN) remains a major cause of morbidity and mortality. There are few FDA approved treatments for LN. In general, they are non-selective and lead to global immunosuppression with significant side effects including an increased risk of infection. In the past 60 years, only one new drug, belimumab was approved for lupus disease with modest efficacy in clinic and not approved for patients suffering for nephritis. Therefore, it is urgent to develop new treatments to replace or reduce the use of current ones. Histone deacetylase 6 (HDAC6) plays a variety of biologic functions in a number of important molecular pathways in diverse immune cells. Both innate and adaptive immune cells contribute to pathogenesis of lupus. Among those cells, B cells play a central role in pathogenesis of lupus nephritis in an anti-body dependent manner through differentiation into plasma cells (PCs). As a result, HDAC6 inhibitors represent an entirely new class of agents that could have potent effects in SLE. Importantly, the available toxicity profile suggests that HDAC6 inhibitors could be advanced into SLE safely. We have demonstrated previously that histone deacetylase (HDAC6) expression is increased in animal models of systemic lupus erythematosus (SLE) and that inhibition of HDAC6 decreased disease. ACY-738 is a hydroxamic acid HDAC6 inhibitor that is highly selective for HDAC6. In our current studies, we tested if an orally selective HDAC6 inhibitor, ACY-738, would decrease disease pathogenesis in a lupus mouse model with established early disease. Moreover, we sought to delineate the cellular and molecular mechanism(s) of action of a selective HDAC6 inhibitor in SLE. In order to define the mechanism by which HDAC6 inhibition decreases disease pathogenesis in NZB/W mice by using RNAseq to evaluate the transcriptomic signatures of splenocytes from treated and untreated mice coupled with applied computational cellular and pathway analysis. In addition, we sought to bridge between the transcriptomic data obtained from the HDAC6 treated mice and human gene expression information to determine the relevance to this target in possibly controlling human lupus. We treated 20-week-old (early-disease) NZB/W F1 female mice with two different doses of the selective HDAC6 inhibitor (ACY-738) for 4~5 weeks. As the mice aged, we determined autoantibody production and cytokine levels by ELISA, and renal function by measuring proteinuria. At the termination of the study, we performed a comprehensive analysis on B cells, T cells, and innate immune cells using flow cytometry and examined renal tissue for immune-mediated pathogenesis using immunohistochemistry and immunofluorescence. We then used RNAseq to determine the genomic signatures of splenocytes from treated and untreated mice and applied computational cellular and pathway analysis to reveal multiple signaling events associated with B cell activation and differentiation in SLE that were modulated by HDAC6 inhibition. Our results showed a reduced germinal center B cell response, decreased T follicular helper cells and diminished interferon (IFN)-γ production from T helper cells in splenic tissue. Additionally, we found the IFN-α-producing ability of plasmacytoid dendritic cells was decreased along with immunoglobulin isotype switching and the generation of pathogenic autoantibodies. Renal tissue showed decreased immunoglobulin deposition and reduced inflammation as judged by glomerular and interstitial inflammation. The molecular pathways by which B cells become pathogenic PC secreting autoantibodies in SLE are incompletely characterized. RNA sequence data showed that PC development was abrogated and germinal center (GC) formation was greatly reduced. When the HDAC6 inhibitor-treated lupus mouse gene signatures were compared to human lupus patient gene signatures, the results showed numerous immune and inflammatory pathways increased in active human lupus were significantly decreased in the HDAC6 inhibitor treated animals. Pathway analysis suggested alterations in cellular metabolism might contribute to the normalization of lupus mouse spleen genomic signatures, and this was confirmed by direct measurement of the impact of the HDAC6 inhibitor on metabolic activities of murine spleen cells. Taken together, these studies show selective HDAC6 inhibition decreased several parameters of disease pathogenesis in lupus-prone mice. The decrease was in part due to inhibition of B cell development and response. RNA sequence data analysis show HDAC6 inhibition decreases B cell activation signaling pathways and reduces PC differentiation in SLE and suggests that a critical event might be modulation of cellular metabolism.
Doctor of Philosophy
Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease by which immune cells mistakenly attacks healthy self-cells in different organs. Kidney inflammation occurs in nearly 50% of patients with lupus resulting in kidney damage leading to end stage renal disease. Lupus nephritis (LN) is major cause of morbidity and mortality associated with SLE. Current treatments for LN consist primarily of immunosuppressants that block the immune response and leave the patients with unwanted side effects including an increased risk of infection. To circumvent the unwanted side effects, we explored a novel mechanism to target the immune response. My project was to determine whether histone deacetylase 6 (HDAC6) inhibition would suppress the autoimmune inflammatory response in lupus. We found that inhibition of HDAC6 was effective at attenuating early LN, probably by down-regulating innate immune response, which suppressed subsequent adaptive immune responses downstream. HDAC6 inhibition affected the innate immune response by inhibiting type I interferon production by plasmacytoid dendritic cells. HDAC6 inhibition affected the cell mediated immune response by decreasing T helper cell and B cell activation. To determine the mechanism by which HDAC6 inhibits immune cells activation, we used RNAseq to reveal HDAC6 inhibition on multiple signaling events associated with the induction of lupus disease. These results suggest that HDAC6 could be a potential therapeutic target in the early stage of LN.

Inhibitors of histone deacetylases (HDAC) are used widely in medicine. ITF2357 is a hydroxamic acid-containing, orally active HDAC inhibitor with anti-inflammatory properties. In endotoxin-stimulated human PBMC, ITF2357 at 50 to 125 nM inhibited the release of TNFalpha and IL-1β by more than 70%, respectively. The induction of IFNgamma by the combination of IL 18 plus IL 12 was also reduced by ITF2357. We report here a Phase I safety and pharmacokinetic trial. After an oral dose of 50 mg, the maximal plasma concentration was 104 nM at 2 hours post dosing with a half-life of 6.9 hours. For the 100 mg dose, the Cmax was 199 nM at 2.1 hours and a half life of 6.0 hours. For a dose of 200 mg, the Cmax was 470 nM after 2.1 hours and a half-life of 6.0 hours. Repeat daily doses of ITF2357 for 7 consecutive days of 50, 100 or 200 mg resulted in nearly the same Cmax and half-life each day as was observed following a single dose. Side effects were limited to reductions in platelets (10-25% of baseline) within the first week after 7 days of treatment, reached a nadir point after 2 to 3 weeks and resolved fully within 2 to 3 weeks. Whole blood was cultured with endotoxin (10 ng/mL). Production of TNFα progressively decreased reaching a nadir 4 hours after oral dosing (45 and 52% of the pre-drug level for 50 and 100 mg, respectively). Similar decreases were observed after 4 hours for IL 1 beta, IL 6 and interferon gamma but not for IL 8. The IL 1 receptor antagonist did not decrease in the same samples. After 24 hours, the levels had returned to base line measurements. In a one week-long multiple dosing schedule, the fall in cytokine production in blood cultures observed on day 7 was nearly the same as that of the first day. We conclude that ITF2357 is a safe and effective therapy for reducing cytokine production following oral dosing.
Gli inibitori delle istone deacetilasi (HDAC) sono stati recentemente introdotti nella terapia medica oncologica. ITF2357 (Givinostat) è un inibitore delle HDAC, attivo per via orale e con proprietà anti-infiammatorie. In colture di PBMC umani stimolati con LPS, ITF2357 alle concentrazioni da 50 a 125 nM ha dimostrato efficacia nell’inibire il rilascio di TNFα ed IL-1β. Anche l'induzione di IFNγ mediante la combinazione di IL-18 ed IL-12 è ridotta in vitro. Il seguente rappresenta uno studio di Fase I relativo alla sicurezza e alla farmacocinetica di ITF2357 in volontari sani. Dopo una dose orale di 50 mg, la concentrazione plasmatica massima è stata di 104 nM a 2 ore dalla somministrazione, con una emivita di 6,9 ore. Per la dose di 100 mg, la Cmax è stata di 199 nM a 2,1 ore e con un’emivita di 6,0 ore. Per la dose di 200 mg, la Cmax è stata di 470 nM dopo 2,1 ore e una t1/2 di 6,0 ore. Ripetute dosi giornaliere di ITF2357 per 7 giorni consecutivi (50, 100 o 200 mg) hanno generato la stessa Cmax ed emivita osservate dopo una singola dose. Gli effetti collaterali si sono limitati ad una riduzione delle piastrine (10-25% del basale) entro la prima settimana di assunzione con un nadir al settimo giorno ed un ripristino completo dopo 3 settimane. Il sangue intero dei soggetti in studio è stato incubato con lipolisaccaride (10 ng/mL). La produzione di TNFα è risultata ridotta raggiungendo un nadir a 4 ore dall’assunzione orale (-45 e -52% comparati con i livelli pre-assunzione per i dosaggi di 50 e 100 mg, rispettivamente). diminuzioni simili sono state osservate dopo 4 ore per IL-1beta, IL-6 ed IFNgamma ma non per IL-8 ed IL-1Ra. Nei campioni di sangue prelevati a 24 ore dall’assunzione, i livelli citochinici sono tornati comparabili al basale. Nello studio con dosi multiple, l’effetto anti-citochinico osservato al settimo giorno è stato sovrapponibile a quello del primo. In conclusione ITF2357 è una terapia sicura ed efficace nel ridurre la produzione di citochine in un modello ex-vivo.

SUMMARYHuman papillomaviruses (HPV) are small, non-enveloped viruses with a dsDNA genome. About 40 HPV types infect the mucosa and skin of the anogenital tract, and they are further subdivided into low-risk HPV (LR-HPV) and high-risk HPV (HR-HPV). Long-term, persistent infections with the latter type could result in the development of cervical cancer. Cervical cancer is the second most prevalent cancer worldwide, and the third leading cause of cancer-related deaths in women. HR-HPV DNA is frequently found integrated in cervical carcinoma tissue. Thus, HR-HPV integration is considered to be mechanistically linked to virus-promoted malignancy. Several complex and sometimes inter-linked signal transduction pathways, collectively termed as the DNA Damage Response (DDR), act to sense damage incurred to DNA and ultimately promote its repair. The central players of the DDR are three proteins of the phosphatidylinositol 3-kinase-like protein kinase (PIKK) family, ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and DNA protein kinase catalytic subunit (DNA-PKcs). Also crucial to the DDR are members of the poly(ADP)ribose polymerase family (PARP). Interactions between histones modified by PARP enzymes, the PARP enzymes themselves and the X-ray repair cross-complementing protein (XRCC-1), are important for efficient DNA repair. Reversible acetylation of histones plays a key role in the regulation of gene expression and in the DDR. The acetylation of histones, as well as that of numerous other cellular proteins, is carried out by histone acetyl transferases (HAT). This enzymatic process exists in a tightly-regulated equilibrium with lysine deacetylation, which is catalyzed by histone deacetylases (HDAC). HDAC modulate the expression and/or function of key proteins implicated in cancer. In addition, many HDAC are overexpressed in most cancers. Various small-molecule inhibitors of HDAC have been developed. HDAC inhibitors (HDACi) have been shown to promote cancer cell cycle arrest, differentiation and apoptosis. In addition, HDACi are well-documented for their synergistic or additive effects on DNA-damaging agents used in cancer therapy. In spite of a growing body of research however, the cellular and molecular mechanisms behind this phenomenon have not yet been fully uncovered. We evaluated the impact of several pan-HDACi and HDACi analogues on the extrachromosomal and genomically-integrated HPV-16 LCR-driven transcription in various cell lines. Using a classical reporter construct strategy, we report that the pan-HDACi Trichostatin A (TSA), valproate (VPA) and sodium butyrate (NaB) are potent inducers of transcription from the HPV-16 long control region (LCR) in its extrachromosomal form. This effect of HDACi was at least in part mediated by three binding sites of members of the AP-1 family of transcription factors within the viral LCR. HDACi analogues 1 through 6 did not modulate extrachromosomal HPV-16 LCR transcription in a statistically-significant way.
Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie)
info:eu-repo/semantics/nonPublished